IBMS/ECTS 2001 - PROGRAM and ABSTRACTS
PLENARY LECTURES
Click on the abstract number to view the plenary session.
NITRIC OXIDE: FROM DISCOVERY TO THE CLINIC
S. Moncada
The Wolfson Institute for Biomedical Research
In 1987 it was discovered that endothelial cells release nitric oxide (NO) and its generation from the amino acid L-arginine was subsequently established. NO is formed by a family of enzymes, the NO synthases, and is involved in many physiological functions. Its formation in vascular endothelial cells maintains a vasodilator tone that is essential for the regulation of blood flow and pressure. Thus NO acts as a homeostatic regulator of vessel wall functions and a decrease in its synthesis or actions contributes to the development of some vascular pathologies, including hypertension, vasospasm and atherosclerosis.
NO is also synthesized in the central nervous system, where it acts as a neuromediator with several physiological functions, including the formation of memory. In the peripheral nervous system, NO is now known to be the mediator released by a widespread network of nonadrenergic, noncholinergic nerves that mediate some forms of neurogenic vasodilation and regulate certain gastrointestinal, respiratory and genitourinary functions. All these physiological actions of NO are mediated by activation of the soluble guanylate cyclase in target cells.
In addition, NO is generated in large quantities during host defence and immunological reactions. When NO is released in this way it contributes to the development of certain pathologies, including septic shock and some forms of inflammation.
One way in which NO may be transformed from a physiological mediator to a pathophysiological entity may be through its actions on mitochondrial function. At low, physiological, concentrations NO inhibits cytochrome c oxidase in a reversible manner which is competitive with oxygen. At higher concentrations it irreversibly inhibits other enzymes in the respiratory cycle, either directly or through interaction with superoxide anion leading to the generation of peroxynitrite. Increased understanding of the biochemical actions of NO and the role it plays in mitochondrial function and gene expression will doubtless lead to novel therapeutic applications.
ROLE OF INFLAMMATORY CYTOKINES IN OSTEOCLAST DIFFERENTIATION AND FUNCTION
N. Takahashi
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan
Since the discovery of the RANK ligand-RANK signaling system, it has been believed that RANK ligand (also called ODF/OPGL/TRANCE) is the sole factor for inducing osteoclast differentiation and function. However, we recently found that two inflammatory cytokines, TNFalpha and IL-1, also stimulated differentiation and function of osteoclasts, respectively, through the mechanism that is independent of the RANK ligand-RANK interaction. When M-CSF-dependent bone marrow macrophages were cultured in the presence of mouse TNFalpha together with M-CSF, osteoclasts were formed within 3 days. Osteoclast formation induced by TNFalpha was inhibited by respective antibodies against TNF receptor 1 (TNFR1) or TNFR2 but not by OPG. We have also shown that purified osteoclast cultured on dentine slices failed to form resorption pits on the slices, but RANKL and IL-1 independently added directly stimulated pit-forming activity of purified osteoclasts. Osteoclasts induced by TNFalpha formed resorption pits on dentine slices only in the presence of IL-1. Lipopolysaccharide (LPS) can trigger a variety of inflammatory reactions. Recent studies have revealed that members of Toll-like receptor (TLR) family, which share sequence homology with the intracellular portion of the IL-1 receptor family, are implicated in LPS signaling. Like IL-1, LPS supported the survival of purified osteoclasts in a dose-dependent manner. LPS similarly stimulated the survival of osteoclasts derived from TNFR1 deficient mice, but not that of osteoclasts derived from C3H/HeJ mice that possess a missense mutation in the TLR4 gene. LPS-induced survival of osteoclasts was not inhibited by adding OPG or anti-M-CSF receptor antibodies, but partially inhibited by IL-1 receptor antagonist. However, LPS failed to induce pit-forming activity of purified osteoclasts. RT-PCR analysis showed that mature osteoclasts strongly expressed TLR4 mRNA. These results suggest that LPS directly acts on osteoclasts through TLR4 and enhances the survival of osteoclasts. Thus, LPS and inflammatory cytokines such as TNFalpha and IL-1 are directly involved in osteoclast differentiation and function through the mechanism independent of RANKL-RANK interaction.
MODULATION OF OSTEOCLAST FUNCTION
H. K. Väänänen
University of Turku, Turku, Finland
Osteoclasts have developed an efficient mechanism to resorb mineralized bone. They use targeted proton secretion and extensive production of several proteolytic enzymes as key mechanisms to dissolve hydroxyapatite and degrade collagen of the extracellular matrix, respectively. Recent studies have suggested that in addition to acid and proteinases osteoclasts may also use reactive oxygen species, ROS, as an additional mechanism to degrade bone matrix.
During a resorption cycle the osteoclast undergoes remarkable changes in the organization of the cytoskeleton and plasma membrane domains. Intensive intracellular vesicular trafficking is needed to create and maintain new plasma membrane domains that are crucial for different functions of the cell during the resorption cycle. Our recent results have demonstrated, for instance, that simultaneous exocytosis and endocytosis takes place in specific membrane domains at the ruffled border and allows continuous resorption and removal of degradation products.
Small GTPases of rab family are key regulators of intracellular membrane trafficking pathways along both the biosynthetic/secretory and endocytotic pathways. We have cloned several members of the rab family from rat osteoclasts and many of them show very specific changes in their subcellular localization during the resorption cycle. Localization and "knockdown" experiments using antisense oligos indicates e.g. that rab 7 is important in the formation of the ruffled border. In addition to the inhibition of rab 7 also disturbing the cholesterol metabolism will lead to the prevention of the ruffled border formation.
Since the resorption cycle is a multistep process it also offers several possibilities to modulate the resorption process. Most of the known resorption inhibitors inhibit osteoclast function. Aminobiphosphonates and clodronate, for instance, disturb protein prenylation and subsequent vesicular trafficking or inhibit mitochondrial ADP/ATP translocator and rapidly induce osteoclasts apoptosis, respectively. Inhibition of bone resorption can also be achieved by direct blocking of vacuolar proton ATPase or proteolytic enzymes. It is thus possible now to inhibit specifically osteoclast function at various stages of the resorption cycle. Possible in vivo and clinical consequences of the use of different osteoclast modulators will also be discussed.
OSTEOPOROSIS IN MEN
E. S. Orwoll
Oregon Health Sciences University and Portland VA Medical Center, Portland, OR, USA
Fractures in men represent an important part of the overall problem of osteoporosis. The nature of osteoporosis in men is in many ways similar to that in women, but distinct differences in fracture epidemiology and male-female differences in important skeletal characteristics indicate that gender effects must be included in consideration of etiology, management and the use of densitometric techniques for detection and monitoring. In general, older men have fewer fractures than women do. The reasons for this apparent protection are several. The age related increase in fractures begins earlier in women, in part because women experience more bone loss and fall more than men do. In addition, there may be biomechanical factors (e.g. relatively larger bone size or a different distribution of bone) that offer men some protection against fractures. These gender differences in epidemiology, function and skeletal morphology probably provide important insights into the basic causation of fracture risk that are important in both men and women.
The causation of osteoporosis in men is multifactorial, apparently including secondary factors (e.g. glucocorticoids, alcoholism, hypogonadism), calcium and vitamin D undernutrition, inactivity, and others. Osteoporosis in men is also commonly idiopathic - an interesting and poorly understood condition that may have genetic roots. Gonadal function gradually declines in men with aging, and the part that change plays in the genesis of age-related bone loss and fractures continues to be uncertain. More data has accumulated to support the importance of gonadal steroids - estrogens as well as androgens - in skeletal homeostasis in older men. The relative roles of estrogen and androgen in bone metabolism in men are gradually emerging.
The prevention and therapy of osteoporosis in men is a particularly unexplored field, but recent findings provide direction for patients and clinicians. Bisphosphonates are effective in reducing glucocorticoid induced bone loss in men and in improving bone density and reducing vertebral fractures in men with primary osteoporosis. Testosterone is capable of increasing bone mass in men with low testosterone levels, but the extent to which fractures are reduced is unknown. There is still much to be learned about the indications, benefits and drawbacks of prevention/therapy for osteoporosis in men.
EPIDEMIOLOGICAL ASPECTS OF OSTEOPOROSIS IN MEN AND WOMEN
H. A. P. Pols
Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
Osteoporosis-related fractures are a major source of both morbidity and costs in the elderly. The most important fractures related to osteoporosis are those of the hip, wrist and vertebrae, although it is believed that most other fractures occurring in the elderly are also related to osteoporosis. In most areas in the world osteoporotic fractures occur more frequently in women than in men. For hip fractures the typical female-to-male ratio is around 2:1. For wrist fractures the female-to-male ratio rises with age from 6:1 at age 55 to 10:1 at ages over 75. For vertebral fractures these ratios are difficult to calculate given the absence of a universally accepted definition of these type of fractures.
A large number of risk factors for osteoporotic fractures have been identified. Age, BMD, previous fractures and frailty related parameters are strong predictors of a future osteoporotic fracture. Between men and women no large differences appear to exist in the predictive power of these risk factors. Based on these factors also prediction rules have been developed in an attempt to identify subjects at highest risk for an osteoporotic fracture.
To increase etiologic insights in the pathogenesis also more experimental approaches have been integrated in epidemiological analyses of osteoporosis. Biochemical parameters of bone turnover have been investigated in a number of population-based studies. In most studies a clear relationship between bone resorption parameters and especially non-vertebral fractures have been found. However, most studies are restricted to women.
Genetic epidemiological studies of osteoporosis have been a fast growing field during the last decade. Both genome scans and candidate gene approaches are used. Although problems exist with the definition of endpoints to be studied (BMD, fractures, frailty?) and the complex nature of the interaction with environmental factors, it is clear that this new epidemiological approach has the potential to provide new insights.
Taken together epidemiological research has moved from a descriptive epidemiology in women only to a much broader approach whereby different tools are used to understand the pathogenesis of the disease in both men and women.
OSTEOBLASTIC CONTROL OF BONE FORMATION
P. Ducy
Baylor College of Medicine, Houston, TX, USA
The molecular mechanisms controlling the differentiation and functions of the osteoblasts begin only to be understood. Recent studies have emphasized the pivotal role of Cbfa1, an osteoblast-specific member of the runt family of transcription factors, in genetically controlling these two processes. Thus, it is important from a biological as well as from a biomedical point of view to understand how Cbfa1 expression is triggered during skeleton development and modulated afterwards. To achieve this goal, my laboratory is taking multiple genetic approaches, ranging from conventional molecular biology techniques to mouse genomic strategies. Progress in our understanding of the genetic control of Cbfa1 expression will be presented at the meeting.
OSTEOBLAST APOPTOSIS AND THE REGULATION OF BONE FORMATION
R. L. Jilka
University of Arkansas for Medical Sciences
Central Arkansas Veterans Healthcare System
Osteoblast number is an important determinant of the rate of bone formation. The majority of osteoblasts die by apoptosis; therefore this process is potentially as important as the proliferation of osteoblast progenitors for determining osteoblast number, and thereby bone formation rate and bone mass. In support of this notion, studies from our group have shown that the bone loss caused by sex steroid deficiency or by glucocorticoid excess in mice is associated with increased osteoblast apoptosis, whereas decreased osteoblast apoptosis is associated with the gain in bone mass obtained with intermittent PTH administration. PTH dose-dependence and time course studies further indicate that that suppression of osteoblast apoptosis is tightly correlated with increased bone formation. Administration of alendronate to mice prevented the increased osteoblast apoptosis caused by excess glucocorticoids, raising the possibility that this anti-resorptive agent may also exert an anabolic effect on the skeleton.
In vivo and in vitro studies by our group and others have indicated that apoptosis is the default fate of osteoblasts in the absence of survival signals provided by binding of integrins to proteins in the extracellular matrix. Matrix metalloproteinase-mediated pericellular degradation of type I collagen also provides survival factors, perhaps by exposure of cryptic integrin ligands or by release of anti-apoptotic proteins bound to the extracellular matrix. Locally produced factors such as IL-6-type cytokines, TGFbeta, and IGFs also prevent apoptosis of osteoblastic cells. Thus, the effects of sex steroids, glucocorticoids and PTH on osteoblast apoptosis may be due to their effects on the biosynthesis of integrins, matrix metalloproteinases, cytokines and growth factors. However, in vitro studies have shown that sex steroids, bisphosphonates, and PTH also rapidly activate intracellular anti-apoptotic signaling pathways that do not depend on changes in gene transcription. Thus, rapid activation of the MAP kinase signaling cascade is required for the anti-apoptotic activity of sex steroids and bisphosphonates, but not of PTH. Instead, the effect of this hormone is mediated by stimulation of cAMP production; and involves protein kinase A-stimulated phosphorylation of the pro-apoptotic protein bad, which results in its inactivation. Elucidation of the signaling pathways and factors governing osteoblast apoptosis may lead to the development of therapeutic agents which increase osteoblast number as a means of stimulating bone formation in osteoporotic conditions.
DIAGNOSTIC EVALUATION OF OSTEOPOROSIS
R. Eastell
Bone Metabolism Group, University of Sheffield Division of Clinical Sciences (NGH), Sheffield, UK
The standard approach to the diagnostic evaluation of the patient with osteoporosis includes characterising the degree of bone fragility, examining for secondary osteoporosis and factors that may influence the choice of treatment. The fractures that characterise osteoporosis usually result from a fall from standing height or less (mild to moderate trauma). The definition of osteoporosis in women is based on a comparison of the femoral neck bone mineral density with that of healthy young women – a value that is 2.5 standard deviations (T<-2.5) below the average for young women is considered to represent osteoporosis. For men, it is undecided whether the young male reference range should be used for this comparison or whether a T-score of –2.5 is an appropriate threshold. It is more common to identify secondary osteoporosis in men than in women presenting with vertebral fracture. The common causes include hypogonadism, alcoholism, and glucocorticoid therapy and so the biochemical investigation should include tests to evaluate these disorders. Biochemical markers of bone turnover tend to be higher in young men than in young women, but lower in older men than in older women. Thus, gender specific reference ranges need to be established. The markers can prove useful in identifying secondary osteoporosis and response to therapy. The choice of therapy may be determined by the identification of modifiable risk factors such as hypogonadism and idiopathic hypercalciuria, and by the presence of coexistent diseases such as prostate disease in men or breast cancer and endometrial disorders in women.
ALL THE KINGS HORSES AND ALL THE KINGS MEN COULD NEVER PUT HUMPTY DUMPTY BACK TOGETHER AGAIN
E. Seeman
Austin & Repatriation Med. Centre, Melbourne University, Melbourne, Australia
Where are we? We know how to design and execute clinical trials but we apply insufficient constraint to inference when design and execution is flawed. Doubt diminishes when the rules of evidence are followed (randomization, blinding, large samples, long study duration, few dropouts, intent to treat analysis, replication). Data generated under these experimental conditions can be presented with transparency and are easy to understand. Inferences can be made confidently because the results, positive or negative, are difficult to challenge on methodological grounds. Anti-spine fx efficacy of alendronate and raloxifene are difficult to refute. Studies of residronate show a consistent reduction in spine fx risk, especially in the first year, but large numbers of dropouts may introduce bias by producing unequal distribution of confounders which may under- or over-estimate efficacy. Non-spine fx rates are also reduced with alendronate and residronate, not raloxifene. The anti-hip fx efficacy data are more difficult to interpret. In the alendronate trials, hip fx was not a primary endpoint, there was consistency in the fx risk reduction across trials but event rates were low and confidence intervals overlapped unity except in the Fracture Intervention Trial. In the residronate trial, hip fxs were the primary endpoint, there were many events, with a significant reduction in fx risk in patients with osteoporosis but dropout rates were large. Estrogen, etidronate, calcitonin, vitamin D metabolites may prevent fxs, but flaws in design, execution, and convoluted analyses (small sample sizes, brevity, unplanned pooling of groups, subgroup analyses, selective presentation of data, dropouts, unblinding, lack of controls) make the results uninterpretable. Attempts to correct design flaws by statistics creates confusion.
Antiresorptive agents slow progression of fragility, they do not restore lost bone, architecture or strength. BMD increases by filling of the remodeling space and more complete secondary mineralization of bone. There is no evidence of trabecular thickening, new trabeculae, cortical thickening by periosteal or endocortical apposition. Some evidence for a less negative or a positive BMU balance is available. Whether this can this restore structure in the presence of slow remodeling seems unlikely. The reduction in fx risk within 12-18 months of treatment may be due to reduced remodeling, filling the remodeling space, increased true BMD of matrix, reduced progression of architectural disruption, increased osteocyte survival. We are far from zero fracture growth; drugs reduce but do not eliminate fx risk, most patients at risk are uninvestigated, untreated, or mistreated, ~50% of all fxs occur in the large population at modest risk (women with normal BMD or osteopenia). Targeted drug therapy is an option for the small high risk group. Moving the population BMD distribution by 0.5 SD is a solution but how is this to be achieved? Fragility fractures are not going away.
MECHANISM OF REGULATION OF 25 OH D3-1ALPHA HYDROXYLASE
S. Kato1,2*, A. Murayama1, J. Yanagisawa1,2, K. Takeyama1,2
1The University of Tokyo, Tokyo, Japan
2CREST Japan Science and Technology, Saitama, Japan
Nuclear Vitamin D receptor(VDR) belongs to the nuclear hormone receptor superfamily and act as ligand-inducible transcription factors. Therefore, most of the biological actions of vitamin D are now considered to be exerted through positive and negative controls of gene expression. Two types of coactivator complexes are shown to be recruited to ligand-bound VDR for activating gene expression, however the molecular mechanism how VDR supresses gene expression remains largely unknown.
Using a newly developed cloning system with the expression library from the kidney of the VDR KO mice (Takeyama et al., Science, 277, 1827, 1997), we cloned the cDNA encoding mouse 25-(OH) vitamin D 1 a-hydroxylase [1a(OH)ase]. No repression by 1a,25(OH)2D3 was observed in the gene expression of the VDR KO mice, establishing function of liganded VDR in the negative feed-back regulation of 1a,25(OH)2D3 production. We then demonstrated that through this enzyme the serum level of 1a,25(OH)2D3 is strictly controlled in response to calciotropic hormones (Murayama et al., Endocrinology, 140, 2224, 1999). We further cloned human 1a(OH)ase gene promoter, and identified a negative vitamin D response element (1anVDRE). The negative response to vitamin D in this nVDRE was observed only in the cell lines from the renal proximal tubular cells. A candidate binding factor is isolated by a yeast one-hybrid screeing system from the cDNA library of the renal cell line.
Further studies of this factor, with mapping the regions in VDR for transcriptional repression, will be presented.
THE MOLECULAR PHARMACOLOGY OF ESTROGEN RECEPTOR AGONISTS, ANTAGONISTS AND SERMS
D. P. McDonnell*, C. E. Connor, J. Hall, C-Y. Chang, M. S. Jansen, J. D. Norris
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
The pharmacological definitions of agonist and antagonist do not appropriately describe the activity of the currently available estrogen receptor (ER) ligands. In large part, this is due to the observation that compounds, such as tamoxifen and raloxifene, which were initially developed as antiestrogens, actually exhibit estrogenic activity in bone and the cardiovascular system. Consequently, these and other pharmacologically related ligands are now classified as Selective Estrogen Receptor Modulators (SERMs). We have probed the molecular mechanism of SERM action and determined that the structure of ER is affected differentially by the nature of the bound ligand and that cells possess proteins or processes that can distinguish between these different conformational states. The identification of a new bone sparing SERM, GW5638, that induces a unique structural alteration in ER and which inhibits the growth of tamoxifen resistant tumors in mice lends support to this hypothesis.
In environments where SERMs and pure antiestrogens function as antagonists, it has been determined that they do so by (a) competitively blocking agonist access and (b) inducing a conformational change within the receptor that prevents it from interacting efficiently with transcription coactivators (i.e. SRC-1, AIB-1 and GRIP-1). These findings suggested to us that it might be possible to develop a new class of ER-antagonists that function by directly blocking the ability of the receptor to interact with transcription coactivators within target cells. This is an ongoing project but the preliminary results obtained thus far indicate that ER-coactivator interactions are bona fide drug targets.
CANCER AND BONE
T. A. Guise
University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
Bone metastases are the most common skeletal manifestation of malignancy. Certain cancers have a propensity to metastasize to bone and cause destruction or new bone formation. The osteolytic or osteoblastic phenotypes are due to the action of tumor-produced factors to stimulate either osteoclast or osteoblast activity and disrupt normal bone remodeling. Thus, the tenacity with which certain tumors grow in bone depends on tumor cell-bone cell interactions as well as the fertile soil of the bone microenvironment.
Osteolytic bone destruction by metastatic cancer is mediated by the osteoclast. Substantial data support a central role for tumor-derived parathyroid hormone-related protein (PTHrP) as a local mediator of bone destruction in osteolytic metastases due to its capacity to stimulate osteoclastic bone resorption. Transforming growth factor beta (TGFbeta), stored in bone matrix and released as a result of PTHrP-stimulated osteoclastic bone resorption, stimulates more PTHrP production by tumor cells and perpetuates a vicious cycle of local bone destruction. The signaling pathways by which TGFbeta increases PTHrP secretion by tumor cells include both Smad and MAP kinase pathways. Tumor-produced PTHrP stimulates osteoclastic bone resorption via osteoblast production of RANK Ligand. Identification of these tumor cell-bone interactions responsible for osteolytic metastases has important therapeutic implications. In a mouse model of breast cancer metastases to bone, therapy directed to neutralize PTHrP, reduce tumor PTHrP production or block the effects of TGFbeta on the cancer cells all significantly reduced the osteolytic bone metastases caused by MDA-MB-231 breast cancer.
On the other end of the spectrum are osteoblastic bone metastases. The cell responsible for the aberrant new bone formation is the osteoblast. Endothelin-1 (ET-1), a vasoconstrictor produced by breast and prostate cancer, is a potent stimulator of osteoblast proliferation. Plasma concentrations of ET-1 are increased in men with advanced prostate cancer. A human breast cancer line, ZR-75-1, which produces ET-1, causes osteoblastic metastases in a mouse model. Treatment with a specific endothelin A receptor antagonist significantly reduced the osteoblastic metastases. Evidence from other models implicates tumor-produced platelet-derived growth factor as another mediator of osteoblastic metastases.
These data indicate that tumor cells selectively interact with the bone microenvironment. Understanding these interactions at the molecular level has identified novel targets for therapeutic intervention aimed at both osteolytic and osteoblastic metastases.
PAGET'S DISEASE FROM A GENETIC PERSPECTIVE
W. Van Hul
Department of Medical Genetics, Univerisity of Antwerp, Belgium
Paget's disease of bone (PDB) is a (multi)focal skeletal disease characterized by an increased bone turnover. The initially increased osteoclastic bone resorption is compensated by consecutive excessive bone formation by osteoblasts. In many parts of the world the prevalence of PDB is only second to osteoporosis affecting up to 5% of individuals over 55 years.
A long lasting hypothesis regarding the pathogenesis suggests PBD to be a slow-virus infection by paramyxovirus. Next to this hypothesis, but not necessarily opposing it, it has long been recognized that a genetic susceptibility is present in at least a subset of PDB patients. Previous linkage studies have suggested the involvement of two chromosomal regions in PDB. Dating from the early period of linkage analysis, two studies suggested a role in the etiology of PDB for the HLA locus on chromosome 6. Since then this has never been confirmed. More recently, a positional cloning effort for the gene causing Familial Expansile Osteolysis, a rare PDB-like inherited condition, revealed activating mutations in the RANK gene on chromosome 18q21-22. However, no mutations were found in PDB patients with the exception of one similar to the FEO-mutations, found in a somewhat atypical Japanese Pagetic kindred. This indicates that the RANK gene is no major PDB susceptibility gene.
Important tools to identify such genes are extended multigenerational families with many affected individuals. Despite the high prevalence of PDB in general, such families are not described numerously. This is mainly due to the clinical presentation of PDB, with a late age of onset, highly variable even within families and a high rate of asymptomatic cases. However, several extended pedigrees have been recruited originating from Spain, the United States, the UK, Australia and New Zealand all showing a autosomal dominant mode of inheritance. Interestingly, in the French-Canadian population in Quebec an extended set of putatively related families has been found. Several ongoing genome searches will most likely soon lead to the localization of further PDB loci. Considering the advanced status of the Human Genome Project, one can be hopeful that these soon will be followed by the characterization of PDB genes.
THE GENETIC BASIS OF NORMAL AND ABNORMAL CRANIOFACIAL DEVELOPMENT
I. Thesleff
Institute of Biotechnology, University of Helsinki, Finland
The knowledge of the genetic mechanisms behind animal development has increased rapidly and it has become apparent that these mechanisms have been conserved to an astonishing extent during evolution. There are several important groups of developmental regulatory genes which play key roles in numerous developmental events including craniofacial morphogenesis. Interestingly, most of these genes are associated with signalling networks regulating cellular communication.
Genes regulating the development of the craniofacial bones and teeth continue to be identified by experimental and molecular genetic studies. For example, more than 200 genes have been associated with the regulation of tooth morphogenesis, and their expression patterns at different stages of tooth development can be viewed in our graphical database at: http://bite-it.helsinki.fi/. The deletion of the function of many of these genes in transgenic mice results in missing teeth as well as in defects in many other tissues including bone. Also in humans, mutations in several regulatory genes have been shown to cause craniofacial defects, such as bone disorders and dental abnormalities, and it can be expected that new genes behind craniofacial defects will be identified with increasing speed. We have analysed the function of the transcription factor CBFA1/RUNX2 which is absolutely required for bone and tooth development and causes the cleidocranial dysplasia syndrome in humans.
FUNCTIONS OF FOS PROTEINS IN BONE CELL DIFFERENTIATION
E. F. Wagner, et al.
Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
c-Fos is a key regulator of bone development, since transgenic mice expressing exogenous Fos develop bone tumors, whereas mice lacking c-Fos are osteopetrotic due to a differentiation block in bone resorbing osteoclasts. We are interested to study how c-Fos and its related protein Fra-1, which is c-Fos inducible, control osteoblast proliferation and osteoclast differentiation (1). We recently found that Fra-1 is an essential gene for mouse development (2) and transgenic mice overexpressing Fra-1 develop the bone disease osteosclerosis, which is due to increased bone formation (3). To test whether Fra-1 can substitute for c-Fos, we generated knock-in mice that express Fra in place of c-Fos. Fra-1 rescues c-Fos dependent functions in bone development which appeared to be gene dosage dependent (4). However, Fra-1 failed to substitute for c-Fos in inducing expression of target genes in vitro. We are using these systems to identify novel Fos target genes by microarrays and with the help of bone-specific conditional alleles of c-Fos and Fra-1, we are studying the molecular mechanisms how Fos proteins govern bone cell development and differentiation.
Since Fos proteins need Jun proteins to activate transcription, we investigated the function of c-Jun in bone cells using the cre/loxP system. Chondrocyte-specific inactivation using col2A1-cre transgenic mice results in severe scoliosis caused by failure of intevertebral disk formation and abnormal vertebral arch development, suggesting that c-jun is a novel regulator of sklerotomal differentiation.
1. Matsuo, K., Owens, J.M., Tonko, M., Elliot, C. Chambers, T.J. and Wagner, E.F. (2000) Osteoclast differentiation by the c-Fos target gene Fra-1, Nature Genetics 24, 184-187.
2. Schreiber, M., Wang, Z.Q., Jochum W., Fetka, I. Elliott, C. and Wagner, E.F. (2000). Placental vascularization requires the AP-1 component Fra1. Development 127, 4937-4948.
3. Jochum, W., David, J.P., Elliot, C., Wutz, A., Plenk, H., Matsuo, K. and Wagner, E.F. (2000) Increased bone formation in transgenic mice expressing the transcription factor Fra-1, Nature Medicine 6, 980-984.
4. Fleischmann, A., Hafezi, F., Elliott, C., Remé, C.E., Rüther, U. and Wagner, E.F. (2000). Fra-1 replaces c-Fos-dependent functions in mice. Genes & Development 14, 2695-2700.
MEDIATING TRANSCRIPTIONAL ACTIVATION BY THE VITAMIN D3 RECEPTOR
L. P. Freedman*, C. Rachez, D. Burakov, M. J. Gamble, W. Yang, C. P. Chang
Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Ligand-binding induces conformational changes in nuclear receptors that facilitate interactions with several types of cofactors critical for transactivation. A distinct set of ligand-dependent, vitamin D receptor (VDR) interacting proteins, called DRIPs (also called TRAPs), constitute a novel cofactor complex. This multi-subunit complex binds to several nuclear receptors and is required for ligand-dependent enhancement by VDR and thyroid hormone receptor in cell-free transcription assays. Although both endogenous SRC-1/p160 coactivators and DRIP complexes bind to the same activation domain of nuclear receptors (AF-2) in response to hormone, they do so as distinct complexes. Moreover, in contrast to the SRC-1/p160 family of coactivators, none of the DRIP subunits contain intrinsic HAT activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they may be both required, in equilibrium with one another or in a sequential, regulated manner, for transcription activation by nuclear receptors. We have completed the identification of all 15 DRIP subunits. At least seven subunits are homologous to proteins described as components of Mediator, a complex that together with SRB proteins associates with RNA polymerase II through its large subunit's carboxyl-terminal repeat domain. This suggests that DRIP, perhaps through Mediator/SRB subunits, might function in part by targeting Pol II holoenzyme to promoters.
Several new, clinically relevant compounds dissociate 1,25(OH)2D3's antiproliferative /differentiation and calcemic activities, but the molecular basis for this has not been clearly elucidated. We tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is due to direct molecular effects on VDR. Two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21Waf1, Cip1, a key VDR target gene involved in growth inhibition, at a concentration 100 times lower than that of 1,25(OH)2D3. The differentiation dose-response closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR, and the receptor's ability to activate transcription in a cell-free system. These results provide compelling links between the 20-epi analogues' efficiency in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation.
REGULATION AND FUNCTION OF VERTEBRATE HOX GENES DURING LIMB DEVELOPMENT AND EVOLUTION
D. Duboule*, M. Kmita, F. Spitz, J. Zakany
Dept. of Zoology and Animal Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland
The function of Hox genes during development and their potential role in the evolution of the vertebrate limb has been studied in two different systems. In mice, single, double as well as triple inactivations (cre-mediated deletions) have been produced to assess the function of these genes during development. The results demonstrate that Hox genes act in a coordinated and cooperative way, where quantitative interactions seem to be at least as important as qualitative ones. This is supported by the molecular aetiology of some human syndromes due to the misfunction of these genes.
In a different approach, we analysed the development of fish appendicular skeletons. Pectoral fins are the appendicular structures which are, in fishes, homologous to the tetrapod forelimbs. We initiated a comparative study in lower vertebrates in the aim of understanding the ontogenetic and phylogenetic relationships between these two structures. These genes were shown, in mammals, to play an important function in limb pattern formation. The expression of these genes during pectoral fin development were analyzed in details and the significance of these results will be discussed in the light of the current theories (mainly derived from paleontological data) which account for the fin to limb transitions.
In parallel, we have used the tools of mouse genetics to investigate the regulatory mechanisms underlying the coordinated expression of Hox genes during limb development. Results will be discussed regarding the characterization of global regulatory controls, i.e. sequences that can control many genes at once, as well as their relevance in the above mentioned phylogenetic context.