Sarah L. Dallas - US grants

DESCRIPTION: A frequent and debilitating complication of breast cancer is the bone destruction that results when the cancer cells metastasize to bone. The underlying hypothesis for the proposed studies is that matrix-bound growth factors in bone may help define it as a fertile ground for the growth of breast cancer metastases and influence their bone-destructive potential. The goal of this proposal is to examine the role of bone matrix-bound transforming growth factor beta (TGFb) in regulating the bone destructive capacity of metastatic breast cancer cells through stimulating production of parathyroid hormone related protein (PTHrP), a powerful bone resorbing factor. Specific Aim 1 will determine whether bone matrix-bound TGFb is responsible for the increase in PTHrP production observed when breast cancer cells are grown on bone matrix. This will be done in vitro through the use of TGFb neutralizing antibodies and the use of bone matrix preparations from mice which lack the gene for TGFb-1. In vitro studies will be confirmed using an in vivo metastasis model to compare PTHrP production and osteolytic lesions by breast cancer cells in normal and TGFb-l deficient mice. In Specific Aims 2 and 3 the molecular mechanism for release of bone-matrix bound TGFb by breast cancer cells will be investigated. Since proteolytic cleavage of the latent transforming growth factor beta binding protein-1 (LTBP-1) is an important mechanism for release of matrix-bound TGFb by bone cells, Specific Aim 2 will determine whether a similar mechanism is used by breast cancer cells. This will be done by measuring release of LTBP-1 and TGFb by breast cancer cells grown on radiolabeled bone matrix using immunodetection methods. In Specific Aim 3 the proteolytic cleavage sites in LTBP-1 will be mapped. Antagonist peptides will then be designed and tested to identify peptides which will inhibit cleavage of LTBP-1 by breast cancer cells and therefore inhibit release of TGFb from bone matrix. Since latent TGFP must be activated in order to exert its effects on the breast cancer cells, Specific Aim 4 will examine the ability of the breast cancer cells to activate matrix-released latent TGFb. This will be done using bioassays for TGFb in conjunction with Western blotting. This study will provide a new approach for studying the complex interactions between breast cancer cells and the bone microenvironment and will address the general question of whether matrix-bound growth factors in host tissues influence the behavior of metastatic cancer cells. Blocking the release of bone matrix-bound TGFb by breast cancer cells may potentially be developed as a new treatment strategy to reduce cancer-associated osteolysis and alleviate the suffering of patients with bone metastatic breast cancer.

DESCRIPTION (Applicant's Description): The candidate's research experience is in the field of bone cell biology, where her main interests have been in growth factors, extracellular matrix proteins and myeloma bone disease. The candidate is at the end of her postdoctoral training and wishes to become an independent investigator, capable of directing high quality academic research in her own laboratory. To do this, she proposes to being her expertise in bone matrix and growth factors into the field of breast cancer. Breast cancer frequently metastasizes to bone and often causes bone destruction, with debilitating complications for the patient. The proposed studies are based on the hypothesis that bone matrix-bound growth factors play a key role in defining bone as a fertile ground for the growth of breast cancer cells. These studies define an area of interest for the candidate which is separate from that of her mentors, but allows her to draw from the expertise she has gained from her postdoctoral training. The candidate would obtain additional training during years 1 and 2 from her co-sponsors, Drs. Theresa Guise and Lynda Bonewald, to prepare her for entry into the field of breast cancer as a productive researcher. By year 3 the candidate will be a fully independent investigator. The candidate is currently in the Division of Endocrinology, which has a large group of workers who are experts in the field of breast cancer. Thus there will be many opportunities for professional interactions with senior colleagues, and for presentation and discussion of data. The candidate would also become a member of the San Antonio Cancer Institute and benefit from the resources provided by this organization. The goal of the proposed studies is to examine the role of bone matrix-bound transforming growth factor beta (TGFbeta) in regulating the bone destructive capacity of metastatic breast cancer cells through stimulating production of parathyroid hormone related protein (PTHrP), a powerful bone resorbing factor. Specific Aim 1 will examine whether bone matrix-derived TGFbeta is responsible for stimulating PTHrP production by breast cancer cells, thereby increasing cancer-associated bone destruction. This will be done using in vitro and in vivo models. Specific Aims 2 and 3 will examine the molecular mechanisms for release of bone matrix-bound TGFbeta by breast cancer cells. Initially it will be determined whether release occurs through proteolytic cleavage of the latent TGFbeta binding protein (LTBP-1) as is the case with bone cells. The proteolytic cleavage sites in LTBP1 will then be mapped and antagonist peptides designed to inhibit cleavage of LTBP1 and the resulting release of TGFbeta from bone matrix. Specific Aim 4 will examine the ability of breast cancer cells to activate matrix-released latent TGFbeta. This study will provide a new approach for studying the complex interactions between breast cancer cells and the bone microenvironment and may lead to the development of new therapies to reduce cancer-associated bone destruction.

The extracellular matrix (ECM) has been viewed as a static three dimensional scaffold that supports cells and tissues. However, our recent molecular imaging studies in living osteoblasts have shown that the ECM is highly dynamic and that ECM molecules form structures that continually undergo movement and deformation, mediated by cell-generated mechanical forces. These studies also suggest a novel role for cell movement in ECM assembly and reorganization. Fibronectin is one of the earliest proteins to be assembled into the ECM and facilitates assembly of other matrix proteins. In the previous funding cycle, using fibronectin null cell culture models and targeted gene deletion in osteoblasts, it was shown that fibronectin is essential for assembly of multiple bone ECM components, including type I collagen, fibrillin-1, Latent TGFβbinding protein-1, decorin and biglycan and is also required for normal mineralization. Fibronectin depletion also inhibits osteoblast differentiation. Fibronectin[unreadable]s effects on differentiation can be rescued by supplementation with BMP2, whereas its effects on ECM assembly and mineralization cannot, suggesting that fibronectin may regulate osteoblast differentiation via ECM targeting of osteogenic growth factors. Based on these observations, the proposed studies are centered around two main hypotheses. The first is that fibronectin is a multifunctional regulator of osteoblast function through its effects as a central orchestrator for assembly of bone ECM proteins and through its role in ECM regulation of growth factor activity. The second is that dynamic cell movement is essential for the assembly and reorganization of bone ECM proteins. To test these hypotheses, in vitro and in vivo approaches will be used in combination with live cell imaging. Aim 1 will define the cascade of assembly of bone ECM proteins and its integration with cell and matrix dynamics. This will be done using fibronectin null osteoblast models in conjunction with live cell molecular imaging of bone ECM proteins and quantification of cell and fibril dynamics by computational analysis. Aim 2 will further define the role of fibronectin in osteoblast differentiation through regulation of osteogenic signaling pathways. Live cell imaging techniques will also be used with osteoblast/osteocyte lineage reporters and fluorescent probes for ECM components to determine how osteoblast differentiation is dynamically integrated with ECM assembly and reorganization. Aim 3 will use novel imaging probes to determine the dynamics of collagen assembly into the ECM of osteoblasts and the role of fibronectin in collagen deposition in vitro and in vivo. The studies will provide fundamental insights, from a dynamic perspective, into the mechanisms of assembly of bone ECM and how the ECM regulates osteoblast function. The data generated will significantly advance our understanding of the molecular and dynamic mechanisms underlying bone formation and have key implications for skeletal diseases such as osteoporosis, arthritis, osteogenesis imperfecta and bone diseases related to ECM proteins.

[unreadable] DESCRIPTION (provided by applicant): Osteocytes make up over 90% of the cells in bone. However, little is known about their function, due to difficulties in accessing these cells in vivo or isolating them for in vitro studies. Although the osteocyte has classically been viewed as an inactive cell, evidence is accumulating that these cells are responsive to mechanical loading and are able to modify their local environment, suggesting that they may be more active than previously thought. Using a transgenic mouse in which green fluorescent protein (GFP) is targeted to osteocytes under control of the dentin- matrix protein-1 (DMP1) promoter, we have performed time lapse dynamic imaging studies on living osteocytes in calvarial organ cultures. Surprisingly, these studies have revealed that, far from being a static cell, the osteocyte is highly dynamic. Osteocytes that were embedded within their lacunae expanded and contracted their cell bodies and extended and retracted their dendrites. Dendritic connections between osteocytes and with motile cells on the bone surface appeared to be transient, with connections being made and broken. These observations raise the possibility that dendrites, rather than being permanent intercellular connections, may be dynamic structures that can be altered in response to stimuli affecting osteocyte function. The central hypothesis for the proposed studies is that osteocytes within bone are highly dynamic cells and that their cell body and dendrite motions are critical to their function and to the embedding process. To test this hypothesis, multidisciplinary approaches will be used, including the use of transgenic mouse models for dynamic imaging of living osteocytes and osteoblasts, together with computational analysis of cell body and dendrite motions. In aim 1 we will use state-of-the-art imaging techniques to determine the dynamic properties of osteocytes and their dendrites in living bone explants. We will determine whether these change with age and/or in response to external stimuli known to affect osteocyte function, such as hypoxia and prostaglandin E2. The effects of inhibitors of cell motility and actin polymerization/ depolymerization on gap junctional signaling as an endpoint of osteocyte function will be determined. In aim 2 we will use a bone forming organ culture model to dynamically image osteoblasts embedding and differentiating into osteocytes. This will be done using calvaria from mice expressing DsRed in osteoblasts and GFP in osteocytes, driven by the Col1a1 and DMP1 promoters, respectively. Mineral deposition will also be imaged to determine how the dynamics of osteoblast embedding/differentiation are integrated with mineralization. The effects of inhibitors of cell motility and actin polymerization/depolymerization on embedding and mineralization will be determined. Successful completion of these exploratory studies will develop new and innovative models to facilitate research into osteocyte function, provide fundamental insights into the process of osteoblast to osteocyte transition and provide a new paradigm for viewing osteocyte dendrites as dynamic interactive structures. This will have major implications for our understanding the role of osteocytes in normal bone and in diseases, such as osteoporosis and osteomalacia. These studies will lay the foundation for an RO1 application in 1-2 years. This research is relevant to public health as it will provide highly novel insights into the function of the osteocyte, a major cell type in bone, which is still not well understood. Osteocyte apoptosis has been implicated in diseases such as osteoporosis and osteonecrosis of the jaw and recent evidence suggests that this cell type plays a major role in regulation of bone formation/mineralization and in regulation of phosphate homeostasis. Understanding the function of this cell type will therefore aid the development of treatments for osteoporosis, osteomalacia and other metabolic bone diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our capacity to understand cellular function in health and disease from the macro to micro to nano and even to the atomic scale depend on the capabilities of advanced instrumentation. The NIH funded projects described in this application require increased imaging resolution and multi-spectral capabilities for their brightfield and fluorescence microscopy applications to advance to the next level. A Zeiss LSM 710 34 channel laser scanning confocal microscope is requested. The instrument will be operated as a shared resource to support the research programs of a core group of NIH funded major and secondary users in the UMKC School of Dentistry, UMKC School of Nursing and University of Kansas Medical Center. The user group has a research emphasis on mineralized tissues and musculoskeletal research. To accommodate the range of project applications, the instrument will be configured for basic confocal microscopy on fixed cell and tissue specimens, and for live cell imaging, multispectral imaging, fluorescence recovery after photobleaching (FRAP) and optical sectioning/3D imaging. One of the projects that will employ this technology is investigating the mechanisms of assembly of bone extracellular matrix (ECM), using time lapse imaging with novel fluorescent probes for various bone ECM components. These studies are providing novel insights into the dynamic nature of bone matrix assembly and the role of cell motility in the assembly process. The role of the osteocyte in regulating skeletal responses to mechanical loading is the focus of three of the participating projects. Until recently, little was known about the function of this elusive cell type, due to its inaccessibility, embedded within bone. With the recent identification of osteocyte differentiation markers and development of transgenic mice with fluorescent osteocyte lineage reporters, it is possible to image these cells within their mineralized environment to an unprecedented level. One project examines the dynamic properties of osteoblasts and osteocytes using lineage reporters combined with time lapse imaging to gain insight into the process by which osteoblasts differentiate into osteocytes. Another project examines the role of the osteocyte protein, E11/gp38, in dendrite formation and in osteocyte responses to mechanical loading. A third project examines the molecular mechanisms by which osteocytes perceive mechanical loading signals and translate them into bone formative responses. Other projects involve calcium imaging in muscle, determining the molecular mechanisms regulating phosphate homeostasis and determining the molecular pathogenesis of bone inflammatory lesions in Cherubism patients. The Zeiss LSM 710 system will advance the progress of these projects by providing an unsurpassed level of resolution that is needed for 3D imaging of osteocyte networks in mineralized tissues. The multi-spectral imaging capabilities of the LSM 710 and its enhanced sensitivity for live imaging, are essential to advance this research to the next level, and will allow for simultaneous imaging of multiple molecular probes, maximizing the information gathered from each experiment. Acquisition of this technology will advance discoveries regarding musculoskeletal health and will have implications for diseases, such as osteoporosis, osteomalacia and inherited connective tissue disorders. PUBLIC HEALTH RELEVANCE: This application is for a Zeiss LSM 710 34 channel laser scanning confocal microscope, to be operated as a shared resource that will support the research programs of a core group of NIH funded major users and secondary users, located in the UMKC School of Dentistry, UMKC School of Nursing and University of Kansas Medical Center. The research emphasis of the core users is on mineralized and musculoskeletal tissues and this state of the art instrumentation will enhance their ability to generate high resolution images of muscle cells and bone cells in culture and in situ within the mineralized tissue to advance the research goals of the participating projects. Acquisition of this technology will result in new discoveries regarding bone and muscle health and will have major implications for diseases of mineralized tissues, such as osteoporosis, osteomalacia and inherited connective tissue disorders.

The goal of the Muscle/Bone Phenotyping Core is to support the research alms of all subprojects within the program project. The overall aim of the program project is to understand the mechanisms underlying crosstalk between muscle and bone that may contribute to the age related decline In muscle and bone mass and function. This global question is addressed by each subproject from a different perspective, including: the effects of muscle on osteoblast and osteocyte function with aging (subproject 1); osteocyte modulation of muscle function during aging (subproject 2); osteocyte control of osteoblast dynamics with aging (subproject 3); and effects of aging on osteocyte responses to mechanical stimulation (subproject 4). The Muscle/Bone Phenotyping Core will provide phenotyping support for all subprojects, which will include In vivo and ex vivo X-ray, densitometric and microCT analysis of skeletal tissues, histological preparation and staining of muscle and mineralized tissues, quantitative histomorphometry and dynamic bone and muscle histomorphometry, immunohistochemistry and in situ hybridization. These techniques are essential for all the subprojects in which muscle and bone phenotypes are being characterized as a function of age in transgenic mouse models with altered osteocyte or muscle function, as well as mice that have been subjected to mechanical loading. In addition to providing these services the core will provide standardized protocols for sample preparation and for all methods within the core to maintain consistency across the subprojects. The core will also act as a resource to provide training in the above techniques for students, postdocs and research personnel conducting research within subprojects 1, 2, 3 and 4. By centralizing these phenotyping methods within a single core, we will standardize these techniques across all the subprojects, accelerate the pace of the research, enhance the education of students and postdocs and enable the research to be completed in a more cost-effective manner.

DESCRIPTION (provided by applicant): Osteogenesis imperfecta (OI) is a genetic disorder in which the bones are extremely brittle and highly susceptible to fracture. Most cases of OI are caused by mutations in type I collagen genes that result in reduced amounts of normal collagen or structural defects of the triple helix, leading to abnormal fibril formation and/or assembly. Th disease spectrum in OI varies from severe forms with intrauterine fractures/perinatal lethality to mild forms without fractures. The current standard of care for OI is bisphosphonate treatment. However, recent concerns over the potential of these drugs to inhibit bone remodeling and impair fracture healing, as well as the lack of knowledge about the long term consequences of bisphosphonate treatment in children bring new urgency to the search for alternative OI therapies. Our laboratory has recently generated a green collagen mouse in which the collagen pro¿2(I) chain is fused with green fluorescent protein (GFP) and is expressed under control of the 3.6kb COL1A1 promoter for expression in osteoblasts in bone. Our preliminary data validating this novel ¿2(I)-GFP-collagen fusion protein have shown that it behaves similarly to the wild type form, is secreted upon addition of ascorbic acid, co-precipitates with collagen ¿1 chains, and is assembled into banded collagen fibril arrays. Mice expressing the GFP-collagen construct show green fluorescent collagen in the bone matrix, tendon, intervertebral discs and skin and appear phenotypically normal. These mice provide a novel and powerful new research tool with which to explore the utility of transplantation of whole marrow, stem cells or induced pluripotent stem cells (iPS) as potential therapies for repair of abnormal collagen in OI. In this exploratory R21, we plan to perform combined bone marrow transplantation of GFPcollagen expressing cells and osteoprogenitors into two mouse models of OI representing moderately severe and mild forms of OI. These include the oim mouse, which does not produce functional pro¿2(I) collagen chains and the G610C mouse, which carries a cysteine mutation in the ¿2(I) chain. The GFP-collagen expressing transplanted cells provide a powerful in vivo readout with which we can assess not only engraftment of donor cells, but also the degree of replacement of mutant (host) bone collagen with donor (GFP-positive) collagen. These innovative studies will determine the extent to which abnormal collagen can be replaced by the collagen from the transplanted cells as a function of time and explore potential approaches to enhance the extent and amount of collagen replacement through treatment with bone anabolic and antiresorptive agents. These studies have the potential to lead to novel or improved treatments for patients with OI.

Osteoporosis and Sarcopenia are diseases of bone and muscle loss that represent a major clinical problem in the aged population. These conditions often occur together, suggesting common pathogenic mechanisms and/or crosstalk between muscle and bone. Cunent treatmente for osteoporosis target osteoclast or osteoblast activity to maintain bone mass, but tiie osteocyte has been overiooked. Exciting recent research has shown that osteocytes are major regulators of osteoblast and osteoclast function and that regulation of the Wnt/3-catenin pathway by osteocytes may play a central role inregulationof bone mass. Our laboratory has taken a unique approach to examining osteoblast-osteocyte interactions using fiuorescence live imaging approaches in bone cell and organ culture models. We have shown that osteoblasts on the bone surface are motile cells and that assembly of ECM proteins In living osteoblasts is a highly dynamic process that is integrated with cell motility. We have also shown that Sclerostin and Wnts, both produced by osteocytes, can alter osteoblast motility and differentiated function. Building on these observations, this project will examine osteocyte control of osteoblast function from a dynamic perspective. The overall hypothesis is that osteocytes regulate bone mass through the Wnt/p-catenin signaling pathway by controlling the motile properties and differentiated function of osteoblasts and that this regulatory process is modulated by muscle- bone crosstalk and is impaired during aging, leading to a compromised skeleton. To address this hypothesis, live cell imaging techniques will be used in young and aged transgenic mouse models expressing fluorescent reporters for osteoblast and osteocyte lineages and GFP-tagged extracellular matiix proteins. The effect of modulation of osteocyte-produced Wnt and sclerostin will be investigated using inhibitors, gene silencing and transgenic approaches. To determine whether crosstalk from muscle alters osteocyte control of osteoblast function, in viti-o models of myoblast differentiation and transgenic and aged models of impaired or enhanced muscle function will be used. These studies may lead to the way to novel therapeutic approaches for preventing loss of bone and muscle mass in the elderiy. RELEVANCE (See Instnictions): Osteoporosis and sarcopenia are diseases of bone and muscle loss that often occur togetiier in the aged population and represent a major public health problem. The goal ofthis research is to determine the molecular and cellular mechanisms that contribute to the co-ordinated development of these conditions. This research may lead to development of new treatment approaches for these diseases.

? DESCRIPTION (provided by applicant): Our ability to understand cellular function in normal health and disease from the macro to micro to nano scale depends on the capabilities of state-of-the-art advanced instrumentation. The NIH funded projects described in this S10 application require enhanced imaging resolution, deeper tissue imaging and the ability to image in live animals for their fluorescence microscopy applications to advance to the next level. A Leica TCS Sp8 MP multiphoton microscope is requested and will be located in the UMKC Confocal Microscopy Core. It will be operated as a shared resource to support the research programs of a core group of NIH funded major and minor users in the UMKC Schools of Dentistry, Medicine, Nursing and Pharmacy as well as in the University of Kansas Medical Center and the Kansas City University of Medicine and Biosciences. The user group has a diverse research emphasis on musculoskeletal research, neural/vision research, developmental biology, dental research, nephrology and drug delivery research. To accommodate the range of project applications, the instrument will be configured for multiphoton imaging on live animals, live cells and tissue explants as well as fixed cell and tissue specimens and for optical sectioning/3D imaging. One of the projects that will employ this technology is investigating the mechanisms of assembly of bone extracellular matrix (ECM), using time lapse imaging with novel transgenic mice expressing a GFP-tagged collagen fusion protein. These studies are providing novel insights into the dynamic nature of bone matrix assembly and the role of cell motility in the assembly process. The role of the osteocyte in regulating skeletal responses to mechanical loading and in regulating muscle function is the focus of three of the participating projects. One project examines the dynamic properties of osteoblasts and osteocytes using lineage reporters combined with time lapse imaging to gain insight into the process by which osteoblasts differentiate into osteocytes. Other projects are focused on the role of mitochondria in degenerative muscle diseases, the role of hyperfiltration in glomerular injury, muscle contractile function and calcium signaling, developmental heart morphogenesis and morphogenetic tissue movements in early embryos. Acquisition of the Leica TCS Sp8 MP multiphoton technology will allow us to take these projects to the next level by providing our investigators with the ability t perform deep tissue fluorescence imaging in live animals and cell and explant culture systems as well as in fixed tissue samples. There is currently no capability for this type of imaging at UMKC or the other participating institutions and therefore this instrument will be a local resource to provide access to investigators who need multiphoton capabilities. Acquisition of this technology will advance discoveries regarding musculoskeletal health, development, aging, vision, dental restorations and drug delivery.

The reduced bone density and quality that occur in osteoporosis and the associated fracture risk is a major clinical problem in the aged population. Current therapies are aimed at modulating osteoclastic bone resorption and osteoblastic bone formation to maintain bone mass and until recently the osteocyte was overlooked. However, osteocytes are now known to be key regulators of bone mass through controlling the activity of both osteoblasts and osteoclasts. An exciting new paradigm in cell-cell communication is that extracellular vesicles (EV) (exosomes and microvesicles) may provide a novel mechanism for commun- ication between cells. These are membrane-bound particles shed from cells that carry a cargo of proteins, mRNAs and microRNAs (miRNAs). They dock with a target cell, delivering their cargo and altering the function of the target cell. Using a novel transgenic mouse expressing a membrane-targeted GFP in osteocytes, our preliminary live cell imaging data has shown that embedding osteocytes release EV from their cell body and dendrites. We have isolated EV from osteocyte-like cell lines and shown that they contain protein, mRNA and miRNA cargo, are enriched for sclerostin and contain RANKL. These EV have potent effects on osteoblasts to induce differentiation towards an osteocyte phenotype. Based on these novel observations, the overall hypothesis for the proposed studies is that osteocyte derived extracellular vesicles (EV) provide a novel mechanism for regulation of osteoblast and osteoclast function and potentially for molecular crosstalk with cells at distant sites from bone. This exploratory R21 will test this hypothesis using complimentary in vitro and in vivo approaches. Aim 1 will determine the role of osteocyte EV in regulating osteoblast and osteoclast function. EV will be isolated from osteocyte cell lines and their effects in in vitro assays of osteoblast and osteoclast differentiation determined. Live cell imaging will determine the kinetics of EV uptake in osteoblasts and osteoclasts and siRNA and miRNA hairpin Inhibitors will be used to define the mediators of EV effects. In vivo experiments will be performed in mice to confirm the functional effects of osteocyte EV on bone cells in vivo. Aim 2 will use intravital imaging in mice expressing a membrane targeted GFP in osteocytes and DsRed or TdTomato reporters in osteoblasts and osteoclasts. We will determine the dynamics of osteocyte EV release in vivo, whether the EV are shed into the circulation and/or released from bone matrix during bone resorption. Aim 3 will examine whether osteocyte EV provide a mechanism for signal propagation by transferring gene expression changes induced by stimuli such as PTH and oxidative stress to a naïve cell population. Proteomics and miRNA profiling will define the cargo of osteocyte EV and its regulation by PTH. These studies may shift paradigms about cell-cell communication in bone and pave the way for exploiting osteocyte EVs as circulating biomarkers and novel therapeutics for diagnosis and treatment of osteoporosis and other bone diseases.

Osteoporosis is a disease of low bone mass and increased risk of fracture. Exercise can increase bone size and help protect against fractures. This project aims to improve understanding of how bone cells detect bone loading. The bone cell thought to be the load detector is called the "osteocyte". Osteocytes communicate with each other and to other cells on the surface of the bone to change bone size to match the loads on the bone. The goal of the work is to determine how bone deformation and fluid flow are detected and changed into a chemical signal that the cell uses to communicate to other cells. Mechanical loading will be related to the biological response of the cell using advanced cell biological methods. The outcomes of this research will determine the role of solid-fluid interaction mechanics in the activation of bone formation to help explain and mitigate age related bone loss. The project will offer local high school students, especially female students, one day research camps and encourage them to pursue engineering and medical education. Undergraduate students will work on various aspects of the project.

The objective is to gain a better understanding of the role of multiscale mechanics - from macro-scale bone strains to micro-scale strains (local bone matrix and lacunar strain and the corresponding fluid flow shear stress on the cell membrane) in mechanotransduction at the osteocyte cellular level. We plan to study these effects using the activation of the Wnt/beta-catenin signaling pathway in osteocytes as a readout for their response to loading. This pathway is known to be important in mediating load related bone formation. The methods include experimental studies using axial loading experiments on mouse whole forearm, novel microscale axial loading experiments on murine ulna sections using the MicroXCT-200 (Carl Zeiss/Xradia) to determine lacunar strains. Newly developed multiplexed 3D confocal microscopy techniques will be used for 3D modelling of osteocytes and their lacunar fluid space for fluid-structure interaction FE models.

ABSTRACT The Muscle-Bone Imaging Core (Core B) will support the research aims of all projects within this program project. The overall aim of the program project is to understand the mechanisms underlying crosstalk between muscle and bone that may contribute to the age related decline in muscle and bone mass and function. Another goal is to determine if muscle-bone crosstalk mediates some of the beneficial effects of exercise on the musculoskeletal system. This global question is addressed by each project from a different perspective, including: the effects of the muscle-derived factor, BAIBA, in old and young osteocytes (Project 1); the effects of osteocyte factors Wnt3a and PGE2/Wnt-?-catenin on muscle with aging (project 2); the role of extracellular vesicles in bone-muscle crosstalk with aging (Project 3); and estrogen receptor-mediated regulation of bone-muscle crosstalk with aging (Project 4). The Muscle-Bone Imaging Core will provide centralized imaging support and methodologies for all projects. This will include confocal and multiphoton 3D and live cell and intravital imaging of muscle and bone as well as histological preparation and staining of muscle tissues and mineralized tissues. It will also support quantitative histomorphometry and dynamic bone histomorphometry, immunohistochemistry, muscle fiber typing and 3D osteocyte confocal imaging as well as live imaging of mitochondrial dynamics and function. The core will standardize and integrate imaging techniques essential for all the projects in which muscle and bone phenotypes are being characterized as a function of age in transgenic mouse models with altered osteocyte or muscle function, as well as mice that have been subjected to exercise training. The Muscle-Bone Imaging Core is critical to the success and outcomes in all four projects. The technical expertise, instrumentation, quantitative analysis and standardized protocols of the Core will provide a centralized shared resource that will accelerate and enhance the research. Core B will coordinate its activities with the Animal Exercise and Analysis Core (Core C) to support the research aims of all the projects and to assist with characterizing the beneficial effects of exercise on the musculoskeletal system and how this is mediated by muscle-bone crosstalk. Successful completion of the research aims of these projects will provide new insight into why osteoporosis and sarcopenia occur together and may identify molecular mediators of common pathogenic mechanisms and of the beneficial effects of exercise, which may pave the way for development of new therapies. As muscle weakness contributes to falls that lead to fractures, new therapies addressing both aspects of this ?muscle-bone loss syndrome? will improve quality of life and reduce mortality in the aged population.

ABSTRACT Osteoporosis and sarcopenia are diseases of aging that frequently occur together and reduce quality of life in the elderly population. Evidence is emerging for signaling crosstalk between bone and muscle via circulating and local mediators, leading to the concept that muscle-bone crosstalk may coordinate age- related degenerative changes. An exciting new paradigm in cell-cell communication is that extracellular vesicles (EV) (exosomes and microvesicles) may provide a novel mechanism for communication between cells. It has also been proposed that circulating muscle-derived exosomes (termed ?exersomes?) may mediate some of the beneficial effects of exercise in the body. EV are membrane-bound particles shed from cells with a cargo of proteins, mRNAs and microRNAs (miRNAs). The EV dock with a target cell, delivering their cargo and altering its function. We have shown that young and aged osteocytes shed EV, which may provide a novel mechanism for regulation of osteoblast function. Live cell imaging suggests osteocytes shed EV from their cell body and dendrites and may shed them into the circulation. Osteocyte EV are taken up by osteoblasts and myoblasts and have potent effects on osteoblasts to promote differentiation towards an early osteocyte phenotype. EV from myoblasts and myotubes are taken up by osteocytes and induce ?-catenin signaling. These findings lead to our overall hypothesis that extracellular vesicles (EV) are important regulators of bone and muscle cell function and provide a novel mechanism for crosstalk between muscle and bone that may regulate age-related osteoporosis and sarcopenia. This hypothesis will be tested using complimentary in vitro and in vivo approaches and using intravital imaging in young and aged mouse models with fluorescent reporters to tag bone and muscle cells. Aim 1 will determine the role of EV in regulating osteocyte-osteoblast reciprocal interactions in vitro and in vivo and how this is altered by aging and exercise. This will be done using EV from osteoblast and osteocyte cell lines and primary cells to determine EV effects on the differentiated function of the reciprocal cell type. Aim 2 will determine the role of EV in regulating muscle-bone crosstalk and how it is altered by aging and exercise. This will be done using EV from myoblast, osteoblast and osteocyte cell lines and primary cells to determine EV effects on the differentiated function of the reciprocal cell types. In both aims, live cell and intravital imaging will determine the kinetics of EV release and uptake in muscle and bone cells in vitro and in vivo. Young and aged mouse models will be used with and without wheel running exercise to determine in vitro and in vivo the effect of aging and exercise on EV release, composition and function. These studies may result in paradigm shifting insight into the mechanisms of molecular crosstalk between bone and muscle and will pave the way for exploiting the potential of muscle and bone derived EVs as circulating biomarkers and as novel therapeutics for age related bone and muscle loss.