Roberto Civitelli - US grants

Intercellular communication is a crucial issue for understanding the pathogenesis of osteoporotic syndromes, which result from unbalanced bone remodeling, wherein the resorption phase prevails. Bone remodeling is coordinated by a network of signals, in most part converging onto the osteoblasts, which thus works as a signal transducer for hormones, and chemical and mechanical stimuli. This, deranged intercellular cross- talk between osteoblasts and other cell types residing on bone, may well result in unbalanced bone turnover. While a great effort has been expended on soluble factors, which convey messages in endocrine, paracrine or autocrine manner to bone cells, no data currently exists on the direct cell-cell communication through gap junctions. Studies described in this application will focus on the mechanisms whereby hormonal, chemical and mechanical stimuli are received, transduced, and transmitted throughout bone cell populations, with emphasis on the relationship between the expression, function and regulation of gap junctions and osteoblast differentiation. Transmission electron microscopy and video image analysis will be used to evidence gap junction structures and test their function in both rat and human bone cell networks. Experiments are also planned to analyze signal propagation between bone cell via gap junctions. Video image analysis will be employed to monitor and quantitate the changes in cytosolic calcium and cyclic AMP. In addition, the role of inositol triphosphate as a signal transmitter, the modulation of intercellular communication by other second messengers and protein kinase phosphorylation, and the transmission of mechanical signals between bone cells will be explored.

DESCRIPTION (provided by applicant): Bone remodeling involves the synchronized activity of osteoblasts and osteoclasts and results in a cyclical succession of bone resorptive and formative phases. This orderly cellular activity requires efficient mechanisms of cell-cell interactions within the bone microenvironment. During the previous funding cycle, we have found that osteob1asts express members of the cadherin superfamily of cell adhesion molecules, in particular cadherin-11 (cadl1) and N-cadherin (Ncad). These two cadherins are an integral part of the phenotypic fingerprinting that defines cells of the osteogenic lineage as they differentiate into fully mature osteoblasts. We also found that disruption of cadherin mediated cell-cell adhesion by either inhibitory peptides or by expression of a dominant-negative cadherin mutant severely compromises the ability of osteoblastic cells to produce matrix proteins and mineralize in vitro. In the next funding period, we propose to extend these observations to in vivo models, and test the central hypothesis that cell-cell interactions mediated by cadherins are required for osteoblast function and bone remodeling in vivo. The proposed experimentation will make use of genetically engineered mice models with modified cadherin expression or function, some of them developed in our laboratory. These models will be used in the following three specific aims that are designed to address three specific questions: 1: Does interference with osteoblast cadherin expression or function lead to reduced bone formation and development of abnormal bone mass development and blunted response to osteoblast stimulation in vivo? 2: Is osteoblast maturation and function compromised by disruption of Ncad and/or cad11 genes? 3: Can other cadherins compensate for lack of Ncad or cad11 in osteoblasts? We anticipate that Ncad and cadl1 serve critical, common functions and thus interference with either Ncad and cad11 expression or function will result in abnormal bone formation, osteoblast activity, and response to intermittent PTH. These studies will disclose a novel mechanism by which the activity of bone forming cells is controlled invivo Alterations of cadherin mediated cell-cell interactions by hormonal imbalances or aging may lead to osteoblast failure.

Skeletal remodeling is characterized by an intercellular communicating network between osteoclastic and osteoblastic precursors and more differentiated bone forming osteoblast and the bone resorbing osteoclast, all of which is controlled by a complex interaction with bone matrix proteins and a paracrine-endocrine-autocrine interplay between local and systemic osteotrophic factors. This multidisciplinary research program will apply the techniques and concepts of cell and molecular biology to analyze cellular models of biological responses to osteotrophic factors, and to pursue the role of cell-cell and cell-matrix interactions in the transduction of these responses. In order to achieve these goals we propose to: (i) analyze the relationships between integrins-matrix protein interactions to the growth and differentiation of osteoblasts and the effects of cytokines and growth factors thereon; and to identify pivotal signal transduction pathways which mediate the interactions between integrins, matrix proteins and osteoblast function; (ii) analyze the role of metalloproteinases and matrix cleavage in osteoclast activation with the goals of identifying the structural requirements for activation, characterizing the osteoclast receptors necessary for activation, and determining the role of vacuolar H+ATPase polarization in the osteoclastic and osteoblastic activity response; (iii) analyze the role of integrins in response to mechanical strain as they participate in the generation of anabolic signals and cytoskeletal changes in the human osteoblast; (iv) to define a profile of osteoclast-derived chemokines and to analyze hormonal, cytokine and matrix-dependent modulation of osteoblast development, migration and function; and (v) to identify and characterize the chemokine receptors on osteoblasts as a function of osteoblast differentiation and activity. A specialized resource center designated as the "Cell Biology Core Laboratory" will provide assistance and consultation in experimental designs, preparation of appropriate cell cultures, immunochemistry,, ELISAs for cytokines, technical support for in situ hybridization analyses, and a centralized repository for cDNA probes.

DESCRIPTION (provided by applicant): Defective bone remodeling is the pathophysiologic basis of most metabolic bone diseases, including post-menopausal and age-dependent osteoporosis. During the tenure of this project, we have demonstrated hat osteoblasts are coupled through functional gap junctions, formed primarily by the gap junction protein, connexin43 (Cx43), and also by connexin45 (Cx45) [new gene designations: Gja1 and G/a7, respectively]. The finding that Gja1 mutations cause the human disease oculodentodigital dysplasia (ODDD), characterized primarily by skeletal abnormalities, further demonstrates that the skeleton represents one of :he main sites of action of Cx43. Indeed, we find that conditional G/a1 deletion in osteoblasts results in significant osteopenia and reduced bone formation rates, and that Cx43 is required for a full response to anabolic signals. The central hypothesis of this renewal application is that osteoblast connexins, Cx43 and Cx45, are essential modulators of skeletal growth and osteoblast function, and are involved in homeostatic responses to hormonal and physical stimuli in the post-natal skeleton. To test this hypothesis, we propose to determine;1: the relative contribution of Cx43 and Cx45 to postnatal skeletal growth and maintenance;2: the cellular and molecular mechanisms of connexin regulation of osteoblast differentiation and function: 3: the role of connexins in the homeostatic response to mechanical load in the postnatal skeleton. The proposed studies will take advantage of novel models of conditional Gja1 and G/a7 deletion, as well as of a new Gja1 ODDD mutant, building upon experimental methods and expertise we have accumulated during the tenure of this grant, and take the experimentation on gap junction biology in bone to a more translational level. This research will study two molecules that allow bone cells to directly communicate with each other thus influencing each others'function and ability to manufacture new bone. Results will allow us to better understand how the skeleton grows and becomes denser after birth, and how these molecules influence the skeletal response to disuse and mechanical load. The results will disclose new mechanisms by which bone is maintained in post-natal life, thus helping devise new therapeutic approaches to prevent bone loss and reduce fracture risk.

DESCRIPTION (provided by applicant): Defective bone remodeling is the pathophysiologic basis of most metabolic bone diseases, including postmenopausal and age-dependent osteoporosis. In previous work, we have found that osteoblasts express members of the cadherin superfamily of cell adhesion molecules, in particular cadherin-11 (Cad11) and N-cadherin (Ncad). Cadherins mediate cell-cell adhesion, but they also intersect the Wnt signaling pathway by stabilizing [unreadable]-catenin on the cell surface. Cell-cell adhesion is also a pre-requisite for assembly of gap junctions and intercellular communication. We have demonstrated that either dominant-negative disruption of cadherin function or recessive null mutations of the Ncad and/or Cad11 genes (Cdh2 and Cadh11) in mice hinders bone formation, leading to low peak bone mass and osteopenia. We also find that cadherin deficiency negatively affects the Wnt/[unreadable]-catenin system and reduces the abundance of intercellular junctions (adherens and gap junctions), in vitro. The central hypothesis of this project is that cadherins (Ncad and Cad11) control osteogenic differentiation by modulating cell-cell interactions in the bone marrow microenvironment, via cell-cell adhesion, communication and signaling. We further hypothesize that Ncad and Cad11 have partially overlapping, yet distinct roles in the osteoblast differentiation program. To achieve this goal, we propose to determine, 1) the relative roles of Cdh2 and Cdh11 in bone forming cell commitment and differentiation and proliferation in the post-natal skeleton;2) the interactions between cadherins (Ncad and Cad11) and Wnt signaling in osteoblast differentiation and function and 3) cadherin dependent organization and function of intercellular junctions (adherens and gap junctions) in osteogenic differentiation. We will use multiple in vitro, ex vivo and in vivo approaches, based on cadherin gene ablation mouse models we have developed, to study the consequences of cadherin deficiency on bone development, bone mass and osteoprogenitor cell recruitment and differentiation. We will also determine the cellular and molecular bases of the osteopenia of cadherin deficient mice. This proposal addresses fundamental mechanisms by which bone turnover is modulated in the bone microenvironment. Understanding the role of cadherins in bone biology is essential to gain a full picture of the molecular network by which bone development and homeostasis are controlled. PUBLIC HEALTH RELEVANCE: Therapeutic options for stimulating bone formation in subjects with bone demineralization, such as osteoporosis, are limited. This research will study two molecules that allow cells in the bone marrow and on the bone surface to come in direct contact, thus influencing each others'function and ability to manufacture new bone. Results will allow us to better understand how bone forming cells develop in adult animals, and will give us new tools to help people with low bone mass and fractures, by maximizing their potential for making new bone.

DESCRIPTION (provided by applicant): The Wnt/¿-catenin system is essential for skeletal development in embryogenesis and regulates bone mass in adult life. However, the mechanisms by which Wnt/¿-catenin signaling stimulates osteogenesis and bone formation in the post-natal skeleton remain nebulous. Recent data suggest that ¿-catenin provides critical regulatory cues at two points during the osteoblast differentiation program; in immature but committed osteoblast precursors ¿-catenin favors maturation into matrix secreting osteoblasts and expansion of the osteoblast precursor pool; in differentiated osteoblasts it inhibits osteoclastogenesis and perhaps terminal osteoblast differentiation. In previous studies, we demonstrated that ¿-catenin interacts with bone morphogenetic protein-2 and 4 (BMP-2/4) in producing new bone. Indeed, we find that BMP signaling is required for full osteogenic stimulation by ¿-catenin, whereas Tcf/Lef-dependent transcriptional activity is not. We also find that ¿-catenin is competitively recruited to either BMP or Wnt signaling pathways, and that the intersection of BMP and ¿-catenin signaling is, at least in part, mediated by Smad4/¿-catenin interactions. The central hypothesis of this project is that the pro-osteogenic action of ¿-catenin originates from its interactions with BMP signaling, and specifically Smad4, in immature osteoblasts, resulting in competitive recruitment of ¿-catenin to either canonical Tcf/Lef-dependent or Smad4-dependent signals. Thus, ¿-catenin can function as stimulator of either proliferation, via Tcf/Lef transcriptional activity, or maturation of immature osteoblasts, via recruitment into Smad4-containing transcriptional complexes. To test this hypothesis we propose to, 1: determine the dependency of ¿-catenin pro-osteogenic action on BMP signaling via Smad4; 2: analyze the role of Smad4 in modulating Wnt-dependent osteogenesis and Tcf/Lef signaling; 3 analyze the molecular interactions between Smad4 and ¿-catenin for osteogenesis. We will use in vivo and in vitro approaches based on inducible, conditional gene ablation or activation models to study whether interference with Smad4 expression alters ¿-catenin pro-osteogenic action and canonical Tcf/Lef-dependent activity. Considering the fundamental role of ¿-catenin in bone cell regulation, understanding the mechanisms by which ¿-catenin delivers either a mitogenic or a differentiation signal is essential to gain a full picture of the molecular network by which bone development and homeostasis are controlled. The proposed studies will also disclose the biologic importance of a molecular interaction that may be used as a new target for bone anabolism.

DESCRIPTION (provided by applicant): Washington University School of Medicine and the affiliated Barnes-Jewish Hospital have a long tradition of excellence in musculoskeletal research, patient care, education and training. During the past 40 years, a broad program in bone biology consisting of members of the Division of Bone and Mineral Diseases and associated investigators from other Divisions and Departments have provided the institutional and human resources in support of investigators and clinicians committed to diseases of mineral metabolism and skeletal disorders. A Mineral and Skeletal Metabolism Training Grant, AR007033, was continuously funded for more than 3 decades, and until 2004, was fundamental to accomplishing our teaching mission. In this new proposal, we seek to re-establish an interdisciplinary, institutional Metabolic Skeletal Disorders Training Program (MSDTP) to mentor and train the next generation of scientists and clinician-scientists in skeletal research, building upon the depth of talent at our institution. This program will offer pre- and post-doctoral training in 5 thematic areas: 1) Biology of the osteogenic lineage;2) Osteoclast biology and inflammatory skeletal disorders;3) Metastatic bone disease;4) Skeletal development and repair;5) Genetics of skeletal disorders. These themes represent the focus of the faculty participating in this training program and reflect common interests and interactions within each research group. The pool of Preceptors selected for this MSDTP are drawn from 5 academic Departments at Washington University (Internal Medicine, Orthopaedic Surgery, Pathology and Immunology, Developmental Biology, and Anatomy and Neurobiology) and reflects broad research interests, including skeletal development, osteoblast, osteoclast and chondrocyte biology, cell signaling, stem cell and structural biology, bone-immune and hematopoietic system interactions, biomechanics, ectopic calcification, as well as mouse and human genetics, bone metastasis and inflammatory osteolysis. The training will consist of 4 components: 1) mentored research training;2) core curriculum coursework;3) career development coursework;4) presentation and reporting skills. The post-doctoral training program will offer either a basic science- or a translational/clinical- oriented pathway. The pre-doctoral training pathway will be integrated with the PhD and MD/PhD programs administered by the Division of Biology and Biomedical Sciences (DBBS), which manages the graduate programs at Washington University. A streamlined but focused administrative structure will manage trainee recruitment, appointment and progress and monitor the program success. The program, which builds on strong interdepartmental and School-wide support, will leverage on existing institutional infrastructure, such as the Division of Bone and Mineral Diseases (for resources and facilities), the DBBS (for trainee recruitment and coursework), and the Institute for Translational and Clinical Sciences (for cores, services and formal training). Training the next generation of physicians and scientists is paramount to the continuous growth of research on skeletal biology. Such research is necessary to understand the genetic and molecular bases of skeletal disorders, and to devise new treatment strategies for diseases such as osteoporosis, inflammatory osteolysis, osteoarthritis, tendon failure, and bone metastasis, which afflict a large proportion of the elderly population.

DESCRIPTION (provided by applicant): Skeletal modeling represents the shaping of bone as the skeleton develops both pre- and post-natally in order to meet the needs of structural integrity and mechanical competence. Like remodeling, modeling requires coordination of osteoblasts (Obl), osteocytes (Ocy) and osteoclasts (Ocl). Unlike remodeling however, modeling osteoclasts and osteoblasts are not anatomically tethered but respectively resorb and form bone in locations which yield appropriate movement of the skeleton through space. During the tenure of this project, we demonstrated that bone forming cells communicate via gap junctions formed primarily by connexin43 (Cx43). The presence of cortical bone abnormalities in mice with Cx43 gene (Gja1) ablation in cells of the osteogenic lineage, and the fact that Gja1 mutations cause the human disease oculodentodigital dysplasia (ODDD), characterized by craniofacial and skeletal abnormalities, substantiate that skeletal homeostasis requires Cx43. In the last funding cycle, we made the unexpected observation that conditional Gja1 inactivation or induction of an ODDD Gja1 mutation in osteogenic cells primarily impairs cortical bone structure, leading to a larger total area, but decreased cortical thickness and compromised bone strength. This phenotype reflects increased endocortical resorption and periosteal apposition, as well as a hypomineralized and structurally abnormal bone matrix, associated with down-regulation of osteoprotegerin (Opg) and Sost in Obl/Ocy. Lack of Cx43 also alters the sensitivity of cortical bone to mechanical loading and unloading in an envelope-specific fashion. Hence, Cx43 in osteogenic cells controls envelope-specific formation and resorption of cortical bone, thus dictating its size, shape, biomechanical properties and responsiveness to mechanical stimuli. We therefore hypothesize that Cx43 is a key modulator of cortical bone modeling in the adult skeleton, and propose the following Specific Aims: 1) Mechanisms of Cx43 regulation of endocortical bone resorption, which will test the hypothesis that Cx43 regulates endocortical bone resorption via modulation of osteoprotegerin (Opg) expression by Obl and/or Ocy; 2) Mechanisms of Cx43 regulation of periosteal bone formation, which will test the hypothesis that Cx43 regulates periosteal bone formation, in part via modulation of Wnt signals; 3) Mechanical regulation of periosteal bone formation via Cx43, which will test the hypothesis that Cx43 modulates cortical bone responses to mechanical loading independently of bone architecture, via cell autonomous actions (Obl specific) and paracrine mechanisms (via the Ocy). Cortical bone (re)modeling is understudied but its importance in bone homeostasis and in the adaptive responses to mechanical factors is critical for maintaining bone strength and resistance to fractures. The proposed experiments will use genetic mouse models wherein Gja1 is selectively ablated or mutated at different stages of osteogenesis. Results will establish the mechanisms by which Cx43 modulates cortical bone and how such mechanisms cause the skeletal abnormalities of ODDD, findings that may be translated to clinical settings and lead to pharmacologic targeting.

? DESCRIPTION (provided by applicant): Washington University School of Medicine and the affiliated Barnes-Jewish Hospital have a 40-year tradition of excellence in musculoskeletal research, patient care, education and training. In 2011, the Washington University Metabolic Skeletal Disorders Training Program (T32) was established under the leadership of Dr. Roberto Civitelli, with the purpose of training the next generation of skeletal investigators and physician. In January 2012, T32 activities were integrated with those of the Core Center for Musculoskeletal Biology and Medicine (P30), directed by Dr. Linda Sandell, into a new Musculoskeletal Research Center (MRC), through a partnership between the Departments of Internal Medicine (Division of Bone and Mineral Diseases) and Orthopaedic Surgery, and support by the Dean of the Medical School. The MRC integrates the complementary aims of this T32 and the P30 into a unified structure that allows efficient utilization of the resources and synergism between the research and education missions of our Center. This T32, henceforth named Skeletal Disorders Training Program (SDTP), offers 3 pre- and 3 post-doctoral positions, and in the first 4 years of funding, a total of 10 trainees (4 graduate students and 6 post-doctora trainees) have entered the program. Of these, two post-doctoral trainees have obtained faculty appointments, one has been awarded a K99/R00 grant; another one, a Hispanic-American, obtained a F32 fellowship. Of the 4 pre- doctoral trainees, one has graduated and started post-doctoral training, and one is pursuing a combined M.D/Ph.D. degree. Together, they have published 28 peer-reviewed manuscripts and received numerous awards and recognitions. This SDTP offers research training in 5 thematic areas, reflecting the focus and common interests of the faculty participating in this training program: 1) Systemic and Local Regulation of the Skeleton; 2) Skeletal Immunology; 3) Tumor- Skeleton Interactions; 4) Genetics and Development of the Skeleton; 5) Skeletal Biomechanics and Repair. Mentors selected for this SDTP are drawn from 9 academic Departments at Washington University. The training consists of 5 components: 1) mentored research training; 2) curriculum coursework; 3) enrichment activities; 4) career development; 5) responsible conduct of research. The pre-doctoral training program is integrated with the PhD and MD/PhD programs administered by the Division of Biology and Biomedical Sciences (DBBS) and the Department of Biomedical Engineering (BME). This T32 builds on strong interdepartmental and School-wide support, and leverages the MRC Cores and infrastructure and other institutional resources, such as the DBBS and BME Department (for trainee recruitment and coursework), and the Institute for Translational and Clinical Sciences (for additional cores, services and career development training). Specific additions introduced during the current funding cycle and innovations proposed going forward are, among others, inclusion of BME students to the pre- doctoral pool, expansion of the mentor pool to faculty with interest in cancer biology, broadening of the educational coursework and enrichment activities, and strengthening career development education. Training the next generation of physicians and scientists is paramount to the continuous growth of research on skeletal biology. Such research is necessary to understand the genetic and molecular bases of skeletal disorders and to devise new treatment strategies for diseases such as osteoporosis, inflammatory osteolysis, osteoarthritis, tendon failure, and bone metastasis, which afflict a large proportion of the elderly population.

Abstract The host microenvironment is necessary for tumor growth and metastasis, and a major determinant of resistance to treatment and relapse. Expression of N-cadherin (Ncad), a calcium-dependent cell-cell adhesion molecule, in cancer associated fibroblasts (CAF) has been reported to favor tumor growth. Ncad is the main cadherin expressed in bone cells, where it functions in cell-cell adhesion, but also regulates signaling and differentiation. In preliminary studies we found that, contrary to expectations, ablation of the Ncad gene (Cdh2) in osteolineage cells ? expressing the osteogenic marker, Osterix (Osx+) ? does not affect bone engraftment of breast cancer cells; however, subcutaneous tumors grow faster and lung metastases develop earlier than in wild type littermates. We also find, unexpectedly, that Ncad is present in previously unrecognized Osx+ cells in extra-skeletal tumors. These cells have a transcriptomic profile more similar to osteogenic cells than to CAF, and favor tumor growth. Furthermore, Ncad in Osx+ cells down-regulates p38 responsive genes, a pro- tumorigenic pathway. In human breast cancer, Osx+ are an index of poor prognosis. These preliminary results demonstrate that Ncad in Osx+ cells is a negative regulator of cancer progression, an effect opposite to Ncad reported action in CAF. We contend that Ncad exerts multiple and even opposite actions on tumorigenesis depending on the cell context where it is expressed, via modulation of specific signaling pathways. Based on these preliminary data, our central hypothesis is that Ncad in pro-tumorigenic Osx+ cells restrains tumor growth by regulating signals that reprogram the tumor microenvironment. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1 ? Modulation of extra-skeletal tumor growth by Ncad in Osx+ cells; testing the hypothesis that Ncad in Osx+ cells restrains tumor growth; loss of Ncad in TAOC increases tumor growth and metastases in mice. Osx+ Ncad+ cells correlate with tumor grading in human breast cancer. Specific Aim 2 ? Mechanisms of Ncad modulation of pro-tumorigenic signals in tumor-associate Osx+ cells; testing the hypothesis that Ncad in Osx+ cells is an upstream regulator of p38 and Pten signaling; loss of Ncad in Osx+ cells results in accentuated expression of p38-dependent pro- tumorigenic factors and decreased Pten dependent signals, leading to tumor microenvironment modification and enhanced tumorigenesis. We will use in vivo approaches, including diphtheria toxin-induced selective cell ablation, parabiosis, lineage tracking, as well as non-biased transcriptomic approaches (single cell RNAseq) to unlock the cellular and molecular mechanisms by which Ncad in extraskeletal Osx+ cells affects tumor growth and metastasis. We will also determine the clinical pathology correlates of Ncad expression in Osx+ cells in human tumors. Results of the proposed studies will lay the foundations for the development of new markers of tumor progression and/or new therapeutic strategies aimed at interrupting environmental support of cancer growth and metastasis by targeting specific cells in the tumor stroma.