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Hormones and Cytokines
THE EFFECT OF CALCITONIN ON HEALING OF TENDON INJURIES. FOLLOW-UP OF TENDON HEALING WITH MRI, HISTOLOGICAL EVALUATION AND MECHANICAL TESTING. EXPERIMENTAL STUDY IN RABBITS.
H. Petrou1*, T. Karachalios2, L. Khaldi2, K. Kalogera2, G. P. Lyritis2
1Athens University, KAT Hospital, Kifissia, Greece
2Laboratory for the Research of the Musculoskeletal Systeme
Purpose: The purpose of this study was: 1) To evaluate the ability of MRI to follow tendon healing after injuries and 2) to determine if Calcitonin enhances the healing of tendon injuries.
Materials and Methods: 36 New Zealand adult male rabbits sustained a standardised full thickness circular injury of their right Achilles tendon, 2 cm above its calcaneal insertion and covering almost 50 percent of the tendon width.
All animals were divided into two equal groups of 18 animals per group and received a high dose of salmon Calcitonin (21 IU/Kgr) i.m. daily or placebo injection from the day of operation till the day of sacrifice.
The healing process was followed up by MRI weekly. Two rabbits from each group were sacrificed on the first week, 7 rabbits on second and 7 rabbits on third week in order to evaluate the healing process histologically and to perform a tensile stretch test.
Results: Animals of both groups showed a signal reduction of T1 and an increase of the T2-weighted signal on the first week without any intergroup difference. The signals were reduced during the second week and all but disappeared during the third. Histologically, on the first week there was an inflammatory response. On the second week we observed a reduction in cells and an increase in collagen fibers. On the third week we observed maturation of collagen. In the Calcitonin group we noticed increased cellularity, more linear orientation of collagen fibers and more mature healing in contrast with group A.
Moreover, tensile strength of the Achilles tendon was performed and tendons of Calcitonin group has better ultimate tensile strength.
Conclusion: 1) MRI provides an accurate, non-invasive method for following-up the healing process of tendon injuries. 2) Calcitonin enhances the healing of tendon injuries and increases the strength of the tendons.
LACK OF ASSOCIATION BETWEEN ALU I POLYMORPHISM OF CALCITONIN RECEPTOR GENE AND BONE MASS IN CZECH POSTMENOPAUSAL WOMEN
I. Zofková1*, K. Zajíèková1, R. Bahbouh2
1Institute of Endocrinology, Prague, Czech Republic
2Faculty of Medicine, Charles University, Prague, Czech Republic
The calcitonin receptor (CTR)gene represents a novel factor in the number of genes affecting bone mineral density (BMD). The objective of this study was to determine whether BMD and parameters of bone remodeling are dependent on the CTR gene polymorphism.
Methods. One hundred and fourteen postmenopausal women (mean age 62,47±8,95 years)(SD) took part in the study (65 were osteoporotic, 16 were osteopenic and 33 women were normal controls). The T and C alleles of CTR gene were designated according to presence of the Alu I restriction enzyme site. BMD (g/cm2) at the femoral neck and the spine were assessed by dual X-ray absorptiometry. Bone remodeling was determined by serum cross-linked telopeptide of type I collagen, and alcaline phosphatase. The statistical analysis was performed using the nonparametric ANOVA (Kruskal-Wallis) test.
Results. The frequencies of the CTR genotypes in the study population were 57% TT, 39% TC, 4% CC. After adjustment for age and body mass, BMD at the femoral neck or BMD at the spine did not differ in subjects with TT, TC and CC allele combinations. Morover, no relation of these genotypes to indices of bone remodeling were found. All these allel combinations were similarly frequent in osteoporotic (55% TT, 40% TC and 5% CC, respectively) as in healthy women (61% TT, 33% TC and 6% CC, respectively) (Pearson´s Chi-square=0.452, df=2, p=0.7978).
Interpretation. The CTR Alu I gene polymorphism has no effect on bone mass or bone remodeling in postmenopausal women. The results warant further investigation in different ethnic cohorts.
CALICITONIN-RELATED PEPTIDES ACTIVATE PROTEIN KINASE C IN HUMAN OSTEOBLAST-LIKE CELLS
I. Villa1*, C. Dal Fiume1, F. Pagani2, S. Rubinacci1, F. Guidobono2
1Bone Metabolic Unit Scientific Institute H San Raffaele, Milan, Italy
2Dept. of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Italy
Calcitonin (CT), Calcitonin gene-related peptide (CGRP) and amylin are considered member of the same family of peptides. Although they have limited structural homology, they share many biological activities. Like CT, CGRP and amylin lower plasma calcium level, reduce bone resorption and stimulate bone formation. In this study we compared the effect of the three peptides on the proliferation of human osteoblast-like cells (hOB) derived from normal human bone. In hOB, CT increased cell proliferation, as measured by tritiated thymidine incorporation, in a dose-related manner with a maximal effect (+120%) at 10-10M. CGRP showed a similar effect at 10-8M (+130%) and amylin produced a maximal effect (+100%) at 10-8M. On the basis of these results we evaluated the signal trasduction pathways involved in hOB responsiveness to the peptides. We examined the cyclic AMP production and the protein kinase C (PKC) activity in hOB cells after incubation with CT, CGRP or amylin. The results showed a fiftyfold increase in cAMP production after CGRP 10-8M whereas CT and amylin did not stimulate cAMP production up to the concentration of 10-7M. All three peptides were able to activate PKC. CT (10-10M) produced a maximal effect after 5 min incubation; after 30 min the effect was still significant, but less pronounced. CGRP (10-8M) activated PKC with the same time course as CT. The maximal activity was reached after 5 min, after 30 min the effect was still significant and decreased to basal level after 60 min incubation. Amylin (10-9M) reached the maximal effect after 60 min and after 120 min incubation the extent of PKC activation was still pronounced. CT and CGRP have shown a more rapid activation of PKC than amylin. The fact that, of the three peptides, only CGRP stimulated cAMP production suggests that cAMP dependent pathways are not involved in hOB proliferation, moreover that CGRP which is coupled to both cAMP and PKC pathways, could activate different receptors in hOB. In conclusion, these findings suggest that activation of PKC pathway are likely to be responsible for the anabolic effects of the CT family of peptides on human osteoblasts.
HIGH EXTRACELLULAR GLUCOSE CONCENTRATION ENHANCES THE SENSITIVITY OF THE CALCIUM-SENSING RECEPTOR
H. Kanazawa*, H. Tanaka, M. Inoue, Y. Seino
Okayama University Medical School, Okayama, Japan
Aim: Human calcium-sensing receptor (CASR) is expressed
in many organs, senses the extracellular calcium concentration, and plays central role of
calcium homeostasis. Although the CASR is also able to change the sensitivity by ion
strength, the regulatory mechanism of its function has not been well documented. To
clarify the other regulatory factors and their mechanisms, we examined the effect of high
glucose condition on the function and conformation of the CASR.
Materials and Method: HEK 293 cells stably transfected with human CASR gene was used for
experiments. Under high (50mM), moderately high (30mM), and normal glucose condition
(4.5mM) with growing medium, cells were incubated for 72 hours prior to assays. We
examined the response of the CASR to the extracellular calcium concentration by inositol
trisphosphate (IP3) measurement by RIA, phosphatydyl inositol hydrolysis using [3H]
inositol, and intracellular calcium measurement by Fluo3 and Fura2. As osmotic control,
D-mannitol was also added to adjust the osmolarity same to high glucose state.
Result: In high glucose condition, extracellular calcium-stimulated IP3 generation was augmented under the high glucose condition. The amplitude of the augmentation depended on the extracellular glucose concentration. D-mannitol did not augmented IP3 response. In high and moderate glucose condition, extracellular calcium dose response curve was shifted left side compared to normal glucose condition by both IP3 measurement and Fluo3 (ED50 were about 2.8mM, 3.8mM, and 4.5mM by Fluo3, respectively). D-mannitol slightly shifted the curve left compared to that of control. Chorine chloride decreased the elevation of IP3 similar to the previous report.
Discussion: Recently the mechanism of the CASR on sensing extracellular ligands has been known. Our data suggested that high glucose condition, not high osmotic condition increased the responsiveness of the CASR to the elevation of extracellular calcium. This might owe to the property of the glucose. Moreover, this phenomenon may suggest a role of extracellular glucose concentration in the relative hypoparathyroidism of diabetic end stage renal failure.
EFFECT OF OXIDATIVE STRESS PROVOKED BY MENADIONE ON INTESTINAL CALCIUM ABSORPTION.
N. Tolosa de Talamoni*, A. Marchionatti, A. Alisio, G. Diaz de Barboza
Laboratorio de Metabolismo Fosfocalcico y Vitamina D "Dr. Canas", Facultad de Ciencias Medicas, Universidad Nacional de Cordoba, Cordoba, Argentina
In a previous work, we have demonstrated that glutathione (GSH) plays an essential role on the intestinal calcium absorption. Since high doses of menadione (MEN) cause oxidative stress in the liver by different mechanisms involving depletion of GSH, we decided to investigate the effect of MEN on intestinal calcium absorption and associated variables. The experimental animal design was the following: four week old chicks were divided in two groups 1) control ones, 2) treated i.p.with MEN at different times and doses. In all of them we measured intestinal Ca absorption, alkaline phosphatase, sucrose and gamma-glutamyltranspeptidase activities, and intestinal carbonyl groups and GSH content. Reactive oxygen species were analysed by EPR in enterocytes exposed to MEN at different times and concentrations. The data revealed that intestinal Ca absorption was inhibited after 30 min of 2.5 micromol of MEN/kg of b.w. administration. This effect correlated with a significant depletion of intestinal GSH. The activity of intestinal alkaline phosphatase was highly reduced, which lasted for several hours and was time and dose-dependent, whereas the sucrose and gamma-glutamyltranspeptidase activities remained unaltered. The carbonyl group content was increased by the MEN treatment. The EPR spectra indicate that MEN produced a signal that characterizes the presence of free hydroxyl radical groups which are obtained after 15 min of exposure of enterocytes to MEN. The results suggest that MEN produces oxidative stress by GSH depletion and appearance of free hydroxyl radical groups, which might attack sensitive proteins of the enterocyte such as alkaline phosphatase and those involved in the transcellular calcium transport.
A NEW MUTATION IN THE EXTRACELLULAR AMINO-TERMINAL DOMAIN OF THE CALCIUM-SENSING RECEPTOR CAUSING FAMILIAL HYPERCALCIURIC HYPOCALCEMIA
S. Mora1*, M. C. Proverbio1, L. Bolzoni1, G. Colussi2, J. Hu3, K. Jones3, A. M. Spiegel3
1Laboratory of Pediatric Endocrinology, IRCCS S. Raffaele, University of Milan, Italy
2Renal Unit, Niguarda Ca’ Granda Hospital, Milan, Italy
3Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, USA
The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular ion homeostasis. Inactivating and activating mutations have been identified to date in the coding region of the CaR that are associated with inherited human hypo- or hypercalcemic disorders. Here we describe an Italian family with familial syndrome of hypocalcemia with hypercalciuria.
The proband is a woman who experienced nocturnal seizure at 42 years of age due to severe hypocalcemia. Normal to low levels of PTH were measured. A CT scan disclosed bilateral basal ganglia calcification. She was treated with oral calcium and cholecalciferol, and she developed hypercalciuria and finally renal failure. Her daughter, a 25 year old woman, experienced abrupt generalized seizures without sphincter relaxation due to hypocalcemia (3.1 mg/dL ionized calcium). Concurrently, hypercalciuria was detected, and a CT scan disclosed faint basal ganglia calcification. The combination of low serum calcium, hypercalciuria, and improper levels of PTH suggested a defect of CaR.
The sequence analysis of PCR amplified genomic DNA revealed in both patients a heterozygous C to A transversion at position 372 in exon 2 of CaR gene, causing a missense mutation N124K in the extracellular amino-terminal domain of the receptor. Mutations found in this region of the receptor have been reported as activating ones. The pedigree suggested an autosomal dominant inheritance.
Data of functional analysis of the receptor construct carrying this mutation in HEK-293 cells indicate that the mutant form of the receptor is expressed like the wild type, but there is a shift toward the left of the dose-response relationship between the Ca2+ and phosphoinositide hydrolysis (IP). This finding suggests an increased sensitivity of CaR for Ca2+, and therefore confirms the activating nature of the mutation.
INCREASED CALCIUM-SENSING RECEPTOR EXPRESSION IN CALCIUM-SUPPLEMENTED RATS WITH RENAL FAILURE
B. D. Kuizon1*, I. B. Salusky1, D. Shoback2, E. Cambay1, H. Jueppner3, W. G. Goodman1
1UCLA School of Medicine, Los Angeles, USA
2Endocrine Research Unit, VA Medical Center, San Francisco, USA
3Endocrine Unit, Massachusetts General Hospital, Boston, USA
Linear growth is diminished in prepubertal children with adynamic bone. Recent data indicate that impaired growth in uremic rats given calcium supplementation results from decreased angiogenesis and matrix degradation in the growth plate cartilage. To further characterize this disorder, 47 Sprague-Dawley rats underwent either sham procedure (Sh) or 5/6 subtotal nephrectomy (Nx). Animals were given either regular diet (Reg) containing 0.8% Ca, 0.7% P or calcium-supplemented diet (Ca) containing 2.5% Ca, 0.7% P for 4 weeks to induce biochemical changes consistent with adynamic bone. At sacrifice, blood was obtained for ionized Ca, PO4 and PTH determinations. Tissues were fixed by transcardiac perfusion using 4% PFA/PBS. Tibiae were obtained, decalcified in 0.1M EDTA-4% PFA/PBS, and embedded in paraffin. In-situ hybridization was done using 35S-labeled riboprobes for collagen types II and X, PTH/PTHrP receptor and matrix metalloproteinase MMP-9/gelatinase B. Histochemical staining for tartrate-resistant acid phosphatase (TRAP), vascular endothelial growth factor (VEGF) and calcium-sensing receptor (CaSR) was done. TUNEL assay was used to assess apoptosis. Ionized calcium levels were higher, 1.49±0.02 mmol/L (SE) vs 1.37±0.02 mmol/L, p<0.001, whereas PTH levels were lower, 35±8 pg/ml vs 202±24 pg/ml, p<0.001, in Nx-Ca than in Nx-Reg animals. Serum phosphorus levels did not differ among groups. Total body length at sacrifice was less in Nx-Ca than in Nx-Reg, 36.9±0.3 cm vs 37.8±0.3 cm, p<0.05. This was associated with marked increases in both the total width of the growth plate, 407±31 um vs 289±11um, p<0.001, and of the hypertrophic zone, 228±28 um vs 131±6 um, p<0.001 in Nx-Ca than in Nx-Reg. Levels of mRNA expression for collagen types II and X, and PTH/PTHrP receptor did not differ among the 4 groups. In contrast, mRNA expression for matrix metalloproteinase MMP-9/gelatinase B, immunoreactivity for TRAP and VEGF, and the percentage of apoptotic terminal hypertrophic chondrocytes were markedly reduced in Nx-Ca than in Nx-Reg. Immunostaining for CaSR was greater in Nx-Ca than in Nx-Reg and staining did not differ between Sh-Reg and Sh-Ca. These results suggest that alterations in CaSR expression may contribute to the diminished apoptosis, matrix degradation and angiogenesis in uremic rats given calcium-supplementation.
IBANDRONATE PREVENTS THE DEVELOPMENT, AND INDUCES REMISSION OF BONE METASTASES IN A NUDE RAT MODEL OF HUMAN BREAST CANCER
M. Neudert1, C. Fischer1, B. Krempien2, M. J. Seibel1*, F. Bauss3
1Dept. of Medicine, Univ. of Heidelberg, Germany
2Dept. of Pathology, Univ. of Heidelberg, Germany
3Roche Diagnostics GmbH, Mannheim, Germany
In vitro studies have shown nitrogen-containing bisphosphonates to exhibit anti-tumoral effects. Employing a newly developed nude rat model using the human breast cancer cell line MDA-MB-231, we have tested the in vivo anti-tumoral/ anti-osteolytic potential of Ibandronate (IBN) in both preventive and therapeutic settings.
Three groups of male nude rats (rnu/rnu Rowett nudes; n=24/ group) were inoculated into both femoral arteries with 105 cells of the human breast cancer cell line MDA-MB-231. Group 1(controls) received no further intervention. Group 2 (IBN–3) received subcutaneous Ibandronate at 10 microg P/kg/d starting on day –3, while group 3 (IBN18) received identical treatment from day 18 onwards. X-rays were taken on days 0, 6, 12, 18, 22, 26 and 30. Osteolytic areas (OA) were measured by computer based X-ray analysis. In each group, 4 animals were sacrificed at days 6, 12, and 18, while the remaining 12 animals were sacrificed on day 30. Femoral/ tibial bones were taken for histological analyses.
In groups 1 and 3, all animals developed femoral or tibial osteolyses by day 18. Between day 22 and 30, OA increased in controls at a growth rate (GR) of 4.8-7.2mm2/ analytical interval (a.i.), whereas in IBN18, OA increased in 2, remained unchanged in 4, and decreased in 5 animals (mean GR: –0.3 to -0.6 mm2/ a.i.). Histology verified an increase in tumor burden in controls, while reductions to complete remissions were seen in IBN18. In group 2 (IBN–3), only 25% of animals developed osteolyses, the proportion of affected bones was 14.3% and remained stable until sacrifice (mean GR: 0.1 to 1.7 mm2/ a.i.). In some animals sacrificed before day 18, sclerotic areas were seen on histology, possibly corresponding to repaired osteolytic lesions.
We conclude that in the present model, Ibandronate prevents the development, and inhibits or even reverts the growth of established osteolyses, thus leading to a significant reduction in tumor burden. Therefore, Ibandronate may exhibit anti-tumor properties in vivo.
THE COMBINATION OF PAMIDRONATE WITH INTERFERON-ALPHA INDUCES BONE FORMATION IN PATIENTS WITH MULTIPLE MYELOMA IN PLATEAU PHASE
E. Terpos*, N. Viniou, G. Vaiopoulos, J. Meletis, X. Yataganas
First Department of Internal Medicine, University of Athens Medical School, Laiko General Hospital, Athens, Greece
Pamidronate is a second-generation aminobisphosphonate that reduces the disease-related skeletal complications when it is used in combination with chemotherapy in patients with multiple myeloma (MM). It also has an antimyeloma effect and probably inhibits apoptosis of primary osteoblastic cells. Interferon-alpha (IFN-alpha) is used for maintenance treatment in MM and recent studies have demonstrated that it decreases bone resorption and it might increase serum osteocalcin (OSC) when it is given in low doses in rats. The aim of this study was to evaluate the effects of pamidronate on biochemical markers of bone resorption [cross-linked N-telopeptide of type I collagen (NTx)], bone formation [bone alkaline phosphatase (BAP), OSC], disease activity [paraprotein, beta2-microglobulin, CRP] and IL-6 in patients with MM in plateau phase under IFN-alpha maintenance. The inclusion criteria specified a plateau phase of MM after an initial response to induction chemotherapy and a maintenance treatment with IFN-alpha for at least 6 months at a dose of 1.5x106 IU/3 times a week, sc. The above biochemical parameters were evaluated in 31 patients (18M/13F, median age 70y) every 2 months before and 1, 3, 6, 9, 12 and 14 months after the initiation of pamidronate administration which was given at a monthly dose of 90 mg iv. The maintenance treatment with IFN-alpha was given for a median time of 13 months before the addition of pamidronate. A significant reduction of NTx and IL-6 was observed from the 3rd month of the combination treatment and of beta2-microglobulin, CRP and paraprotein from the 6th month, while BAP and OSC were significantly increased from the 6th month. These changes continued during the 14-month period of follow-up. Multivariate analysis showed a significant negative correlation between changes of BAP, OSC and NTx with patients' age. A greater increase of bone formation markers and a greater decrease of NTx levels were observed in younger patients. These results suggest that in addition to the inhibition of osteoclastic activity pamidronate in combination with IFN-alpha was shown to induce bone formation in patients with MM in plateau phase indicating the presence of operating bone remodeling mechanisms when the disease activity is low.
MARKERS OF BONE RESORPTION IN PATIENTS WITH BREAST CANCER RECEIVING PAMIDRONATE
E. Terpos1*, J. Palermos2, M. Vassilaki3, G. Papadopoulos4, G. Vaiopoulos1, J. Meletis1
1First Department of Internal Medicine, University of Athens Medical School, Laiko General Hospital, Athens, Greece
2Department of Immunology, 251 General Air Force Hospital, Athens, Greece
3Hygeia Hospital, Athens, Greece
4Department of Medical Oncology, 251 General Air Force Hospital, Athens, Greece
Bisphosphonates are potent inhibitors of osteoclastic activity and they are used in combination with anticancer treatment in patients with breast cancer and bone metastases, resulted in a significant reduction of skeletal events. The aim of this study was to evaluate the markers of bone resorption [pyridinoline (PYD), deoxypyridinoline (DPD) and N-telopeptide cross-links of type I collagen (NTx)] in patients with breast cancer receiving pamidronate and to correlate the levels of these markers with skeletal complications. Sixty-seven patients with newly diagnosed osteolytic lesions from breast cancer randomized to receive pamidronate (group I) or placebo (group II) in addition to anticancer treatment. Pamidronate was administered in 35 patients at a monthly dose of 90 mg (iv) and group II included 32 patients. The above biochemical markers were evaluated at baseline and every month for 9 months. At the initiation of this study 31/35 (88.5%) patients of group I and 28/32 (87.5%) of group II had elevated levels of NTx, while the frequency of increased levels of PYD and DPD were 31.4% and 40%, respectively, in group I, and 28.1%, 40.6%, respectively, in group II. Over the period of 9 months of the study there was no significant difference in PYD and DPD values between the patients of two groups (p=0.077 and 0.062, respectively). However a significant reduction of NTx values was observed in group I compared with group II from the 2nd month of treatment (p<0.02) that continued for the next 7 months of follow-up. With respect to the 32 placebo patients only 7 (21.8%) experienced a decrease in NTx values compared to 29/35 (82.8%) patients of group I who had normal NTx values at the end of 9 months. The observed proportion of patients with bone disease progression was 6/35 (17.1%) in group I versus 17/32 (53.1%) in group II (p<0.02) while there was no difference between the two groups according to patients' fractures. These results suggest that NTx is a sensitive marker for monitoring bisphosphonate therapy of bone disease in patients with breast cancer and the pamidronate administration in addition to anticancer treatment reduces significantly the progression of osteolytic lesions in these patients, improving the quality of their life.
MONITORING METASTATIC BEHAVIOR OF HUMAN BREAST CANCER CELLS IN MICE WITH SPECIES-SPECIFIC PCR AND BIOLUMINESCENT REPORTER IMAGING
G. van der Pluijm1, A. Wetterwald2, J. Buijs1, B. Sijmons1, E. Gautschi2, I. Que1, B. Stadler3, M. Cecchini2, C. Lowik1*
1Leiden Univer. Medical Center, Dept. of Endocrinology, Leiden, The Netherlands
2Gene Therapy Lab., Dept. of Clin. Res. and Urology Clinic, University Hospital, Bern, Switzerland
3Institute of Immunology and Allergology, University Hospital, Bern, Switzerland
Breast cancer metastasizes frequently to the skeleton and causes considerable morbidity. Injection of human MDA-MB-231 breast cancer cells into the left heart ventricle of immunodeficient mice produced multiple osteolytic bone lesions and soft tissue metastases. However, micrometastases are not readily detectable and more sensitive methods are required to detect minimal disease states. Here we describe the use of species-specific PCR (ssPCR) and bioluminescent reporter imaging (BRI) for monitoring the tumor metastatic in vivo. We used a CCD camera connected to the Argus-20 image processor (C-2400/VIM, Hamamatsu) to monitor bone metastasis by tumor cells transfected with the luciferase reporter gene. MDA-MB-231 cells were stably transfected with a pCMV-plasmid containing the firefly luciferase gene (MDA-231/luc+). After intracardiac inoculation with MDA-231/ luc+ cells, the mice were monitored weekly for the development of bone and soft tissue metastases. For this, the mice were anaesthesized, injected with D-luciferin and photon emission was measured. Micrometastases developed predominantly in the hind limbs and the axial skeleton. Distinct photon emission was detected already after 24 days in bone (detection limit ±10,000 cells in bone and 1000 cells subcutaneous). ssPCR revealed significantly increased expression of VEGF-A, -B and PTHrP by the cancer cells in bone metastases when compared to soft tissues metastases suggesting a causal role in the observed preferential skeletal metastasis. In conclusion, BRI and ssPCR can be used for quantitative detection and growth of micrometastases and may become extremely useful to study the pathogenesis of bone metastasis in living animals, to monitor expression of targeted gene vectors and to determine the efficacy of novel therapeutic agents.
BREAST CANCER CELL OSTEOPONTIN EXPRESSION, CONNEXIN PROFILE AND GAP JUNCTIONAL COMMUNICATION WITH OSTEOBLASTIC CELLS CORRELATES WITH METASTATIC POTENTIAL
H. J. Donahue*, M. M. Saunders, Z. Li, E. Kunze, A. Mastro, D. R. Welch
Musculoskeletal Research Laboratory, Department of Orthopaedics, Pennsylvania State Uiversity, Hershey, PA, USA
Breast cancer cells preferentially metastasize to bone. However, the mechanism by which this occurs is unknown. We have previously demonstrated that the expression of the gap junction (GJ) protein connexin (Cx) 43 correlates with breast cancer cell metastatic potential. Furthermore, previous studies suggest that expression of osteopontin (OPN) may also be correlated with increased metastatic potential and we have shown that inhibiting Cx43 expression in bone cells results in upregulation of OPN. Therefore, in this study we examined the relationship between GJ function, Cx expression, OPN expression and metastatic potential. We utilized the non-tumorigenic and non-metastatic breast epithelial cell line Hs578Bst, a metastatic breast cancer cell line MDA-MB-435 (435), these cells expressing the gene for the metastasis suppressor BRMS1 (435-BRMS1), control transfectants (435/pvc) and a human fetal osteoblastic cell line (hFOB). Gap junctional intercellular communication (coupling), as revealed by fluorescent dye transfer, was barely detectable between 435 (or 435/pvc) and themselves, a feature common in tumor cells, whereas 435-BRMS1 were well coupled to one another. However, 435, 435/pvc and 435-BRMS1 were all well coupled to hFOB. Cx43 mRNA was detected in Hs578Bst and, to a lesser degree, in 435-BRMS1 but we were unable to detect Cx43 in either 435 or 435/pvc. On the other hand, abundant levels of Cx32 were detected in 435 and 435/pvc but not in Hs578Bst or 435-BRMS1. OPN mRNA levels, assessed by quantitative real time RT-PCR and normalized to 18S RNA, in Cx43 deficient 435 and 435/pvc cells were 100-fold and 50-fold, respectively, higher than those in Hs578Bst cells (p<0.003, n=3). However, OPN expression in 435-BRMS1 was similar to that in Hs578Bst. Our results suggest that breast cancer cells expressing BRMS1, a metastasis suppressor gene, express a connexin profile, Cx43+/Cx32-, similar to normal breast tissue and can communicate with themselves via GJ. Metastatic cancer cells, however, are Cx43-/Cx32+ but, while unable to communicate with themselves, retain the ability to communicate via GJ with osteoblasts. Furthermore, metastatic Cx43-/Cx32+ cancer cells express OPN to a much greater degree than do Cx43+/Cx32- cells suggesting that the Cx43-/Cx32+ expression profile may contribute to metastatic potential by facilitating OPN expression.
HISTOMORPHOMETRIC ANALYSIS OF BONE REMODELLING IN PATIENTS WITH METASTATIC BREAST CARCINOMA
S. Vukmirovic Popovic1*, N. R. Colterjohn2, S. Lhotak1, W. C. M. Duivenvoorden1, G. Singh1,3
1Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
2Hamilton Health Sciences Corporation
3Hamilton Regional Cancer Centre, Cancer Care Ontario, Hamilton, Ontario, Canada
We examined normal and pathological bone resorption (BoRe) induced by metastatic tumour cells in femoral bone biopsies from 33 patients with metastatic breast carcinoma and 19 normal controls. Quantitative histomorphometry was performed on 20 biopsies from patients with pathological femoral fracture and 13 biopsies from patients with no fracture. Increased BoRe was observed in metastatic bone disease, as seen by eroded surface (ES), osteoclast surface (OcS) and number of osteoclasts per bone surface (NOc). Increased BoRe was accompanied by non-significant increases in bone formation (BoFo) parameters. Patients with pathological fractures appeared in a more advanced disease stage, they had higher resorption parameters, but a significant decrease of osteoblast number compared to non-fracture group. In spite of the increased BoRe rate, cancellous bone volume (BV) was not significantly lowered due to an increase in woven bone production. Although the fracture group also showed extensive pathological woven BoFo, a significant drop in lamellar BoFo and a concomitant significant decrease in BV was observed. The non-fracture group included 6 patients with no radiological evidence of metastasis. They showed higher tumour volume (TuV) (27.26% versus 14.44% of total volume in the remainder of the group), a significant increase in ES compared to controls, but no significant increase in NOc and no correlation between OcS and ES. When patients were divided into two groups according to TuV (<40% and >40%) we found increased ES in both, but whereas NOc in the group with TuV>40% was close to control, it was significantly increased in the group with TuV<40%. Presumably, when more bone marrow is replaced by tumour cells, BoRe is carried out by other cells (such as tumour cells, tumour-associated macrophages). Different degrees of osteoclastic BoRe and biphasic BoFo response in the different stages of bone metastasis from breast carcinoma is needed and this would enable us to study the effects of anti-resorptive drugs. It appears that BoRe is a process that is dependent on the disease stage. Tumour cells may be involved in BoRe in late stage disease.
P155 T
Withdrawn
CHARACTERIZATION OF CAPACITATIVE CALCIUM INFLUX IN TUMORAL HUMAN EPITHELIAL BREAST CELLS
G. Picotto, A. Russo de Boland, R. L. Boland*
Dept. Biologia, Bioquimica & Farmacia - Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
Little is known about the regulation of intracellular calcium Ca2+ levels ([Ca2++]I) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumoral cell line MCF-7 and its responsiveness to the osteotropic agent ATP. Besides estrogen receptors (ER), it has been reported that these cells also express ATP receptors, type P2Y2, coupled to G proteins and phospholipase C (PLC) activation. Depletion of intracellular calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca2+, resulted in a rapid and transient elevation in [Ca2+]I; after recovery of basal levels, calcium readdition (1.5 mM) to the extracellular medium resulted in increased Ca2+ influx (2-fold over basal), reflecting pre-activation of the CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same calcium store is involved in their response. Moreover,
IP3-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, a PLC specific inhibitor. Compound 2-APB (50 microM), antagonist of the CCE pathway and Gd3+ (50 microM) completely prevented ATP-stimulated capacitative Ca2+ entry. CCE in MCF-7 cells resulted to be highly permeable to Mn2+ and to the Ca2+ surrogate Sr2+. Mn2+ entry sensitivity to Gd3+ matched that of the Ca2+ entry pathway. 17ß-estradiol which binds to ER-like proteins residing at the cell surface and exerts non-genomic effects in MCF-7 cells, blocked the ATP response in these cells. Taken together these studies represent the first evidence of the existence of a agonist-responsive CCE pathway in MCF-7 cells, raising the possibility that this could be an alternative way through which different extracellular signals can regulate calcium homeostasis. Future experiments should be designed to investigate the mechanism by which 17ß-estradiol influences this transduction pathway. It may throw light into the steroid mode of action in other target cells involved in regulation of mineral metabolism, e.g. osteoblasts.
THE PRELIMINARY STUDY OF RELATIONSHIP BETWEEN MALIGNANT TUMOUR AND OSTEOPOROSIS
M. H. Bai
The Orthopaedics Institute of Lanzhou General Hospital, PLA, 730050, PR China
Objective: To investigate the relationship between malignant tumour and osteoporosis and to provide the basis for osteoporosis prevention and treatment.
Methods: Calcaneal parameters were measured in 32 normal subjects (c group) aged 40-72 years (23 males and 9 females) using UBIS 3000 quantitative ultrasound (QUS). Also serum Calcium (Ca) levels were determined.
Results: A half of patients in M group had low Broadband Ultrasound Attenuation (BUA) and speed of sound (SOS). In M group, Z score values of BUA and SOS were significantly lower than those for c group (BUA, -1.5±2.76 vs.1.24±0.85; SOS,-1.63±3.49 vs.0.47±1.09 P<0.01) and serum levels of Ca did not change obviously.
Conclusions: We suggest that the bone loss is associated with malignant tumour (p<0.05). Calcium and vitamin D supplementation is necessary in patients with malignant tumour in order to prevent osteoporosis.
FUNCTIONAL IMAGING OF CARTILAGINOUS BONE TUMORS USING POSITRON EMISSION TOMOGRAPHY (PET) SCAN
F. Y. Lee*, M. Scanlan, M. Ho, M. Parisien, F. Feldman, H. M. Dick
Columbia University, New York, USA
Cartilage tumor is the most common tumor in the bone. It is characterized by abnormal proliferation of neoplastic chondrocytes with variable degrees of mineralization. Grading of enchondroma and chondrosarcoma is often very difficult based on plain x-rays, computed tomography, magnetic resonance imaging and bone scan. Although the anatomic location of the lesion is well demonstrated on the currently available imaging studies, the biologic activity of the tumor is still poorly represented. This dilemma often makes decision of adequate treatment difficult.
The purpose of the proposed study is to find out whether metabolic activity of cartilage tumors can be assessed using PET scan. Our hypothesis is that higher grade cartilage neoplasms have higher degree of metabolism reflected by increased uptake of radioisotope labeled glucose.
Sixteen lesions were evaluated by PET scan and underwent pathologic examination after surgical excision. The glucose metabolic uptake in the articular cartilage and growth plate served as internal controls. There were 6 benign enchondromas, 4 grade I chondrosarcomas and 6 high grade chondrosarcomas. There was no uptake in benign cartilage tumors on PET scan although bone scan showed nonspecific uptake. Grade I cartilage tumor showed very mild uptake which was not statistically different from enchondromas. Bone scan showed increased uptake in grade I chondrosarcoma which is not distinguishable from benign enchondromas. High grade chondrosarcomas showed increased uptake on PET scan which is distinct from enchondroma or grade I chondrosarcomas. PET scan showed the lesion both in the bone and soft tissue in patients with metastatic lesions in the lung and other bones while bone scan did not show the lesions in the lung.
Our data showed that high grade cartilage tumors are metabolically more active than low grade or benign lesions. Functional imaging of cartilage tumors using PET scan provided unique information which correlated well with histologic grade.
ASSESSMENT OF MAST CELLS MAY HELP IN THE DIFFERENTIAL DIAGNOSIS OF REACTIVE LESIONS AND PRIMARY BONE TUMOURS
C. J. Joyner*, T. Yang, A. Reed, N. A. Athanasou
Nuffield Department of Orthopaedic Surgery, University of Oxford, UK
Introduction: Mast cells (MC), the tissue-based effector cells in allergic diseases, are thought to be involved in new blood vessel formation and marrow fibrosis. Increased MC numbers have been reported in post-menopausal osteoporosis, hyperparathyroidism and chronic periodontitis. In fracture callus, too, large numbers of MC are present, especially during the onset of remodelling where it is believed they may be responsible for osteoclast recruitment and/or differentiation.
Given this association of MC with osteoclastic activity, fibrosis and angiogenesis, we decided to examine MC activity in lesions where these processes are prominent features, notably Paget's disease of bone and fibrous dysplasia. A number of the malignant and benign bone- and other matrix-forming tumours were also examined.
Methods: Histological sections were prepared from archival paraffin blocks and immunostained by using a commercial monoclonal antibody against mast cell tryptase with routine detection. Stained cells were counted in random fields (100 x magnification) within affected areas of tissue.
Results: Tissue affected by fibrous dysplasia, Paget's disease of bone and eosinophilic granuloma was found to contain high densities of MC, approaching those found in hyperparathyroid bone. In marked contrast, the numbers of MC within bone-forming tumours both malignant and benign were low. In some specimens of osteosarcoma which included the margin of the tumour, the surrounding tissue was found to contain very large numbers of mast cells (up to 280).
Conclusion: The marked difference in the extent of MC infiltration between the reactive and tumour-like lesions examined (e.g. fibrous dysplasia) and bone-forming tumours suggests that an assessment of MC numbers may be helpful in distinguishing these lesions.
PROTECTION OF NORMAL HUMAN BONE CELLS FROM APO2L/ TRAIL AND CHEMOTHERAPEUTIC DRUG COMBINATION-INDUCED APOPTOSIS
D. M. Findlay1*, S. Bouralexis1, G. J. Atkins1, F. Chai2, S. Hay1, M. T. Clayer2, A. Evdokiou1
1Dept. of Orthopaedics and Trauma, Royal Adelaide Hospital, The University of Adelaide, Adelaide, SA, Australia
2Dept. of Medicine, Dept. Orthopaedics, The Queen Elizabeth Hospital, The University of Adelaide, Woodville, SA, Australia
Although significant progress has been made in the treatment of osteogenic sarcoma, resistance to therapy and systemic toxicity remain as major problems. APO2L/TRAIL is a tumour necrosis factor (TNF) family member, which can induce apoptosis in tumour cells following ligation to death domain-containing receptors DR4 and DR5. This effect is mediated at least in part through the caspase-8/FADD pathway, which is disrupted by the cytosolic antagonist FLIP. Expression of three "decoy" receptors, DcR1, DcR2 and osteoprotegerin (OPG) protects normal cells, although the way in which they do so is not fully understood.
Normal human bone cells (NHB), cultured from trabecular bone fragments, and osteogenic sarcoma cell lines, were treated with recombinant TRAIL. TRAIL was able to induce apoptosis in only one of 6 osteosarcoma cell lines tested, as assessed by cytosolic caspase-3 activity and nuclear (DAPI) staining. However, TRAIL used in combination with the chemotherapeutic agents, doxorubicin, cisplatin and etoposide, acted synergistically to induce apoptosis in osteogenic sarcoma cells. These data suggest the possibility that doses of commonly used anti-cancer drugs could be lowered when used in combination with recombinant TRAIL to provide a more effective treatment of osteogenic sarcoma, with fewer side effects. Importantly, neither TRAIL alone, nor in combination with cytotoxic drugs, had a significant apoptotic effect on NHB. RT-PCR analysis of mRNA expression in treated NHB revealed upregulation of DR4, DR5, caspase 8, FLIP and DcR2 mRNA in response to TRAIL-drug combinations. While expression of decoy receptors per se does not correlate with protection of cells from TRAIL-mediated apoptosis, linear regression analysis revealed a strong assocaition between expression of mRNA levels of DR5 and DR4, DR5 and DcR2, DR5 and FLIP in these cells. This suggests that pro-apoptotic signals generated by cytotoxic agents in NHB, which lead to increased expression of DR4 and DR5, are countered by a concomitant increase of ‘protective’ molecules such as DcR2 and FLIP. The coordinated expression of TRAIL and its receptors, as well as downstream molecules involved in apoptotic pathways, may represent a natural buffer against apoptosis-inducing agents in normal cells.
PTHrP PRODUCTION BY BREAST CANCER CELLS STIMULATES OSTEOCLAST ACTIVITY AND BONE RESORPTION
M. T. Gillespie1*, R. J. Thomas1, J. J. Yin2, J. Elliott1, M. Dallas2, Y. Cui2, T. A. Guise2
1St. Vincent's Inst. Med. Res., Melbourne, Australia
2Univ. Texas Hlth. Sci. Ctr., San Antonio, USA
Bone destruction in breast cancer is mediated by the osteoclast. Several breast cancer cell lines may induce osteolytic lesions in animal models that mimic the metastatic process in clinical breast cancer. RANKL expressed by osteoblasts in association with M-CSF is responsible for the differentiation of hematopoietic cells into osteoclasts. We have investigated the possibility that breast cancer cells might use this pathway in promoting osteoclast formation in vitro and osteolytic lesions in vivo, and determined the effect of PTHrP expressed by breast cancer cells in these processes.
Expression of RANKL, its decoy receptor OPG and signaling receptor RANK were assessed in the breast cancer cell lines T47D, MDA-MB-231 and MCF7 as well as in unselected primary human breast tumors. All tumors and lines expressed OPG and RANK, but no expression of RANKL was noted. In accordance, these three cell lines could not act as stromal cells to support osteoclast formation from murine co-cultures, comprising osteoblastic stromal cells and hematopoietic cells. The ability of MDA-MB-231 and MCF7 parental and both cell types overexpressing PTHrP to support osteoclast formation were assessed further by their addition to murine co-cultures. In cultures where breast cancer cells overexpressed PTHrP, no exogenous osteotropic factors were required to stimulate osteoclast formation, whilst in cultures with parental breast cancer cells osteotropic agents were required. Concomitant with the ability of the PTHrP overexpressing cells to promote osteoclast formation in vitro was an upregulation of RANKL mRNA expression by mouse calvarial osteoblasts cocultured with breast cancer cells overexpressing PTHrP.
Finally, we assessed the ability of the MDA-MB-231 and MCF7 parental and overexpressing cells to induce osteolysis in a nude mouse metastatic model. Osteolytic lesion number and area on radiographs were greater in mice inoculated with either MDA-MB-231 or MCF7 clones which overexpressed PTHrP compared with the respective parental lines. Histomorphometric analysis demonstrated increased osteoclast numbers in both PTHrP-overexpressing MDA-MB-231 and MCF7 clones compared with the parental lines. Further in MDA-MB-231 cells overexpressing PTHrP, the 139 amino acid isoform of PTHrP increased osteolytic lesions relative to those induced by MDA-MB-231 cells expressing either the 141 or 173 aa isoforms.
These data indicate that breast cancer-induced osteolysis results from osteotropic factors of breast cancer origin, such as PTHrP, that acts on osteoblastic cells to induce RANKL, a mediator of osteoclast formation.
GENES THAT REGULATE BREAST CANCER METASTASIS TO BONE
M. D. Tavaria1*, M. Lelekakis1, A. Natoli1, E. Sloan1, P. Ho2, D. Hards2, T. J. Martin2, J. M. Moseley2, R. L. Anderson1
1Peter Mac Callum Cancer Institute, Melbourne, Australia
2St Vincent's Institute of Medical Research, Melbourne, Australia
Over a century ago Paget noted that breast tumours have a proclivity for destructive bone metastasis, yet the mechanisms underlying this phenomenon are yet to be fully understood. One of the factors impeding progress in this regard has been the lack of suitable animal models that mimic the entire metastatic process.
We have previously reported the initial characterisation of an orthotopic mouse model of breast cancer metastasis to bone (1). The model is unique in that primary breast tumours spontaneously metastasise to bone from the mammary gland. These metastatic tumour deposits cause osteolysis and hypercalcemia, which are characteristic of human metastatic breast cancer. Increasing evidence suggests that many tumour cells are capable of the early steps of the metastatic process, but only a small percentage, those that respond to secondary site-specific proliferative signals, go on to form overt metastases. Our model provides a powerful new tool for investigating mechanisms and kinetics of breast cancer metastasis to bone and for testing novel anti-metastatic agents.
Parathyroid hormone related protein (PTHrP) was first identifed as the factor mediating humoral hypercalcemia of malignancy (HHM) and has since been implicated in enhancing tumour mediated osteolysis in experimental models. We have used our model to further investigate the role of PTHrP in breast tumour metastasis to bone. Using a real-time PCR assay to measure metastatic tumor burden, we find that inhibition of PTHrP expression in tumour cells that metastasise to bone reduces tumour burden. We are currently investigating the ability of PTHrP to promote bone metastasis in other tumour cells. In addition, we are using the model to investigate the ability of known inhibitors of bone resorption, such as bisphosphonates, to prevent metastatic bone resorption.
Using DNA microarrays, we have identified a number of other genes that are candidates for promoting tumour cell homing to bone. These genes include cell adhesion molecules, proteases and growth factors. Manipulation of gene expression in our tumour model is now being used to determine the requirement for these genes in metastasis to bone.
(1) Lelekakis et al., (1999) Clin. Exp. Met. 17: 163-170
BONE SIALOPROTEIN EXPRESSION IN CELL LINES OF HUMAN BREAST AND PROSTATE CANCER
B. Fohr1*, M. F. Young2, L. W. Fisher2
1Department of Medicine I, University of Heidelberg, Germany
2Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, Bethesda, MD, USA
Bone sialoprotein (BSP) is a highly posttranslationally modified glycoprotein, featuring several acidic regions, three tyrosine-rich repeats and a C-terminal RGD-site for integrin binding and (cell) attachment. BSP is most abundant in bone matrix; other sources of BSP include dentin, chondrocytes, placenta and osteotropic carcinomas. Significant expression of BSP can be found in breast cancer, where expression patterns seem to be correlated with disease severity. Accordingly, prostate cancer shows a BSP expression often correlated with the histological subtype.
Curiously, screening breast (MCF-7, MDA-231, MDA-435, LCC15) and prostate (DU145, CRL1435, CRL1740) cancer cell lines by western and immunohistochemistry for BSP expression did not result in detectable quantities of BSP. Only in a squamous cell carcinoma line, cell-associated BSP could be detected by immunohistochemistry, whereas conditioned media supernatant of the same cell line was negative for BSP. Employing a high expression adenoviral system, encoding for human BSP under the control of EF-1, the breast and prostate cell lines were investigated for BSP expression. Compared to BSP made by human bone marrow stromal cells, western blotting of conditioned media showed that BSP released by the cancer cells was degraded into fragments, similar to those described earlier for UMR BSP.
Thus, BSP produced by these cancer cells might either be subjected to endogenous proteases or/and to incomplete posttranslational processing. Since BSP is found to be elevated in cancer patient’s sera, efforts have been made to establish BSP as a diagnostic marker for metastatic bone disease. However, discerning whether this BSP is derived from bone resorption (e.g. due to metabolic bone disorders) or released from tumor cells has not been possible yet. Further investigations are necessary to determine if BSP fragments are present in cancer patient’s sera and if there is a relevant ratio to intact BSP, which can aid in differential diagnosis increased resorption vs. bone metastases in future assays.
MARKERS OF BONE TURNOVER AND ITS RELATION WITH CYTOKINES ASSOCIATES WITH MULTIPLE MYELOMA AND MONOCLONAL GAMMOPATHIES OF UNKNOWN SIGNIFICANCE
B. Suquía1*, J. M. Hernández2, J. Sánchez3, P. Amantegui1, R. M. Fisac2, J. A. Queizán2, J. Fernández Calvo4, A. Barez5, J. A. Navajo1, J. F.San Miguel1
1Hospital Universitario Salamanca
2Hospital General Segovia
3Universidad Autónoma Madrid
4Hospital Clínico Valladolid
5Hospital "Ntra. Sra. Sonsoles" Ávila
We analyzed the relationship between serum cytokines and markers of bone turnover for two related diseases with and without bone damage: Multiple Myeloma (MM) and Monoclonal Gammopathies of unknown significance (MGUS) for a greater knowledge on pathogenesis of MM's bone damage.
MATERIAL AND METHODS: Prospective study of patients with MM (108 cases) and MGUS (59 cases) that attended hospitals of the Community of Castilla-León (Spain). At the time of diagnosis, serum levels of cytokines traditionally related with tumour activity (IL-6, sIL-6R, OSM, TNFalpha) as well as others implied in MM's bone damage (IL-1beta and TNFbeta) were determinated by ELISA(Quantikine-R&Dsystem). Markers of bone formation: serum bone-sepecific alkaline phosphatase (bAP) by ELISA (ALKPHASE-Metra Biosystems), N-Mid Osteocalcine by ELISA (CIS ESPAÑA), markers of bone resorption: pyridinolin and d-pyridinolin crosslinks (BIO-RAD) by HPLC were also determined.
For statistical analyses nonparametric test (U-Mann Whitney, Kruskal-Wallis, median test and Spearman's coefficient) were performed. OSMc has been analyzed by Fisher's test.
RESULTS AND CONCLUSIONS: Apart from the free forms, all resorption markers are useful to distinguish between MM and MGUS. Significance increases considerably when we introduce a factor (DPD/bAP) wich relates resorption to formation. Some cytokyines typically related to tumour growth are also useful. According to the results, OSMc is somewhat related with osteoclast activation. We did not find significant differences between MM without osteolysis and MGUS using markers of bone turnover.
EXTRACELLULAR CALCIUM INDUCED ESTROGEN-LIKE ACTIVITY IN MCF-7 BREAST CANCER CELLS
F. Journé*, J. C. Dumon, N. Kheddoumi, I. Laïos, Y. Ma, G. Leclercq, J. J. Body
Laboratory of Endocrinology and Breast Cancer, Brussels, Belgium
Bone tissue is the most common site of distant metastasis in breast cancer. Tumor cells are known to stimulate directly or indirectly osteoclast-mediated bone resorption and they could resorb bone matrix by themselves at a late stage of disease. This enhanced bone resorption leads to the release of growth factors which will further stimulate breast cancer cell growth (vicious cycle) but also to the release of bone mineral, especially calcium, whose effect on breast cancer cells has been little studied.
We have examined steroid receptor content of MCF-7 cells in the presence of increasing quantities of extracellular calcium. We tested two ranges of calcium concentrations: 0.5 to 3 mM and 5 to 20 mM whose upper limits correspond, respectively, to concentrations which can be observed in the serum of cancer patients and in the skeletal microenvironement. For this purpose, we have used a culture medium with low carbonate and phosphate concentrations to the growth of MCF-7 cells under high calcium concentrations.
Concerning estrogen receptors (ER), high calcium concentrations (10 and 20 mM) induced a significant decrease of the [3H]-oestradiol binding capacity of ER (whole cells assays) concomitantly with a decrease in peptide expression (Western blotting). Moreover, in MVLN cells (MCF-7 cells stably transfected with the estrogen response element cloned upstream the luciferase reporter gene), we observed that calcium led to a dose-related reproducible increase of ER transcriptionnal activity. We also detected a weak increase in [3H]-Org 2058 binding capacity, a specific ligand for progesterone receptor (PgR; whole cells assays) when extracellular calcium was increased; determination of PgR protein amount (Western blotting) confirmed this increase.
These results suggest that calcium may act as an estrogen-like compound, acting most probably onto the membrane since enhancement of intracellular calcium concentrations by the ionophore A23187 failed to show similar effects.
Our results suggest that calcium released during the process of metastatic bone destruction could modulate breast cancer cells growth and function and be important in the pathogenesis of tumor-induced osteolysis.
CLINICAL EVALUATION OF A NEW ASSAY FOR URINARY L-GALACTOSYLHYDROXYLYSINE (L-GHL): A PROMISING MARKER FOR CANCER PATIENTS
J. C. Dumon1*, N. Kheddoumi1, F. Journé1, R. B. Boga2, J. Mistry2, J. J. Body1
1Laboratory of Endocrinology and Breast Cancer
2Diagnostic Systems Laboratories, Inc., Webster, TX 77598, USA
We have evaluated a new enzyme immunoassay for urinary L-galactosyl hydroxylysine (L-GHL) which is a marker of collagen degradation. The assay detection limit is 0.5 µg/ml. We have evaluated 5 groups of patients: 73 healthy premenopausal women (PreMP); 76 healthy postmenopausal women not taking hormone replacement therapy (HRT) (PostMP); 34 osteoporotic patients (T score < -2.5); 11 under HRT (OPsis + HRT) and 23 without specific therapy (untr. OPsis); and 36 patients with breast cancer and bone metastases (BM+). L-GHL concentrations (in µg/mg urinary creat.) in the 5 groups are tabulated below:
The difference between the 5 groups was significant (ANOVA, P<0.0001), but disappeared when the BM+ group was not included. Along the same line, when we compared to the upper limits of normal in the PreMP or the PostMP groups, there was no elevated value in the OPsis + HRT group and only 1 in the group with untreated OPsis, whereas 44% of the patients in the BM+ group had elevated values as compared to 73% and 55% for total Pyr and DPyr, respectively. However, the degree of increase in L-GHL levels was much higher than for Pyr and DPyr (14- and 10-fold higher for the median values as compared to PostMP women).
We then correlated L-GHL levels to the concentrations of the serum bone formation markers Alk Phos, BAP and BGP, of the urinary bone resorption markers hydroxyproline, free and total crosslinks measured by HPLC (total Pyr, total D-Pyr, free Pyr, free D-Pyr). When considering all values, non-parametric correlations were significant only with total Pyr (rs=0.24, P<0.001) and free Pyr (rs=0.21, P<0.01).
In conclusion, this new assay for L-GHL appears to be quite promising for the evaluation and maybe the monitoring of tumor bone disease, but its specificity must still be established.
| GROUPMean±SEMMedian(2.5-97.5th perc.) | |||
| PreMP | 0.084±0.001 | 0.05 | (0.01-0.23) |
| PostMP | 0.087± 0.019 | 0.05 | (0.01-0.30) |
| Opsis + HRT | 0.073±0.014 | 0.08 | (0.01- 0.30) |
| Untr. OPsis | 0.099±0.030 | 0.04 | (0.01- 0.23) |
| BM+ | 0.706±0.214 | 0.135 | (0.1-4.25) |
BIOCHEMICAL EVALUATION OF BONE TURNOVER AND BONE COLLAGEN DEGRADATION IN A LARGE SERIES OF PATIENTS WITH BONE METASTASES FROM BREAST CANCER
J. C. Dumon*, B. Siwek, N. Kheddoumi, F. Journé, I. Mancini, I. Cstoth, J. J. Body
Laboratory of Endocrinology and Breast Cancer, Brussels, Belgium
Bone resorption is known to be increased in patients with bone metastases but detailed biochemical evaluations in large series of patients are lacking. We evaluated bone turnover in a large series of patients with breast cancer and bone metastases. Parameters of bone resorption included the measurement by HPLC (Bio-Rad) in fasting second morning urinary specimens of total Pyridinoline (tPyr), free Pyr (fPyr), total deoxypyridinoline (tDPyr) and free DPyr (fDPyr). Parameters of bone formation included serum osteocalcin (BGP; "intact" assay of Biosource) and the bone isoenzyme of Alk Phos (BAP; Hybritech assay). Normal values were determined in 105 healthy premenopausal women (PreMP) who did not take any drug known to affect bone metabolism. We evaluated 87 healthy postmenopausal women (PostMP) not taking HRT and 117 patients with breast cancer and bone metastases (BM+) evaluated before bisphosphonate therapy. Mean (±SEM) values in the three groups are tabulated below.
In patients with bone metastases, non-parametric correlations were significant between the two markers of bone formation (BGP vs BAP: rs=0.26, P=0.03), between the four markers of bone resorption (rs values comprised between 0.76 and 0.91, P<0.0001), but also between both sets of markers (rs values between 0.27 and 0.35, P<0.01). Bone remodelling balance was calculated according to the following formula: ((Tscore BGP + Tscore BAP)/2) – ((Tscore tPyr + Tscore tDPyr)/2). The score was not significantly different than zero in the PostMP group (mean±SEM= -0.30±0.13) but was significantly decreased in the BM+ group (-2.34±0.72; P<0.0001). Moreover, the ratios fPyr/tPyr and fDPyr/tDPyr in the BM+ group were 0.49±0.02 and 0.61±0.03, respectively, as compared to 0.37±0.01 and 0.44±0.02, respectively, in the PostMP group (P<0.01 for both). In conclusion, our study indicates that there is a disequilibrium in bone remodelling in favor of bone resorption in patients with bone metastases from breast cancer, although some coupling persists. Moreover, the fact that the ratios free/total crosslinks were increased suggests that the pattern of bone collagen degradation is abnormal in patients with bone metastases.
| Markers | PreMP | PostMP | BM+ |
| tPyr, nmol/mmol Creat. | 46.4±1.7 | 65.9±3.0 | 153.9±17.9 |
| fPyr, nmol/mmol Creat. | 19.2±0.8 | 24.6±1.0 | 77.8±11.4 |
| tDPyr, nmol/mmol Creat. | 8.6±0.4 | 13.6±0.6 | 29.0±3.2 |
| fDPyr, nmol/mmol Creat. | 4.0±0.2 | 6.0±0.3 | 17.5±2.2 |
| BGP, ng/ml | 8.3±0.3 | 11.7±0.8 | 15.2±1.7 |
| BAP, ng/ml | 7.9±0.5 | 9.9±0.4 | 21.1±1.7 |
CLINICAL AND BIOCHEMICAL EFFECTS OF A SHORT-TERM IBANDRONATE TREATMENT IN CANCER PATIENTS WITH OPIOID-RESISTANT METASTATIC BONE PAIN
J. C. Dumon*, I. Mancini, J. J. Body
Laboratory of Endocrinology and Breast Cancer, and Clinic of Suppportive Care
We have evaluated in an open pilot trial the clinical and biochemical effects of 4 consecutive daily infusions of 4 mg ibandronate (in 250 ml saline solution IV over 2 hours) in 14 patients with painful bone metastases insufficiently controlled by optimal analgesic therapy. We assessed pain by a visual analog scale (VAS, scale from 0 to 10), analgesic consumption by the morphine equivalent daily dose (MEDD), quality of life and pain intensity by a VAS and the Edmonton Functional Assessment Tool (EFAT, scale from 0 to 30). Biochemical evaluation included parameters of bone resorption (Pyr and Dpyr measured by HPLC; upper limits of normal in postmenopausal women are 98.4 and 23.3 nmol/mmol Creat., respectively) and parameters of bone formation (BGP and Alk Phos). All parameters were determined at least on days 0, 7, 21 and 42. There was a rapid improvement in bone pain which persisted at least until day 42 (P<0.01), whereas analgesic consumption did not change (cf MEDD). Parameters of quality of life improved more gradually (P<0.05 from day 21 until at least day 42). Biochemical parameters of bone turnover followed a different pattern. Urinary Pyr levels fell from 253±45 to a nadir on day 7 of 218±44 nmol/mol creat (P<0.05) and were back to baseline levels at day 42. The changes in Dpyr levels were similar, from 36.3±5.9 before therapy to 25.8±6.4 at day 7 and 37.1±18.0 nmol/mol Creat. at day 42.There were no significant changes in bone formation parameters. We found no correlation between the levels of biochemical parameters of bone turnover before or after therapy and any of the evaluated clinical parameters. In conclusion, this pilot trial suggests that a short-term ibandronate treatment at relatively high doses exerts significant analgesic effects and improves quality of life in patients with various tumors and opiod-resistant bone pain. These effects were not evidently linked to the inhibition of bone resorption.
| Parameters | Day 0 | Day 7 | Day 21 | Day 42 |
| VAS QOL | 6.4±0.5 | 4.7±0.7 | 3.9±0.6 | 3.0±0.6 |
| VAS PAIN | 6.1±0.5 | 3.0±0.3 | 3.0±0.6 | 2.8±0.7 |
| MEDD | 522±185 | 574±240 | 555±254 | 500±232 |
| EFAT | 8.5±1.7 | 8.0±1.5 | 5.8±1.1 | 4.7±1.6 |
| Pyr | 253.4±44.6 | 218.1±44.1 | 226.0±49.3 | 256.0±53.9 |
| DPyr | 36.3±5.9 | 25.8±6.3 | 33.9±14.3 | 37.1±11.2 |
COMPARATIVE STUDY OF THE SENSITIVITY OF BIOCHEMICAL MARKERS OF BONE TURNOVER IN PATIENTS WITH BONE METASTASES FROM BREAST CANCER
J. C. Dumon*, B. Siwek, N. Kheddoumi, F. Journé, J. Barlé, J. J. Body
Laboratory of Endocrinology and Breast Cancer Research and Clinical Chemistry
Modern markers of bone turnover will probably have an increasing role in the management of patients with breast cancer-induced osteolysis. Measurement of crosslinks by HPLC has been progressively replaced by direct measurement of peptide-bound crosslinks using easier immunoassay techniques in osteoporosis but there are very few comparative data in cancer patients. Moreover, the effect of the increase in bone turnover after menopause is often overlooked, although this can evidently affect markers specificity for tumor bone disease.
We measured the urinary excretion of crosslinks by HPLC (total and free: tPyr, tDPyr, fPyr, fDPyr), of hydroxyproline (OH-Pro), of the telopeptide NTx (Ostex), and the serum levels of total Alk Phos, of its bone isoenzyme (BAP, Hybritech) and of intact osteocalcin (BGP, Biosource). Normal values were determined in 105 healthy premenopausal (PreMP) women and in 87 healthy postmenopausal (PostMP) women not taking HRT. We compared the percentage of increased values ("sensitivity") of the various markers in 46 women with breast cancer and bone metastases, as compared with the upper limits of normal in PreMP and in PostMP women: Parameters of bone formation were less often increased than parameters of bone resorption, especially when comparing the values to PreMP women. Among bone resorption parameters, NTx and tPyr were most often elevated when the values were compared to PreMP women. However, when compared to PostMP women, NTx appeared to be the least sensitive marker of bone resorption. NTx was thus significantly less often elevated than tPyr and fPyr (P<0.01 and P<0.05, respectively). DPyr was not more sensitive than Pyr and measurement of free crosslinks gave a similar information than the measurement of total crosslinks. Most importantly, because of the interfering effect of menopause, our data suggest that the direct measurement of peptide-bound crosslinks cannot currently replace the more cumbersome determination of crosslinks by HPLC in patients with bone metastases.
| Markers | v. PreMP | v. PostMP | P value |
| BAP | 31% | 28% | 0.11 |
| BGP | 52% | 26% | 0.04 |
| OH-Pro | 40% | 34% | 0.39 |
| tPyr | 65% | 57% | 0.09 |
| t Dpyr | 57% | 40% | 0.67 |
| f Pyr | 59% | 55% | 0.20 |
| f Dpyr | 57% | 43% | 0.80 |
| NTx | 72% | 28% | <0.0001 |
CHANGES IN OPG GENE EXPRESSION AND SECRETION IN HUMAN OSTEOBLASTIC CELLS, ACCORDING TO DONOR AGE AND SKELETAL SITE. EFFECTS OF 1,25(OH)2D3 AND PTHrP
P. Martinez1*, M. Garcia1, I. Del Valle1, C. Guillen2, A. Cortazar2, P. Esbrit2, M. E. Martinez1
1Biochemistry Division La Paz Hospital
2Bone and Mineral Metabolism Laboratory (Fundacion Jimenez Diaz)
Osteoprotegerin (OPG) is a soluble osteoblastic protein, which inhibits osteoclast differentiation. A decrease in OPG production by osteoblasts could be related to the increased bone resorption observed with aging. In the present study, we evaluated the changes in OPG in human osteoblastic cells (hOB), depending on donor age and the skeletal site. We also assessed the effects of 1,25(OH)2D3 and PTHrP in these cells.
Human OB were isolated from trabecular bone samples from donors undergoing knee (n=13, 5 <50 yrs, 8 >50 yrs) or hip (n=11, 4 <50 yrs, 7 >50 yrs) arthroplastia. Cells were cultured for variable time periods in FBS-free medium, with or without 1,25(OH)2D3, or PTHrP (N- and C-terminal fragments), each at 10-8molar. OPG was measured in the cell-conditioned medium by ELISA (Immunodiagnostik, Germany). Cell total RNA was extracted by the Trizol method. OPG mRNA levels were assayed by RT-PCR. 18S was coamplified as a constitutive control.
In basal condition, OPG mRNA and protein secretion, increased up to 48 hours in hOB. Both OPG levels were similar in hOB cells at each skeletal site. However, OPG secretion increased with aging in these cells at the hip. Addition of 10-8molar 1,25(OH)2D3 increased 2-fold OPG, both mRNA and protein secretion. An inconsistent effect of 10-8molar PTHrP (1-36) on OPG mRNA was observed in these hOB. However, PTHrP (107-139), at 10-8molar, increased OPG mRNA about 2-fold at 6 hours in these cells. Moreover, a similar response pattern to this peptide was observed for OPG mRNA in human osteosarcoma cells MG-63.
Conclusions: OPG secretion increased with aging in hip hOB (mainly cortical bone). Both 1,25(OH)2D3 and the C-terminal PTHrP fragment induces an increase in OPG expression in human osteoblastic cells. The interaction between OPG and both 1,25(OH)2D3 and PTHrP in the human bone microenvironment could be a mechanism to modulate bone turnover.
EFFECTS OF POLYETHYLENE AND ALPHA-ALUMINA PARTICLES ON IL-6 EXPRESSION AND SECRETION IN PRIMARY CULTURES OF HUMAN OSTEOBLASTIC CELLS
A. M. Rodrigo1*, M. E. Martínez2, L. Saldaña1, G. Vallés1, P. Martínez1, J. L. González-Carrasco3, J. Cordero4, L. Munuera4
1Investigation Unit. La Paz Hospital.
2Biochemical Division. La Paz Hospital.
3National Center for Metallurgical Research (CENIM)
4Orthopaedic Department. La Paz Hospital.
The effect of two biomaterials, polyethylene and alpha-alumina, on interleukin-6 (IL-6) secretion and expression has been studied in human osteoblasts in primary culture. Human osteoblastic cells were derived from fresh trabecular bone explants removed during total knee arthroplasty. On reaching confluence, cells were subcultured in 6 well plates; polythylene or alpha-alumina particles were added to some and the rest were left as controls. The resulting subcultures were incubated until confluence. The IL-6 mRNA levels were assessed by reverse transcription (RT) followed by PCR. IL-6 secretion was measured in the conditioned medium. The IL-6 expression was increased in the presence of both biomaterials. Maximum expression ocurred in response to a dose of 50 mg particles per well with both biomaterials and was greater after polyethylene particle addition than after alpha-alumina particle addition at this dose. The maximum IL-6 secretion elicited by alpha-alumina was produced at 10 mg particles per well while maximun response with polyethylene required 50 mg per well. At a dose of 10 mg per well, alpha-alumina particles induced more secretion than 10 mg of polythylene particles. Nevertheless, at a dose of 50 mg per well maximun secretion was produced with polyethylene particles. In conclusion, polyethylene as well as alpha-alumina increased both the expression and the secretion of IL-6 in human osteoblastic cells in primary culture and stimulation from polyethylene is stronger than that from alpha-alumina.
INTERLEUKIN-6 EXPRESSION IN OSTEOBLASTIC CELLS IS MODULATED BY BOTH HOMOLOGOUS AND HETEROLOGOUS MECHANISMS INVOLVING PARATHYROID HORMONE-RELATED PROTEIN
C. Guillen1*, P. Martinez2, M. E. Martinez2,3, N. Del Valle2, P. Esbrit1
1Bone and Mineral Metabolism Lab. Fund J Díaz
2Research Unit, Hosp. La Paz
3Biochemistry Division, Hosp. La Paz
The N-terminal region of parathyroid(PTH)-related protein(PTHrP) has PTH-like features in bone metabolism. Thus, PTHrP (1-36) stimulates bone resorption by inducing osteoblasts to synthesize several osteolytic cytokines, e.g., interleukin-6 (IL-6). IL-6 and its soluble receptor (sIL-6R) stimulate IL-6 expression in rat calvaria osteoblastic cells. In the present study, we determined whether this autoregulatory loop operates in osteoblastic (hOB) cells from human trabecular bone fragments, and in rat osteoblastic osteosarcoma cells UMR-106. We also evaluated the possible effect of IL-6, with and without its soluble receptor, on PTHrP gene expresion in these cells. RT-PCR was carried out with cell total RNA and specific primers for either PTHrP or IL-6, and GAPDH, as a constitutive control. Nuclear factor-kappa B (NF-kB) binding activity was measured in cell nuclear extracts, using EMSA analysis.
IL-6 (100 ng/ml) maximally increased 2-fold its own mRNA at 2 h, decreasing thereafter, in hOB cells. Addition of 125 ng/ml sIL-6R superinduced (4-fold) this response for at least 24 h. Moreover, either IL-6 (100 ng/ml) or sIL-6R (125 ng/ml) maximally increased 2-fold PTHrP mRNA at 2 h in hOB cells. However, this response neither increased further nor lasted longer when both IL-6 and sIL-6R were added together to hOB cells. In UMR-106 cells, sIL-6R, but not IL-6, maximally increased 2-fold both IL-6 and PTHrP mRNA at 6 h. The stimulatory effect of IL-6 on its own expression, but not on PTHrP expression was diminished by NF-kB inhibitors (PDTC, dexamethasone, partenolide). In these cells, similarly to previous findings in hOB cells, both PTHrP (1-36) and PTHrP (107-139) increased 2-fold IL-6 mRNA. Moreover, each PTHrP peptide, at 100 nM, maximally increased NF-kB binding to nuclear extracts from either hOB or UMR-106 cells. The NF-kB inhibitors abolished the effects of each PTHrP peptide on both NF-kB activation and IL-6 mRNA in these cells.
Conclusions: These findings demonstrate that IL-6 and/or its soluble receptor rapidly autoregulates IL-6 and increases PTHrP expression in hOB cells. The present data also indicate that the interaction between IL-6/sIL-6R and PTHrP expression might be part of a metabolic loop to maintain IL-6 levels in osteoblasts.
IN VIVO EFFECT OF INSULIN LIKE GROWTH FACTOR-II (IGF-II) ON DE NOVO BONE FORMATION IN THE PRESENCE OF HYDROXYAPATITE IN RABBIT FEMUR
E. Damien*, T. MacInnes, P. A. Revell
Royal Free and University College Medical School, Department of Histopathology, Osteoarticular Research Group and IRC in Biomedical Materials, London NW3 2PF, UK
In vivo studies have demonstrated that growth factors (GFs) are candidates for future use in orthopedic surgery. In clinical situations, enhanced bone formation and healing could lead to improved results of surgical procedures. Growth stimulation of periimplant osseous tissues by GFs increases the indication for and success of implant use. Hydroxyapatite as a biomaterial for implants or for coating of implants is known for its good biologic interaction with bone.
We hypothesise that the addition of recombinant human IGF-II to hydroxyapatite increases de novo bone formation. In the investigation reported here IGF-II, known to stimulate osteoblast proliferation in vitro(1), has been used to induce bone healing and new bone formation in vivo in rabbits. IGF-II (0.5 mirog/ implant) adsorbed on to the surfaces of the macro and micro pores of cylindrical porous hydroxyapatite (PHA) (4.5±0.4 mm diameter, 6.6±1.2 mm length) was implanted bilaterally into the cancellous bone of the lower femur. New bone formation and serum levels of IGFs were compared among treatment groups (basal, sham, PHA, and PHA+IGF-II) at 1, and 3 weeks post-treatment.
IGF-II treatment significantly increased the following:- (a) amounts of osteoid/ woven bone formation as shown by toluidine blue staining of sections; (b) mineralisation of the new bone in the interface, surrounding trabecular bone and within the pores of the implants as revealed by Goldner and von Kossa staining methods and electron microscopy; (c) lamellar bone formation and mineralisation rate as demonstrated by fluorochrome labeling; (d) IGF-I and -II protein production as measured in the serum by ELISA (DSL, UK).
In conclusion, new bone formation and mineralisation are increased in vivo by IGF-II supplementation of PHA implants. These results provide a basis for further studies on the possible future clinical usage of GFs to enhance bone formation in fracture healing and the response to prosthetic-bone implants.
(1) Damien et al., 2000 J Bone Miner Res 15;2169-2177.
EFFECTS PRODUCED BY 17-BETA ESTRADIOL, RALOXIFENE AND TAMOXIFEN ON IL-6 RELEASE AND GENE EXPRESSION BY HUMAN OSTEOBLASTS IN CULTURE
C. Méndez-Dávila1*, C. García-Moreno1, M. L. Traba1, C. Turbí2, C. De la Piedra1
1Fundación Jiménez Díaz, Biochemistry Laboratory, Madrid, Spain
2Lilly Laboratories, 28108. Alcobendas, Spain
The hypothesis that estrogens protects human bone by decreasing the production of interleukin-6 (IL-6) by osteoblasts and lowering osteoclast activity by this way, has been discussed controversially, although this fact seems to be demonstrated in rodents models. We investigated whether 17-beta estradiol (ES) and two different selective estrogen receptor modulators (SERMs), tamoxifen (TA) and raloxifene (RA) were able to regulate both constitutive and Interleukin-1(IL-1)-stimulated IL-6 gene expression and synthesis by human osteoblastic cells.
Osteoblasts were obtained from trabecular bone of female patients undergoing orthopedic surgery. The cells were cultured as described by Nacher et al (Rev. Esp. Enf. Metab. Oseas 2:3, 1993). Osteoblasts at confluence were cultured in serum- and phenol red-free DMEM containing 0.1% BSA for 24 h. 10nM ES (Sigma), 10nM RA (Lilly), 10nM TA (Zeneca) or vehicle (ethanol) were added with and without IL-1beta (1ng/ml). After 24 h, levels of L-6 in culture medium were determined (IRMA, Medgenix).
To study IL-6 expression, osteoblastic cells were cultured up to confluence in Petri dishes. The medium was replaced with serum- and phenol red-free DMEM containing 0.1% BSA. After 24 h, the cells were treated with 10nM ES, 10nM RA or 10nM TA for 1, 2, 3 and 6 h, and with these agents and IL-1beta (1ng/ml) for 3 h. Total cellular RNA was isolated by the method of Chomczynsky and Sacchi (Anal Biochem 162:156, 1987). IL-6 mRNA levels were assessed by reverse transcription followed by polymerase chain reaction.
As expected, IL-1beta induced an increase in IL-6 expression and secretion. However, the constitutive or IL-1-stimulated IL-6 expression and production were not altered by treatment with 10nM ES, 10nM RA or 10nM TA.
These findings suggest that the beneficial effects of ES or RA on bone mass in post-menopausal women may not be mediated by a decrease in osteoblastic IL-6 expression or secretion. TA, used for breast cancer treatment, also did not exert any significant action on IL-6 human osteoblast production. Other mechanisms may be implicated in the effects of estradiol and SERMs on bone mass.
CYTOKINES, OSTEOPROTEGERIN AND OSTEOPROTEGERIN-LIGAND IN VITRO AND HISTOMORPHOMETRIC INDICES OF BONE FORMATION IN PATIENTS WITH DIFFERENT BONE DISEASES
H. Siggelkow1*, T. Eidner2, G. Lehmann2, V. Viereck3, D. Raddatz1, G. Hein2, M. Hüfner1
1Dep. for Gastroenterology and Endocrinology, University Hospital of Goettingen, Germany
2Dep. of Internal Medicine, Friedrich-Schiller- University, Jena, Germany
3Dep. of Obstetrics and Gynecology, University Hospital of Goettingen, Germany
Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with osteoporosis is scarce and few attempts have been performed to correlate such data to histomorphometric parameters of the same patients. Therefore, we investigated eleven patients refered to our clinic for bone biopsy with different forms of osteoporosis and analyzed IL-1, IL-6 and TNF-a protein release and gene expression in primary osteoblast cultures. Compared to controls five patients showed normal cytokine protein release, whereas six patients showed much higher levels of IL-6 (26 fold) and TNF-a (84 fold). Surprisingly, statistical comparison of the two groups did not reveal relevant differences for clinical or histomorphometrical parameters. Multiple regression analysis revealed that bone resorption, as measured by the eroded surface, was significantly affected by TNF-a, less by IL-6. In addition, OPG-L gene expression significantly affected the eroded surface and the number of osteoclasts. Finally, the formation parameter osteoid volume and osteoid surface were negatively influenced by TNF-a.
In conclusion, in an in vitro-ex vivo model of a heterogenous group of eleven patients with different forms of osteoporosis, cytokine levels in conditioned medium affected bone resorption and bone formation significantly, as measured by histomorphometry. TNF-a seemed to be the dominating cytokine, its effect on bone resorption being mediated by OPG-L. Discrepancies between low TNF-a and IL-6 levels and high OPG-L expression in two patients demonstrated that also other factors must play a role in this model.
EFFECTS OF TNF-ALPHA ON 1,25-(OH)2D3 ACTIVITY IN OSTEOBLASTS
X. Y. Zang*, Y. B. Tan, Z. Y. Chen, Z. L. Pang
Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, PR China
PURPOSE The present study was designed to examine the effects of TNF-alpha on 1,25-(OH)2D3 activity in osteoblasts.
METHODS The human osteoblast model from died fetal long bones was used after 10-8M 1,25-(OH)2D3 treatment for 4 days without or together with TNF- alpha (50u /ml) added for the last 72 h of culture. The total cell number (neutral red method of Lowik) and cell protein amount (dye-binding method of Bradford) were measured as index of cell growth. Cell ALP activity (Gray modified method of Bessey-Lowry), osteocalcin (RIA) and IL-6 (ELISA) secretions were used to indicate the states of differentiation and functional activity of culture.
RESULTS Treating cell with 10-8M 1,25-(OH)2D3 alone for 4 days, the total cell number and the amount of protein per well were decreased about 20% compared with the control group at the corresponding time point. While TNF-a was added for the last 72 h of culture, the inhibition of cell growth was almost impaired. The 1,25-(OH)2D3-decreased cell number was nearly returned to the control value, so was the amount of protein. After 10-8M 1,25-(OH)2D3 treatment, ALP activity, osteocalcin production and IL-6 secretion increased respectively by 530%, 410% and 360%. Addition of TNF-alpha for last 72 h ALP activity and in osteocalcin production returned almost control levels, while IL-6 increased by 950%. TNF-alpha alone had no effect on basal osteocalcin production, but directly increased IL-6 secretion.
CONCLUSIONS These results suggested:
1) TNF-alpha controlled bone turnover not only by its action alone but also by interaction with systemic hormones such as 1,25-(OH)2D3.
2) Effects of TNF-a on 1,25-(OH)2D3 activity in osteoblasts were complex, with both antagonized and synergy actions.
3) TNF-alpha impaired the stimulatory effects of 1,25-(OH)2D3 on osteoblastic function for bone formation, but not on IL-6 secretion.
FRACTURE HEALING SLOWS WITH AGE WITH PROLONGED EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IN THE RAT
R. A. Meyer*, B. J. Desai, M. H. Meyer, S. Porter, J. F. Kellam
Dept. Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA
Skeletal fractures are followed by a burst of mRNA expression of both matrix genes and a variety of cytokine genes. It is not clear which of these regulatory genes are needed for bone healing. In young rats at 6 weeks of age, there is rapid radiographic and biomechanical healing of femoral fractures with gene expression reduced back to baseline by 4 weeks after fracture. With increasing age, progressively more time is needed for fracture healing. We hypothesized that a gene needed for bone synthesis would have a prolonged expression time in the fracture callus of these adult rats to accompany the prolonged healing time. To test this, female Sprague-Dawley rats at 6 and 26 weeks of age were anesthetized, and an intramedullary rod was placed in their left femora. A closed mid-shaft femoral fracture was induced. Rats were euthanized at 0 (intact), 1, 2, 4, 6, 8, and 10 weeks after fracture, and the fracture calluses were harvested. Total RNA was prepared, and gene expression was measured by reverse transcription - polymerase chain reaction (RT-PCR). The RNA was quantified by a phosphor imager. Six rats were done per age per time point. All young rats healed radiographically by the fourth week, while the adult rats required 8-10 weeks. All genes studied increased in mRNA expression after fracture reaching a peak at 1-2 weeks after fracture. All genes in the young rats and most genes in the adult rats subsided to baseline by 4 weeks after fracture. Only bone matrix genes (osteocalcin and type I collagen) and VEGF, a gene associated with angiogenesis, have prolonged expression at 4 and 6 weeks after fracture in the adult rats. Indian hedgehog, TGF-B1, and BMP-2, -4, -6, and -7 had the same expression pattern in young and adult rats with a peak at 1-2 weeks after fracture and subsidence to baseline by 4 weeks after fracture. In conclusion, fracture healing is slower in adult rats than in young rats. Despite this, only VEGF and bone matrix genes have prolonged mRNA expression. It is not clear what stimulates this bone growth to achieve fracture healing.
ANALYSIS BY DIFFERENTIAL DISPLAY PCR OF THE MOLECULAR MECHANISM BY WHICH LEUKEMIA INHIBITORY FACTOR MEDIATES INHIBITION OF OSTEOPROGENITOR CELL DIFFERENTIATION
D. Falconi*, J. Dreisziger, M. Kwok, J. E. Aubin
Department of Anatomy and Cell Biology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Leukemia Inhibitory Factor (LIF) is a multi-functional cytokine known to have both catabolic and anabolic actions on bone tissue. We have shown previously that LIF plays a critical role in osteoblast development in rat calvaria (RC) cell cultures, blocking osteoblast development in a differentiation stage-specific manner at the late pre-osteoblast stage (Malaval et al. (1995) Endocrinology 136:1411; Malaval et al. (1998) J. Bone Miner. Res. 13:175). This corresponds to a time at which endogenous LIF production by osteoblasts has dropped to its lowest levels, suggesting that levels of LIF must be finely balanced for osteoblast development to occur. Given that the receptors for LIF are expressed at all differentiation stages, but that LIF inhibitory effects are observed only during a short window of time, our hypothesis is that LIF regulates a very specific gene or genes, during the inhibition-sensitive window, that is/are responsible for arrest of osteoblast differentiation. To test this hypothesis, osteoblasts were isolated from calvariae of 21 day old fetal rats and cultured under differentiation conditions [in the presence of ascorbic acid (50 microg/ml) and beta-glycerophosphate (10 mM), but without dexamethasone]. LIF (5 nanog/ml) was added to the RC cultures for different times; the identification of the sensitive period was confirmed by Von Kossa staining of treated and control cultures. Total RNA was purified from treated and control/untreated cultures, reverse-transcribed and subjected to differential display PCR screening to compare the transcriptomes of osteoblastic cells either treated or not with LIF. This method allowed us to identify RC genes regulated differentially (either positively or negatively) by LIF in control/untreated cultures versus sensitive and non-sensitive developmental time windows. Several known and unknown genes were detected by this differential display PCR and the expression patterns were confirmed by semi-quantitative RT-PCR. Amongst the known genes, members of multiple different functional categories (nuclear factors, matrix molecules, membrane proteins, secreted proteins) have been isolated and some are described for the first time as osteoblast products. Of genes identified, several are predicted candidate genes for mediating the LIF-inhibitory effects on osteoblast development, an hypothesis that we are currently testing.
NEW PREPARATION IMPROVING BONE REGENERATION
F. Baranovskij, V. Gonchar*, V. Kravetz, V. Rugal
Institute of Haematology, St Petersburg, Russia
The problem of regulation of bone tissue regeneration is high actual. Previous experiments shown that xenogenic immunoglobulins to osteocytes (Osteolin) are able to restore normal structure of rat's femur in treatment noncomplified mechanic fracture. Volunteer patients 3-4 years ago got traumatic fractures of long bones, where complicated by osteomyelitis. Course treatment included 3 intravenous injections Osteolin daily in dosage 0.1 mg/kg. Got over inflammation, stoppage of purulence and sclerostenosis of fistulas where obtained during 2 months. It was found complete restoration of tissue defects to 6-month after Osteolin injections. X-ray monitoring shown normal bone structure with forming cortical plate. There where no marked any complications and recidivations of illness from curing to present time. Any additive drugs were not applicated. Osteolin may be useful in treatment not only fractures but another pathology of bone tissues.
THE EFFECT OF INSULIN-LIKE-GROWTH FACTOR-I ON THE PROLIFERATION AND DIFFERENTIATION OF TRABECULAR BONE DERIVED CELLS IN VITRO.
J. F. Evans, D. Sima, C. Tapiador, J. K. Yeh*
Winthrop- University Hospital, Mineola, NY, USA
In the hypophysectomized (HX) rat, pituitary hormone deficiency leaves many hormonal receptors without available ligand leading to possible changes in their regulation. This study tested the effect of IGF-I treatment on trabecular osteoblasts derived from both intact and HX rats using medium supplemented with FBS with or without charcoal treatment in order to investigate receptor mediated changes in proliferation and differentiation. Trabecular bone cells from both age-matched intact and HX Sprague-Dawley rats were plated in both alpha-MEM supplemented with 10% FBS (normal serum) and in alpha-MEM supplemented with charcoal treated FBS (growth factor deplete serum). Half the cultures of each group were treated with 200ng/ml IGF-I from the initiation of the cultures and at every medium change. Under normal serum, the HX cultures had a proliferation rate 2.26X greater than the controls at day 7 but no increase in proliferation was detectable with IGF-I treatment. The control cultures experienced a detectable increase in proliferation in response to IGF-I treatment. This possibly indicates that IGF-IR sites were saturated allowing no further stimulation of proliferation with the addition of IGF-I. Charcoal treated serum significantly reduced proliferation in both groups with the reduction greater in the HX. A significant increase in proliferation is also seen in both groups in response to IGF-I with significantly greater response in the HX. This could suggest that the reduction of mitogenic agents in the serum liberated the IGF-IR on the cell surface with greater numbers in HX cultures. The differentiation results reflect the expected inverse relationship between proliferation and differentiation. The HX cultures grown under normal serum were unable to reach the potential of their control counterparts. IGF-I treatment of both groups lowers the OP expression. Charcoal treated serum increases differentiation in both groups reflective of its reduction of proliferation. In conclusion, there is a possible up-regulation of IGF-IR in vivo in the HX rat, which can be attributed to their extended deprivation of hypophyseal hormones, especially GH. This up-regulation is reflected by the proliferative and differentiation responses of trabecular osteoblasts derived from these animals to IGF-I treatment in vitro under typical and growth factor deplete conditions.
INCREASED LEVELS OF THE STIMULATORY IGF BINDING PROTEIN-5 (IGFBP-5) IN EXTRACTS OF BONE MATRIX FROM OSTEOARTHRITIS AND PAGET'S DISEASE
S. J. Brown1,3*, S. Mohan2, P. Magnusson2, M. W. J. Davie1, C. A. Sharp1
1Charles Salt Research Centre, Oswestry, UK
2Musculoskeletal Disease Center, JL Pettis VA Medical Center, Loma Linda, USA
3Chester Centre for Stress Research, Chester, UK
The insulin-like growth factors IGF-I and IGF-II are the most abundant growth factors produced by bone cells and are stored in human bone through their interaction with the IGF binding protein-5 (IGFBP-5). These components have been implicated in the regulation of bone formation both in vitro and in vivo. Because IGFBP-5 acts to fix IGFs in bone and has also been shown to exert stimulatory effects on bone formation via both IGF dependent and IGF-independent mechanisms, we proposed the hypothesis that increased bone formation in patients with metabolic bone diseases is mediated in part by increased IGFBP-5 production. To examine this hypothesis, we have measured levels of IGF-I, IGF-II and IGFBP-5 in extracts of trabecular bone powders taken from femoral heads at autopsy from elderly subjects with no overt bone disease (n=10), after hip arthroplasty for end-stage osteoarthritis (n=16) and Paget's disease (n=6). IGF system components were extracted from bone by demineralization with EDTA in the presence of 4M guanidine-HCl and protease inhibitors and measured using specific valid RIAs. Data are expressed as ng/mg dry bone powder. Comparisons were made between normal and pathological bone using the Mann-Whitney U test (* represents, P<0.05).
In general, bone matrices contain about 3-fold more IGF-II than IGF-I. Mean levels of IGFBP-5 were increased in bone extracts of both OA (p=0.001) and Paget's (p=0.026) patients compared to normal subjects.
Conclusions: 1) These data provide the first evidence for the increased levels of IGFBP-5 in extracts of bone from OA and Paget's disease patients. 2) These findings and the previous findings that serum levels of stimulatory IGFBP-5 correlate with bone formation markers are consistent with the hypothesis that increased IGFBP-5 production may in part contribute to increased bone formation in patients with metabolic bone diseases.
| IGF-I | IGF-II | IGFBP-5 | |
| Normal | 0.062±0.02 | 0.208±0.08 | 0.761±0.38 |
| OA | 0.086±0.04 | 0.204±0.05 | 1.780±0.75* |
| Paget's | 0.049±0.02 | 0.226±0.04 | 2.068±1.24* |
QUANTITATIVE RT-PCR FOR IGF-1 MRNA IN ADULT RAT BONE
H. W. van Essen, N. Bravenboer*, P. Lips
Dept Endocrinology, VU Medical Center, Amsterdam, NL
Mechanical stress is a determinant for bone strength and bone architecture and IGF-1 seems to play an important role in the response of bone cells to mechanical stress. Our objective was to develop a technique to quantitatively analyse IGF-1 mRNA levels in bone in response to mechanical loading. Real-time PCR is a technique that is able to quantitatively analyse specific mRNAs in small samples of RNA, whereby a dual-labelled fluorescent probe is broken down during the PCR, resulting in an increase in fluorescence. A Ct-value is calculated based on the cycle at which the fluorescence increases above a threshold value. This Ct-value is proportional to the amount of RNA input.
With RNA from rat tibia-shaft RT-reactions were performed using the specific primers for IGF-1 and the housekeeping gene PBGD. Real-time PCR was performed according to PE-Biosystems protocols on the ABI 7700 Sequence Detector System. Primers and probes were designed with the Primer Express software (PE Biosystems). To validate the method we measured the specificity, reproducibility and accuracy of the IGF-1 and PBGD RT-PCRs separately and the efficiency of both RT-PCRs.
With gel electrophoresis both PCRs showed a single product indicating that the PCR is very specific. The standard error of RT-PCR quadruplicates was < 1% in both assays. Standard curves of serially diluted tibia shaft RNA showed a high correlation between the amount of total RNA and the Ct-value, both for PBGD and for IGF-1 (see figs.). Standard curves for target and housekeeping gene can be omitted if the efficiency of both RT-PCRs is near equal. Analysis of both standard curves, tested in a single experiment, showed that above an input of 5 ng total RNA the two curves were identical.
We have developed a reproducible and accurate real-time RT-PCR method to quantitatively monitor changes in IGF-1 mRNA. This gives us a tool to investigate quantitative changes of growth factor gene expression after mechanical loading or other kinds of intervention in bone.
PLASMA CATECHOLAMINES AND PARATHYROID HORMONE DURING ENDURANCE EXERCISE
J. Guillemant1*, C. Accarie2, G. Pérès3, S. Guillemant1,2
1Faculté de Médecine Pitié-Salpêtrière, Paris, France
2EPHE, Nutrition Hydrominérale, France
3Médecine du Sport, Hôpital de La Pitié, Paris, France
In vitro studies have shown that PTH release from bovine parathyroid cells is stimulated by catecholamines by a mechanism independent of extracellular calcium. This was confirmed by direct infusion of catecholamines in man. Independent studies have shown that physical exercise increases plasma catecholamines and that under definite circumstances endurance exercise is able to increase PTH secretion. We decided to check the correlation of plasma catecholamines with serum PTH in eleven young well-trained elite triathletes investigated just before (T0), during at 30 min (T1) and 60 min (T2) and 30 min after the end (T3) of a 60 min ergometer cycling exercise at 80% VO2max which had been previously shown to highly stimulate PTH secretion. Blood was collected for measurement of intact PTH, ionized calcium, epinephrine, norepinephrine and dopamine. The results are presented in Table 1. During exercise PTH and catecholamines increased abruptly whereas ionized calcium remained unaffected. Ionized calcium decreased 30 min after the end of the exercise. While no correlation could be found between PTH and ionized calcium, a very significant positive relationship could be demonstrated between PTH and epinephrine (r=0.761; p=0.0001), norepinephrine (r=0.733; p=0.0001) and dopamine (r=0.461; p=0.0016) suggesting a stimulatory role of plasma catecholamines on the sport-induced PTH response independently of serum ionized calcium concentrations.
| Variables | T0 | T1 | T2 | T3 |
| PTH (pg/ml) | 21.7±6.5 | 50.2±12.1 | 54.0±17.5 | 29.6±11.4 |
| Calcium (mmol/L) | 1.234±0.026 | 1.235±0.021 | 1.240±0.024 | 1.19±0.03 |
| Epinephrine (pg/ml) | 105±41 | 384±187 | 628±240 | 124±50 |
| Norepinephrine (pg/ml) | 659±259 | 2313±810 | 3044±588 | 653±216 |
| Dopamine (pg/ml) | 117±80 | 303±213 | 410±267 | 171±93 |
| Table 1: Biochemical and hormonal
variables during a 60mn ergometer cycling exercise at 80% VO2max. Values are mean±SD. |
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PTH AMELIORATES THE ACIDOSIS-INDUCED SKELETAL GROWTH INHIBITION VIA THE INSULIN-LIKE GROWTH FACTOR 1 (IGF1)
G. Maor1*, R. Goldberg1, J. Green2
1Technion, Haifa, Israel
2Department of Nephrology, Rambam Medical Center, Haifa, Israel
Chronic metabolic acidosis (CMA) exerts an adverse effect on bone metabolism and skeletal growth. Chronic acidosis in children, as for example in uremic acidosis, is very often accompanied by skeletal growth retardation. We have shown previously, using in vitro model for endochondral ossification, that acidosis-induced morphological deterioration was accompanied by a state of resistance to GH and IGF1. Given the pivotal role played by parathyroid hormone (PTH) in regulating skeletogenesis and its being markedly affected by CMA, we studied the involvement of PTH in CMA-induced growth retardation. We used an organ culture of murine mandibular condyle (6 days old ICR mice) as a model for in vitro endochondral ossification system. Condyles were incubated in BGJb medium at either neutral pH (pH~ 7.4) or acidic pH (pH~7.15 achieved by adding 2mM HCl to the medium) and in the presence or absence of 10-10M PTH. After 24, 48, 72 and 96 hours, condyles were thoroughly washed, fixed and routinely processed for paraffin embedding. Sections were evaluated for histology (by H&E staining), immunohistochemisty (IHC), and in situ hybridization (ISH). Following 3-4 days in acidic conditions, there was a marked reduction in the chondrocytic population suggesting a defect in the process of differentiation while proliferation seemed to be unaffected. This was accompanied by a reduction in the levels of IGF1, IGF1R and PTH receptors. Incubation in the presence of 10-10M PTH ameliorated the morphological damage induced by the acidosis, which was reflected in the recovery of the expression of cartilage-specific proteoglycans. PTH (10-10M) also upregulated its own receptors in the acidotic condyle as well as IGF1 and IGF1R. In conclusion, these results suggest that PTH reverses the suppressive effect of acidosis on GH/IGF1 axis in skeletal growth centers. PTH also abolishes the negative effect of acidosis on skeletal differentiation.
PARATHYROID HORMONE SIGNALING IN RAT INTESTINAL CELLS
C. Gentili, S. Morelli, R. L. Boland*, A. Russo de Boland
Dept. Biologia, Bioquimica & Farmacia - Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
In a previous study, we demostrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca2 in these cells, in the present study we evaluated the involvement of cAMP, Ca2 and protein kinase C (PKC) on PTH–induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5-15 microM), an AC activator, or Sp-cAMP (50-100 microM), a cAMP agonist, and effectively suppressed by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca2 (EGTA 0.5 mM), chelation of intracellular Ca2 with BAPTA (5 microM), or blockade of L-type Ca2-channels with verapamil (10 microM) significantly decreased PTH-activation of MAPK. Furthermore, similar degree of phosphorylation of MAPK was elicited by the Ca2 mobilizing agent thapsigargin, the Ca2 inophore A23187, ionomycin and membrane depolarization with high K+. Inclusion of the calmodulin inhibitor fluphenazine (50 microM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca2+ play a role upstream in the signalling pathway leading to MAPK activation by PTH in rat enterocytes.
INTRACELLULAR LOCALIZATIN OF GREEN FLUORESCENT PROTEIN-PTH RECEPTOR CONSTRUCTS IN CULTURED LLC-PK1 CELLS: TARGETED MUTAGENESIS OF THE PUTATIVE NUCLEAR LOCALIZATION SEQUENCE
E. Patterson1*, L. Canaff2, G. N. Hendy2, A. B. Hodsman3, L. J. Fraher1,3, P. H. Watson3
1Department of Biochemistry, University of Western Ontario, London, Canada
2Calcium Research Laboratory, McGill University, Montreal Canada
3Department of Medicine, University of Western Ontario, London, Canada
In previous studies, we have shown that the type I PTH/PTHrP receptor (PTHR) can be localized to the nucleus of cells within rat liver, kidney, uterus, ovary and gut. Similarly, nuclear localization of the PTHR was observed in the cultured osteoblast-like cells MC3T3-E1, UMR106, ROS 17/2.8 and SaOS-2. In MC3T3-E1 cells, nuclear accumulation of PTHR immunoreactivity was associated with the timing of mitotic division. A putative bipartite nuclear localization signal (NLS), from residues 446-473, was identified in the protein sequence of the mature PTHR. In this study, a series of PTHR constructs was made in the Enhanced Green Fluorescent Protein (EGFP) expression vector (Clontech), transiently transfected into LLC-PK1 porcine kidney cells and the resultant mutant protein expression followed by fluorescence microscopy. Constructs were made that included the entire PTHR sequence or wildtype (WT), had the putative NLS deleted (-NLS), had the signal sequence deleted (-SS), had both the NLS and signal sequence deleted (-NS) or had the entire PTHR except the putative NLS deleted (+NLS). Control constructs include empty vector and empty vector in which the GFP protein has been deleted. LLC-PK1(clone 46) cells were grown to approximately 70% confluence on glass coverslips in 6-well plates. This particular clone was a gift from Dr. R. Bringhurst and shows no biochemical response in vitro to parathyroid hormone. Transient transfections were accomplished using Lipofectamine 2000 (Gibco) following the manufacturer's protocol. Optimization of transfection was done using the WT construct. Fluorescence microscopy of LLC-PK1 cells transfected with the WT construct showed abundant fluorescent signal throughout the cells with distinctly fluorescing plasma membranes and nuclei. Fluorescence was particularly intense in mitotic cells. Deletion of the signal sequence did not affect nuclear fluorescence of the PTHR construct. In contrast, cells transfected with the NLS deficient PTHR construct showed reduced nuclear fluorescence. These results indicate that the putative NLS at residues 446-473 of the mature PTH receptor plays a role in targeting the PTHR-GFP constructs to the nucleus of transiently transfected LLC-PK1 cells.
PERIPHERAL PTH1 RECEPTOR GENE DELIVERY IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR) DOES NOT AFFECT BLOOD PRESSURE BUT INCREASES PLASMA RENIN ACTIVITY
S. Fritsch1, T. Massfelder1, M. Grima2, N. Taesch1, A. Eichinger1, B. Escande1, M. Barthelmebs1, J. J. Helwig1*
1University Louis Pasteur Medical School, E 0015 INSERM, Strasbourg, France
2University Louis Pasteur Medical School, Department of Pharmacology, Strasbourg, France
In a parallel abstract, Massfelder et al. brought evidence for downregulation of PTH1 receptor (PTH1R) and upregulation of PTHrP in intrarenal arteries of SHR, as compared to normotensive rats. In these studies, replenishment of renal vessels with PTH1R by somatic human (h)PTH1R gene delivery, decreased SHR renal vascular tone in vitro, owing to endogenous vasodilatory PTHrP. These observations suggested potential therapeutic effects of somatic hPTH1R gene delivery to decrease blood pressure in SHR. To test this possibility, the full length of hPTH1R DNA (1.9 kb) under the control of the CMV promoter in the pcDNA1.1 vector was generated. The naked DNA (0.5mg) construct was delivered into SHR via a single intravenous injection. Control-treated SHR received 0.5mg empty pcDNA1.1.
As reported in the parallel abstract, 3 weeks after injection, the expression of hPTH1R mRNA was identified in all main organs, including heart, aorta and intrarenal arteries. Systolic blood pressure (SBP) was measured blindly in control- and hPTH1R-transfected SHR, by the sphygmomanometric tail-cuff method. SBP was not affected in PTH1R-transfected SHR (n=12) as compared to controls (n=12), before (210±6 vs. 210±10 mmHg), as well as 7 (239±13 vs. 218±10 mmHg) and 14 days (224±10 vs. 238±12 mmHg) post-injection. We reported previously that PTHrP directly interacts with juxtaglomerular cells to stimulate renin release. We therefore hypothesized that the absence of effect of peripheral PTH1R gene delivery on SBP was due to the simultaneous activation of the renin-angiotensin system. To test this hypothesis, plasma renin activity (PRA) was measured before, 1, 4, 7, 10, 14 and 18 days following plasmid injection in PTH1R-transfected SHR animals (n=5), and control animals (n=5). PRA was expressed as ng angiotensin-I generated from rat angiotensinogen during 1hr incubation in the presence of converting enzyme inhibitors. Results showed that PRA was significantly increased (p<0.05) by 56±7, 53±15 and 38±11% at 7, 10 and 14 days post-injection, respectively, in PTH1R-transfected SHR animals.
These results demonstrate a direct interaction between PTH1R density and PRA. They also suggest that a putative hypotensive effect of peripheral PTH1R gene delivery on SHR SBP might be obscured by a simultaneous stimulation of the RAS.
FUNCTIONAL IMPORTANCE OF MAZ FOR HUMAN PTH/PTHrP RECEPTOR (PTHR1) PROMOTER P2 CONSTITUTIVE ACTIVITY
C. Leroy1, D. Manen2, R. Rizzoli2, M. Lombès3, C. M. Silve1*
1INSERM U426, Faculté de Médecine Xavier Bichat and IFR02, Paris, France
2Division des Maladies Osseuses, HUG, Genève, Suisse
3INSERM U478, Faculté de Médecine Xavier Bichat and IFR02, Paris, France
Expression of the human PTHR1 gene is controlled by at least three promoters, P1 (upstream), P2 and P3. P2 is moderately active in numerous tissues and is involved in the broad expression pattern of PTHR1 gene. P2 sequence is TATA-box less, G+C rich, and contains consensus motifs for widely expressed transcriptional regulators, including several Sp1 and two MAZ-specific consensus sequences. To explore MAZ importance for P2 activity, wild-type human P2 promoter (wt-P2) and P2 with a mutated MAZ-site at position -45 (mu-MAZ) were inserted upstream to a luciferase reporter gene in a promoter-less expression vector (pGL3-Basic). Luciferase activity, measured after transient transfections in human osteoblast-like SaOS-2 cells, showed that mu-MAZ activity was lower than that of wt-P2 (1.6 vs 5.9 fold-increase respectively, p<0.001). Expression of MAZ mRNA in SaOS-2 cells was demonstrated by RT-PCR technique. Co-transfection with a MAZ expression vector did not significant modify wt-P2 reporter activity. In contrast, it significantly increased mu-MAZ reporter activity (3.5 and 1.8 fold-increase respectively with and without MAZ, p<0.05). This supports the possibility that the decrease in mu-MAZ reporter activity, which presumably results from a decrease binding of endogeneous MAZ-protein to mu-MAZ site, can be partially overcome by overexpression of MAZ-protein. Similar results were obtained in rabbit renal distal tubule cells. The binding of increasing amounts of SaOS-2 nuclear protein extracts to a 21 bp DNA fragment encompassing the -45-MAZ site was monitored in EMSA. Using a wild-type labeled oligonucleotide, two bands were detected. The fast-moving band was observed at the lowest protein amount (0.2 microgr). Using the same oligonucleotide mutated at the MAZ site, the fast-moving band intensity was markedly reduced without any change in the slow-moving band. Competition of wild-type and mutated oligonucleotide binding with unlabeled Sp1 consensus oligonucleotide markedly reduced the slow-moving band intensity, with a concomitant increase in the fast one intensity. These EMSA results are in agreement with the possibility that the factors involved in the fast and low moving complexes are MAZ and Sp1 respectively. Similar results were obtained using Hela cell nuclear extracts. Taken together, the results of these studies suggest that MAZ binding sites are important for the constitutive activity of PTHR1 P2 promoter.
MECHANISM OF THE EFFECT OF RETINOIC ACID ON PARATHYROID HORMONE STIMULATED ADENYLATE CYCLASE ACTIVITY
E. A. Gonzalez*, C. L. McConkey, K. J. Martin
Saint Louis University
PTH plays a major role in the regulation of skeletal metabolism both directly, by its effects upon the osteoblast as well as indirectly, by its effects on the production of growth factors and cytokines by the osteoblast. We have shown that PTH increasess EGF receptor expression in osteoblast-like cells by a cyclic AMP dependent mechanism and this was blocked by treatment with retinoic acid (RA). The present studies investigate the mechanisms involved in the inhibitory effect of RA on PTH actions. We used confluent cultures of UMR 106-01 cells with or without treatment with all-trans RA for 48 hours, after which intact cells were tested for cAMP response to PTH as well as for 125I PTH binding. Membranes were prepared from control and RA treated cells for the study of adenylate cyclase (AC) and cell extracts were analyzed for enzymes affecting GTP biosynthesis. In intact cells, cAMP production in response to PTH was decreased by all-trans RA resulting in values that were 25.1±1.6% of those in control cells. PTH binding decreased slightly in response to RA. Membranes from RA treated cells showed decreased PTH stimulated AC. Forskolin stimulated AC activities were identical. Addition of GTP or Gpp(NH)p, a non-hydrolyzable analogue of GTP, completely restored the response to PTH in the membranes. Treatment of intact cells with pertussis toxin, to inactivate Gi, did not alter the inhibitory effect of RA. Since the effect of RA was corrected by GTP, we examined the effect of RA on the levels of IMP dehydrogenase, the rate limiting enzyme for GTP biosynthesis, and GMP reductase which counteracts the effect of the synthetic enzyme. RA increased GMP reductase activity by 240.9±24.2% at 48 hours and decreased IMP dehydrogenase activity to 67.5±8.8. These data indicate that RA results in a markedly impaired response to PTH in intact cells as well as in isolated membrane preparations, and can be corrected by GTP. The RA induced alterations in the GTP biosynthetic pathway in a direction that favors decreased GTP biosynthesis provide a mechanism for the inhibitory effect of RA on PTH actions.
EARLY PHASE OF CALCIPENIA-INDUCED PARATHYROID HORMONE SECRETION IS BLUNTED IN PERFUSED PARATHYROID GLANDS OF STREPTOZOTOCIN-DIABETIC RATS
O. Mokuda*, R. Okazaki, Y. Sakamoto
Teikyo University, Ichihara, Japan
To study effects of diabetes mellitus on parathyroid hormone (PTH) secretion, the rat parathyroid glands were perfused via the bilateral carotic arteries. Diabetic rats showed plasma glucose levels above 30 mM, but no change in plasma PTH concentration, 14 days after an administration of 70 mg/kg of streptozotocin. Krebs-Ringer bicarbonate buffer containing 0.1% bovine serum albumin was used for perfusate. Perfusate calcium was lowered from 2.5 mM to 0.5 mM. In the normal rat parathyroid glands, PTH secretion was promptly evoked by calcipenia (from below 0.25 ng for 2.5 min in the perfusion with normal calcium to 0.73±0.17 ng during perfusion time 0 - 2.5 min), and slightly decreased (0.51±0.12 ng during calcipenia time 2.5 - 5 min), and then increased (0.75±0.16 ng during calcipenia time 7.5 - 10 min). Diabetes abolished the early phase of PTH secretion (below 0.25 ng during calcipenia time 0 - 2.5 min), but did not affect the late phase of PTH secretion (0.64±0.14 ng during calcipenia time 7.5 - 10 min). Insulin treatment with 25 U/kg daily for 14 days completely normalized the early phase of PTH secretion. It is suggested that an insulin-deficient diabetes, not quantitatively but qualitatively, impairs the PTH secretion.
NUCLEAR IMPORT OF THE IMPORTIN B1-RECOGNIZED TRANSPORT SUBSTRATE PTHrP OCCURS VIA MICROTUBULES
R. J. Thomas1, M. H. C. Lam1,2, K. Lakoski-Loveland3, S. Schilders4, M. Gu4, T. J. Martin1, D. A. Jans2, M. T. Gillespie1*
1St. Vincent's Inst. Med. Res., Melbourne, Australia
2John Curtin School Med. Res., Canberra, Australia
3Inst. Reproduction & Development, Melbourne, Australia
4Swinburne Uni of Technology, Melbourne, Australia
PTHrP has been reported to localise to the nucleus in a variety of cell types and as a result of such localisation, PTHrP has been suggested to have a nuclear role. Evidence suggests that upon phosphorylation of Thr85 by CDC2 and CDK2 that the nuclear localisation of PTHrP was decreased. Nuclear protein import requires that transport substrates contain nuclear targeting sequences recognized by members of the importin family, which mediate subsequent interaction with both nuclear pore components and the guanine nucleotide-binding protein Ran to effect translocation into the nucleus. Nuclear/nucleolar localisation of parathyroid hormone-related protein (PTHrP), whose nuclear targeting sequence is recognized specifically by importin beta1, is critical to its regulatory functions in cell proliferation and apoptosis. In this study we use the technique of fluorescence recovery after photobleaching (FRAP) to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus, and to visualise directly the transport of PTHrP into the nucleus in living cells. PTHrP was demonstrated to colocalize with microtubules within a cell. Furthermore, PTHrP could associate with microtubules in vitro and this was enhanced by the presence of importin beta1. The dependence of PTHrP nuclear import on microtubules was demonstrated by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. The results demonstrate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity. We propose that the microtubule network facilitates intracellular transport of PTHrP synthesised within the cell and exogenous PTHrP that is internalised into the cell to target to the nucleus to perform its nuclear functions.
ALUMINIUM INHIBITS NOT ONLY PTH RELEASE BUT ALSO PTH SYNTHESIS (MRNA OF PRE-PTH). IN VIVO AND IN VITRO RESULTS
J. L. Fernandez-Martin*, M. Naves, C. Diaz-Corte, M. T. Fernandez-Coto, J. B. Cannata
Bone and Mineral Research Unit, Instituto Reina Sofía de Investigación, Hospital Central de Asturias, Oviedo, Asturias, Spain.
Aluminium reduces parathyroid hormone (PTH) levels in chronic renal failure (CRF). In previous studies we and others have suggested that aluminium inhibited PTH release rather than PTH synthesis. The aim of these two complementary studies ("in vivo" and "in vitro") was to assess the effect of aluminium on PTH synthesis by means of quantifying PTH mRNA by Northern blot analysis.
The "in vivo" study was performed in Wistar rats with CRF. They were maintained on a high dietary phosphorous intake. Five weeks after surgery, the rats were randomly divided into two groups and loaded with aluminium (AlCl3) or placebo administered i-p for two consecutive days. After two weeks, serum calcium, phosphorous, creatinine, PTH and aluminium were measured. The parathyroid glands were removed and PTH mRNA was measured by Northern Blot. The "in vitro" study was carried out in parathyroid glands from rats cultured "in vitro" with citrate (control) or aluminium citrate (10 and 100 microM) and both at the same concentration of Ca2+ (1.4 mM). PTH secreted to the medium and mRNA of PTH in the glands were measured.
Results: In vivo: At the end of the study the "aluminium group" had higher serum aluminium levels than placebo and lower serum PTH levels (53%, p<0.05). No significant differences were found for calcium, phosphorous, renal function or body weight. PTH mRNA expression was also lower in the "aluminium group" than in the "placebo group" (57%) and inversely correlated with serum aluminium. In vitro: The PTH released to the medium by the glands cultured in the presence of aluminium was lower than the control glands (33.6% and 22.4% for 10 and 100 microM of aluminium respectively). The mRNA of PTH was also lower in the glands cultured with aluminium (24.6% and 41.6% for 10 and 100 microM of aluminium respectively).
In both studies we found that aluminium reduced PTH release as well as PTH mRNA. The latter demonstrates that aluminium also inhibits PTH synthesis.
LONG-TERM STUDY OF FUNCTIONALLY AND VIABILITY OF RAT AND HUMAN PARATHYROID GLANDS
M. Naves1*, J. Menárguez3, J. R. Polo3, R. Jofré3, J. Anguita3, M. T. Fernández-Coto2, J. L. Fernández-Martín1, J. B. Cannata1
1Bone and Mineral Research Unit. Instituto Reina Sofía de Investigación
2Biochemistry Unit. Hospital Central de Asturias, Oviedo
3Hospital Universitario Gregorio Marañón, Madrid, Spain
To date "ex vivo" viability and functionally of parathyroid tissue have been studied in short-term (up to 8 hours). The aim of this study was to establish a model to investigate in parathyroid tissue functionally and viability during longer periods (up to 7 days).
The study was divided in two phases: I) response to calcium and calcitriol in parathyroid glands from rats throughout 7 days (daily). II) To study functionally and viability of human parathyroid tissue obtained from patients with secondary hyperparathyroidism (n=5).
The parathyroid glands from rats were extracted, pooled in groups of 8-10 and studied without any other manipulation. Meanwhile, human parathyroid glands were cut into small pieces of approximately 1 mm3. In both cases parathyroid tissue was cultured with 2 ml of medium in a basket containing a permeable membrane under shaking at 37degC.
In phase I, the functionally of the parathyroid glands throughout 7 days were assessed performing different experiments (n=5): in the absence of calcitriol, in the presence of calcitriol (10-9M) and changing daily the calcium concentration (0.4 and 1.2 mM).
In phase II, fragments were cultured with two concentrations of calcitriol (10-8 and -9M). Other fragment (control group) was cultured with no calcitriol. The incubation period was 60 hours. The response of the parathyroid gland was expressed in picog of iPTH secreted to the medium per microg of total DNA.
Results of phase I demonstrated the "ex vivo" parathyroid tissue maintains its functional capacity to respond to calcium changes during 4 days. In addition, calcitriol 10-9M was also capable to reduce significantly iPTH secretion and synthesis (mRNA by Northern Blot).
In phase II, human parathyroid glands showed a viability higher than 80% before and after the period of study. Calcitriol 10-8M reduced iPTH in all 5 cases (24 to 64% reduction), lower concentrations of calcitriol 10-9M showed reduction in iPTH only in 3 cases.
In summary, these results demonstrate this model can be used to study the "ex vivo" parathyroid gland response in periods longer than 8 hours (up to 4 days).
DIFFERENCES IN ENDOTHELIAL NOS AND NEURONAL NOS KNOCKOUT MICE - STUDIES OF BEHAVIOUR AND URINARY NITRATE OUTPUT WITH OESTRADIOL ADMINISTRATION
N. Moradi-Bidhendi1,2*, L. Mancini1, P. Forte2, M. Cafferkey2, M. O'Shaughnessy3, P. Huang4, N. Benjamin2, I. MacIntyre1
1Bone Metabolism, William Harvey Research Institute, London, UK
2Dept. of Clinical Pharmacology, St. Barts and The Royal London SMD, London, UK
3Dept of Histochemistry, Imperial College School of Medicine, London, UK
4Cardiovascular Research Centre, Harvard Medical School, MA 02129, USA
Oestrogen deficiency is one of the most important factors in the pathogenesis of osteoporosis. The mechanism by which oestrogen prevents bone loss is incompletely understood. It has been suggested that its actions may be mediated through nitric oxide (NO). It has been shown that NO mediates 17-oestradiol induced increases in osteoblast proliferation and also that NO donors abolish ovariectomy induced bone loss. NO is synthesised from L-arginine by the nitric oxide synthase (NOS) enzymes. There are two constitutive isoforms of NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS).
We have studied the total urinary nitrate output of eNOS and nNOS knockout (KO) mice with and without oestrogen (17beta-E2) administration. eNOS(-/-) KO, nNOS(-/-) KO and c57/bl/6 wild-type control mice were maintained on a diet of low nitrate milk and water for five days with urine samples collected every 24 hrs. Urinary nitrate output was then measured using an automated Griess assay.
The results obtained have shown that female nNOS(-/-) mice have a significantly lower nitrate output than female wild-type and eNOS(-/-) mice. Female eNOS(-/-) mice responded to 17beta-E2 in a similar manner to the wild-type mice producing a two-fold increase in urinary nitrate output compared to untreated controls. Interestingly, however, urinary nitrate output of female nNOS(-/ -) mice was not increased with 17beta-E2 administration.
These results suggest that in the mouse most urinary nitrate is derived from nNOS rather than eNOS. It is likely that the increase in urinary nitrate following oestradiol depends on activation of the nNOS enzyme by 17beta-E2 since the effect is abolished in nNOS KO mice.
We have also examined the behavioural differences between eNOS(-/-) and nNOS(-/-) KO mice. We have observed that male nNOS(-/-) KO display a highly aggressive behaviour compared to male eNOS(-/-) KO mice. Female nNOS(-/-) KO mice also displayed aggressive which was not seen in the female eNOS(-/-) KO. It is possible that oestrogen also plays a role in the differences seen in behaviour.
BONE TISSUE IS A TARGET FOR LUTEINISING HORMONE (LH)
S. J. Yarram1*, K. Westby1, P. Roche2, M. Perry1, J. R. Sandy1, J. P. Mansell1
1The University of Bristol, UK
2The MAYO Clinic, Rochester, USA
Luteinising hormone (LH) and chorionic gonadotrophin (CG), members of the cystine knot superfamily (Hearn & Gomme 2000) share a number of topological features to other family members known to influence bone matrix metabolism (e.g., activins and BMPs). LH secretion is markedly increased (between 4 & 10 fold) following menopausal onset, a period in which bone turnover is uncoupled leading to osteopaenia and an increased risk of developing osteoporosis. We propose that prolonged exposure of LH, as would occur for decades post-menopause, may be partly responsible for the demise of bone tissue. In support of this hypothesis chronic exposure of LH on the adrenal gland has recently been found to precipitate abnormalities in adrenal cortex function (Kero et al. 2000). Here we report the identification of LH receptors upon both primary and transformed osteoblast cells. Incubation of these cells with hCG, a natural homolog of LH which binds to the LH receptor and elicits LH responses suggested the presence of functional receptors; the cells proliferated in response to the ligand. In addition, radiolabelled murine calvarial tissue underwent resorption, dose-dependently, on receipt of hCG providing further evidence of receptor functionality, this time in whole bone. These novel findings support a role for LH in bone metabolism, data which may aid our understanding of the mechanism responsible for post-menopausal bone loss.
Hearn MT and Gomme PT. J Mol. Recognit 2000; 13 (5): 223-278.
Kero J, Poutanen M, Zhang F, Rahman N, McNicol A, Nilson J, Keri R and Huhtaniemi, I JCI 2000; 105 (5): 633-641.
CHARACTERISATION OF THE EFFECTS OF DEXAMETHASONE ON MOUSE BONES
F. McLaughlin1*, J. MacKintosh2, B. Hayes1, A. McLaren1, P. L. Salmon3, J. A. Humphreys3, G. Brown1, S. Farrow1
1Cell Biology, GlaxoWellcome Medicines Research Centre, Stevenage, UK
2Respiratory Systems, GlaxoWellcome Medicines Research Centre, Stevenage, UK
3AEA Technology, Harwell, Didcot, Oxfordshire, UK
Recent gene knock-out and transgenic studies have identified the potential for using mice to study effects of genes on bone. For example, the osteoprotegerin (OPG) knock-out mouse develops severe osteoporosis (Bucay et al. 1998), whilst an OPG transgenic mouse develops severe osteopetrosis (Kong et al. 1999). We have been interested in characterising the effects of anti-inflammatory compounds, such as the glucocorticoid dexamethasone, on mouse bone. The main advantages of the mouse are that experiments can be carried out using much smaller amounts of compounds than other models, and that there are several mouse models of inflammation. We treated mice for 3 weeks with a high and low dose of dexamethasone, injected daily. At the end of the study we analysed the effects of dexamethasone on trabecular bone by dynamic and static bone histomorphometry and micro-CT. Three different sites were studied by histomorphometry - distal femur, proximal tibia and lumbar vertebra. Dynamic histomorphometry showed a sharp dose-dependent decrease in bone turnover rate in response to dexamethasone, at all sites, by around an order of magnitude in the high dose group. In the lumbar vertebral body only there was a modest decrease in osteoid surface and a non-significant decrease in trabecular bone volume and trabecular thickness. Micro-CT analysis of the tibia showed decreased cortical volume due to high dose dexamethasone treatment, but no structural effect on trabecular bone. It is of interest that bone volume remained unchanged or fell slightly, in parallel with a sharp fall in turnover - frequently a reduced turnover rate increases bone volume, as in therapies for osteoporosis. In these cases turnover is decreased by lowering activation rate of new remodelling units. Simulations of bone remodelling - assuming coupling of resorption and formation - suggest that reduced turnover together with trabecular bone loss could would result from a general slowing down and elongation of the remodelling cycle - this could be the mechanism of action of dexamethasone on trabecular bone.
