Gerard Karsenty, Md - US grants

Alterations in the synthesis of extracellular matrix components including type I collagen occur in a number of fibrotic diseases such as scleroderma, ostearithritis as well as osteoporosis. Studies of the regulatory mechanisms which control the expression of the two type I collagen genes are critical to understand these alterations. In most physiological and pathological conditions, the expression of the alpha1(I) and alpha2(I) collagen genes are coordinately expressed. This coordinate regulation can be explained by the interaction of DNA-binding proteins with common cis-acting elements in the promoter region of both genes. One sequence specific DNA-binding protein that binds similar cis-acting elements located at approximately the same distance from the transcriptional start site in the promoter regions of the alpha1(I) and alpha2(I) mouse collagen genes, has been purified to homogeneity. This factor acts as a transcriptional inhibitor and is called Inhibitory Factor 2 (IF-2). IF-2, a metallo protein that requires zinc ions to bind to DNA, is present in a large variety of cells and presumably participates in the control of expression of several genes. We propose to purify IF-2 to homogeneity, to obtain amino-acid sequences for this protein and to clone cDNAs corresponding to the factor. We will use the purified bacterially expressed protein to generate polyclonal antibodies against IF-2. Using the purified protein and the cDNA clone we plan to perform, at a later time, a structure-function analysis of the molecule, to study the molecular mechanism of transcriptional repression by this transcription factor. We expect that the characterization of transcription factors like IF-2 and the molecular studies of its function win increase understanding of the mechanisms which control the expression of the type I collagen genes in physiological situations and in pathological situations and probably other genes and will bring new information about the regulatory mechanisms used by eukaryotic cells to synthesize specialized proteins.

The mechanisms preventing inappropriate calcification of tissues such as the growth plate cartilage or the arteries are poorly understood. The growth plate is a specialized cartilaginous structure responsible for the longitudinal growth of the skeleton. Early calcification of the growth plate leads to an arrest of skeletal growth. one protein that is synthesized by both vascular smooth muscle cells and chondrocytes, is secreted into the extracellular matrix, and is suspected to have mineral- binding properties is matrix gla protein (MGP). To address the possible function of MGP as an inhibitor of calcification we have generated MGP- deficient mice. The MGP-deficient mice have spontaneous calcification of their arteries and of the cartilage in the proliferation zone of the growth plate cartilage, leading to blood vessel ruptures, short stature, osteopenia and fractures. This phenotype indicates that MGP acts as an inhibitor of calcification of the arteries and growth plate cartilage and by that means influence longitudinal growth of the skeleton. We propose: -To rescue the lethal vascular phenotype of the MGP-deficient mice so as to determine the long-term effect of the absence of MGP on bone and cartilage formation and calcification. -To perform a histomorphometric and ultrastructural study of the growth plate in MGP-deficient mice to monitor chondrocyte proliferation and hypertrophy, cartilage calcification and longitudinal bone growth. -To perform a cellular analysis in vitro of the cartilage phenotype of the MGP-deficient mice. -To analyze the effect of MGP overexpression in osteoblasts on bone matrix mineralization in transgenic mice and in tissue culture. -To analyze the effect of MGP overexpression in osteoblasts on bone matrix mineralization in transgenic mice and in tissue culture. -To study human skeletal dysplasias that produce a similar or related phenotype as the one observed in MGP deficient mice.

Extracellular matrix (ECM) mineralization is a physiologic process in bone and teeth and a pathologic one everywhere else in the body. Pathologic ECM mineralization often has deleterious consequences in conditions such as coronary artery disease and osteoarthdtis two diseases for which no curative treatment is available. Despite the biological importance of ECM mineralization the molecular mechanisms restricting it to bone physiologically remain unknown. In particular gene deletion experiments failed to identify osteoblast-specific genes necessary to initiate bone mineralization while they demonstrated the existence of inhibitors of pathologic ECM mineralization. Two of these inhibitors, Npps and Ank, act by producing inorganic pyrophosphate, an inhibitor of ECM mineralization, and exporting it outside the cells. However, the observation that Npps and Ank are expressed in osteoblasts (data not shown), further complicates our understanding of bone mineralization. The absence of osteoblast-specific proteins initiating bone mineralization together with the expression in osteoblasts of mineralization inhibitors like Npps and Ank that affect phosphate metabolism led us to test the following hypothesis: could the spatial restriction of ECM mineralization to bone be explained, at least in part, by coexpression in osteoblasts of genes that are not osteoblast-specific but that affect phosphate metabolism and ECM assembly? The specific aims are: -To induce ectopic ECM mineralization by ectopic expression of tissue non-specific alkaline phosphatase -To raise TNAP levels in blood to determine if this alone can induce ectopic ECM mineralization -To determine whether ECM mineralization in the growth plate is a molecular determinant of longitudinal growth of the skeleton -To use genetic means to identify substrates for TNAP in bone -To address the role of calcium ions in the initiation of ECM mineralization. We believe that this analysis will greatly enhance our understanding of cartilage and bone ECMs mineralization. This may open novel research avenues to understand the mechanisms leading to the closure of the growth plate at the end of puberty. Finally, it could provide new therapeutic avenues to explore diseases characterized by abnormal or ectopic ECM mineralization such as osteoarthdtis (OA).

DESCRIPTION (provided by applicant): Elucidating the transcriptional control of osteoblast differentiation is a prerequisite to achieve a better understanding of vertebrate skeletal development in the craniofacial region. Major progress has been made in the last ten years in identifying transcription factors acting as determinant which between mesenchymal cells and osteoblast progenitors in contrast we still have little knowledge how transcription factors control osteoblast terminal differentiation and function. To address this problem we have used a combination of molecular, human and mouse genetic approaches to identify ATF4 as a regulator of osteoblast terminal differentiation and function and the kinase of osteoblast terminal differentiation and function and the kinase RSK2 as a regulator of ATF4 transactivating function. That RSK2 is inactivated in a human skeletal dysplasia termed Coffin Lowry Syndrome that effects primarily the craniofacial region illustrates the biological importance of this regulatory loop. Because ATF4 regulates expression of osteoblast-specific genes and Type I collagen production post-transcriptionally it raises questions about how it executes all its function. Moreover, that RSK2 kinase activity is regulated by extracellular signals suggests that this regulatory loop may explain some of the bone anabolic effect of secreted molecules. Finally, having in hand three osteoblast-specific transcription factors allows us to ask questions about their relationship during development and after birth. To address these questions the specific aims are: To determine to which extent the skeletal phenotype of Atf4-deficient mice is secondary to a decrease in amino acid import. To analyze whether ATF4 is a transcriptional mediator of known extracellular regulators of bone formation. To compare the effect of an osteoblast-specific versus a ubiquitous over-expression of Atf4 in mice. To determine whether Runx2, Osterix and ATF4 interact genetically to affect osteoblast differentiation. To determine whether Atf4 can rescue the osteoblast differentiation defect of Runx2- or of Osterix-deficient mice. To establish in vivo that ATF4 is the physiological target of RSK2 in osteoblasts.

DESCRIPTION (provided by applicant): The uncovering that leptin regulated bone mass revealed the existence of a control of bone physiology by adipose tissue. Besides the emerging molecular complexity of this regulation its very existence raised a novel question. Do osteoblasts and through them bones regulate in turn expression of leptin and/or of any other adipocyte - derived hormones? To address this question we embarked in a broad-based effort to generate through E.S. cells technology multiple mouse mutant strains each of them lacking one osteoblast enriched gene. We then will analyze their energy metabolism. In the course of there studies we generated through classical and in an osteoblast-specific manner mice lacking Esp. Esp encodes a protein tyrosine phosphatase expressed in osteoblasts and in Sertoli cells of the testis. The only detectable phenotypic abnormalities of any kind one could detect in Esp-deficient mice were an hypoglycemia, an increase in insulin secretion, and an increase in adiponectin secretion resulting in an increase in insulin sensitivity. That these results were obtained in mice lacking this phosphatase only in osteoblasts establishes that bones, through a mechanism we propose to study, regulate energy metabolism. We propose in this application to use a combination of classical physiology, cell-based, molecular, biochemical and genetic approaches to begin elucidating, through Esp biology, this novel function of bones. The specific aims are: - To demonstrate that the phenotype of Espob-/- mice is due solely to an increase in adiponectin and insulin secretion. - To determine whether Espob-/- mice are resistant to diet induced obesity and type 2 diabetes. - To determine genetically whether Espob-/- mice are resistant to atherosclerosis - To determine whether or not OST-PTP acts through its extracellular domain

[unreadable] DESCRIPTION (provided by applicant): The fact that an adipocyte-derived hormone like leptin regulates bone remodeling raises the project that bone cells may in turn influence adipocyte biology. While testing this hypothesis we identified a gene, Esp, encoding a tyrosine phosphatase whose osteoblast-specific deletion results in an increase in insulin and adiponectin secretion by pancreatic cells and adipocytes respectively. Looking for substrates of the Esp gene product we noted that Osteocalcin-deficient mice had a phenotype that is the mirror image of the one observed in Esp-deficient mice. Osteocalcin -/- mice display a decrease in insulin and in adiponectin secretion. Moreover, removing one allele of osteocalcin sufficed to correct the entire metabolic phenotype of the Esp -/- mice. Based on these and additional published preliminary data we have shown that osteocalcin is a hormone regulating insulin secretion and sensitivity we now intend to foster our molecular understanding of how osteocalcin regulates energy metabolism. To achieve this goal we propose the following specific aims: 1. To demonstrate that gamma carboxylase is a target of OST-PTP, the Esp gene product in vivo 2. To generate mice lacking, in osteoblasts only, the Vitamin K epoxy reductase C1, another enzyme involved in carboxylation of osteocalcin 3. To determine whether bones, through osteocalcin, are responsible of the increase in insulin secretion observed in absence of leptin 4. To define how insulin signaling in osteoblasts regulates osteocalcin expression or bioactivity. PUBLIC HEALTH RELEVANCE. Type 2 diabetes and obesity is a growing public concern. Here we identified a potential new hormone regulating glucose metabolism. This hormone may be an important therapeutic tool in the fight against the metabolic syndrome. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our laboratory has proposed 10 years ago that there is a common endocrine control of bone mass, appetite and reproduction. We have in the last decade embarked in a two-pronged approach to verify this hypothesis. In the first aspect of our work we showed that the adipocyte - derived hormone leptin regulates bone mass via a central relay. In the second aspect of this work we then showed that the osteoblast-derived hormone osteocalcin favors glucose handling by the body and enhances energy homeostasis. Taken together, these two aspects of our work cover the first arm of our hypothesis namely that there is a common control of bone and energy metabolisms. More recently we came across another phenotype in the Osteocalcin-deficient mice that may allow us to establish an endocrine link between bone remodeling and reproduction. Indeed, Osteocalcin-deficient male mice are subfertile, have significantly smaller testes, decreased sperm counts and display a 60 to 80% decrease in circulating testosterone levels compared to wildtype littermates. These and additional preliminary data presented in this application suggest the hypothesis that osteocalcin may regulate not only energy metabolism but also reproduction. To further test this hypothesis we propose to use a combination of molecular, genetic and morphologic approaches. Our specific aims are: - To perform a thorough molecular and morphological characterization of the fertility phenotype of male Osteocalcin-deficient mice - To determine genetically and pharmacologically whether it is the carboxylated or the uncarboxylated form of osteocalcin that regulates spermatogenesis - To demonstrate genetically that it is through its osteoblast expression that osteocalcin favors sperm production and testosterone synthesis. - To use genetic epistasis to demonstrate that a putative osteocalcin receptor expressed on Leydig cells mediates osteocalcin function on sperm production and testosterone synthesis.

? DESCRIPTION (provided by applicant): We have been interested in recent years in the biology of the most abundant adipokine, adiponectin. In addressing what could be the functions(s) of adiponectin in animals fed a normal diet we considered the fact that adiponectin appears during evolution with bone and is regulated by the bone-derived hormone osteocalcin. We then asked whether adiponectin was regulating bone mass. Our results obtained in vivo showed that 1) adiponectin inhibits bone formation by acting directly on osteoblasts while it favors bone formation by inhibiting the sympathetic nerves through its signaling in the brain; 2) none of the known receptors for adiponectin seem to mediate any of these two functions and adiponectin does not recruit AMPK and does not regulate ceramide activity in osteoblasts; 3) the signaling pathway triggered by adiponectin in osteoblasts suggested that the receptor for this hormone in osteoblasts is a receptor tyrosine kinase. Further analysis presented in this application strongly suggest, based on molecular and biochemical grounds, that the receptor for adiponectin in osteoblasts and possibly in neurons of the locus coeruleus may be the discoidin receptor 2 (DDR2), a RTK with a slow kinetic of phosphorylation. Based on preliminary findings presented in this application we now propose to test this hypothesis in vivo. The specific aims of this application are: To demonstrate that DDR2 mediates adiponectin signaling in osteoblasts. To demonstrate that in vivo FoxO1 lies downstream of DDR2-dependent signaling in osteoblasts. To determine in vivo the biological significance of the interactions occurring between DDR2, AdipoR1 and T-cadherin. To demonstrate in vivo that DDR2 and/or DDR1 mediates adiponectin signaling in neurons of the locus coeruleus.

? DESCRIPTION (provided by applicant): The regulation glucose metabolism by the bone-derived hormone osteocalcin naturally raises the question of why bone would regulate glucose metabolism in the first place. One possible way to address this question is to determine the function of glucose in osteoblasts. In addressing this question we showed that osteoblasts uptake considerable amounts of glucose in an insulin-independent manner through the glucose transporter Glut1. Glucose is necessary in osteoblasts for collagen synthesis and therefore for bone formation. In addition, by preventing its ubiquitination glucose favors Runx2 accumulation in osteoblasts, expression of Runx2 target genes such as Osteocalcin and thereby whole-body glucose homeostasis. The importance of the function of glucose in osteoblasts begs in turn the question of the regulation of expression of Glut1 in cells of the osteoblast lineage. Our preliminary experiments indicate that Runx2 is a major regulator of Glut1 expression in vivo and therefore point toward a feed forward regulation between Glut1-dependent glucose uptake in osteoblasts that favors collagen synthesis, bone formation and Runx2 accumulation and Runx2 that favors Glut1 expression. We hypothesize that this feed forward loop acts as an amplification mechanism allowing proper bone formation during embryonic development and after birth. To test this hypothesis we propose the following specific aims: To determine through molecular means whether Runx2 regulates Glut1, Glut2 and Glut3 expression in osteoblasts directly or indirectly. To demonstrate through genetic means that Runx2 and Glut1 act synergistically to determine the onset and extent of bone formation. To determine through several genetic means to what extent raising blood glucose levels of pregnant Runx2+/- female mice can rescue the bone formation defects of their Runx2- deficient progeny.

? DESCRIPTION (provided by applicant): Capitalizing on 15 years of research in multiple laboratories in this area the goal of this Program Project application is to provide a comprehensive and mechanistic understanding of the cross-talk existing between bone and the brain by addressing four issues. First, the discovery that brain-derived serotonin favors bone mass accrual by inhibiting the activity of the sympathetic nervous system raises questions about the transcriptional mechanisms regulating brain serotonin accumulation and catecholamine synthesis. Second, the role played by factors coming from the mothers in the offspring brain development and cognition raises the question that a similar influence for bone mass acquisition may exist. Third, that Tph1, the initial enzyme in the synthesis of gut-derived serotonin is also needed for melatonin synthesis in pineal gland raises the question of the possible role of melatonin as a regulator of bone mass accrual. Our hypothesis is that one extracellular cue limiting the influence of the sympathetic tone on osteoblast functions could be melatonin. Fourth, if we now look at the relationship between bone and the brain from the point of view of bone, the powerful but inhibitory regulation of bone mass accrual by the brain raises the question of whether bone limits this deleterious influence by acting directly in the brain. In addressing these fundamental questions of bone biology and whole-organism physiology the laboratories of the three project leaders of this application have generated in the last five years enough preliminary data to justify their respective projects. The three major questions tackled by this Program Project are: * To determine the influences of bone remodeling via osteocalcin on the metabolic status and cognition of mice as they age. * To demonstrate that one mechanism by which the longevity-associated protein Sirtuin 1 regulates bone mass is through its functions in the brain. * To define the role of melatonin in bone remodeling during growth, aging and gonadal failure and determine whether maternal by-products of Tph1 are determinants of peak bone mass and future bone health of the offspring.

Project Summary The influence of muscle derived molecules on bone remodeling has been for a long time a topic of intense research in bone biology. Recently we have provided suggestive evidence that interleukin-6 (IL-6) synthesized in, and secreted in the general circulation by myoblasts, is a powerful and positive regulator of the release of the bone-derived hormone osteocalcin. Moreover, classical studies of bone cell biology had proposed twenty five years ago that IL-6 may regulate osteoclast differentiation and some aspects of bone remodeling. In view of both of these classical and more recent observations, the question arises as to know whether muscle-derived IL-6 is a biologically important regulator of bone remodeling at rest or during exercise, in young and/or older mice. A second question is to know whether muscle-derived IL-6 is a significant regulator in vivo of the release of osteocalcin and therefore of the biological activity of this osteoblast-specific secreted molecule. We intend to address these two related questions of bone biology in vivo and through the use of several mouse models of cell-specific deletion for either IL-6 in myoblasts or its receptor in osteoblasts, osteocytes and osteoclasts. To achieve these goals, the Specific Aims of this application are: ? Determine the role of muscle-derived IL-6 in regulating exercise response and bone mass in vivo. ? Delineate the relative influence of IL-6 signaling in cells of the osteoblast or the osteoclast lineage on exercise capacity and bone mass in vivo. ? Determine whether acute or chronic delivery of recombinant osteocalcin is sufficient to increase exercise capacity in wild-type mice as they age.

Project Summary ? Administrative Core The Administrative Core will be responsible for coordinating the scientific and financial activities of the Program Project, daily administration and collaborative research activities of the overall Program Project. The scientific coordination will be supervised by the Program Project Director, Dr. Karsenty. The duties of this core, which are described in the body of the application, will include: - Scientific leadership to foster the overall progress of research, scientific focus, integrity, and ethical conduct and support to the Projects and Core B. - Administrative support and budget management. - Establishing policies for effective utilization of Core B, and archiving of copies of Core B assembled data. - Supervision of procurement and personnel reports. - Coordinating institutional authorizations for recombinant DNA, radioisotopes, and vertebrate animals. - Organization of project-related records, such as publications and invited seminars by the Program Project Investigators. - Preparation of progress reports to the NIH and other necessary correspondence with NIA program staff. - Scheduling monthly meetings of the Program Project investigators, and annual visits of the External Advisory Committee. - Facilitating and providing data management and services. - Bringing internationally-known scientists to Columbia to consult with the Project Leaders. - Scheduling of meetings and talks of visiting scientists. - Facilitation of Institutional Review Board submissions. - Maintenance of overall progress of research, scientific focus, integrity, and ethical conduct.