Thomas L. Clemens - US grants

Vitamin D acts on the intestine through a mechanism that is steroid hormone-like, to increase calcium transport. The interaction of 1,25-(OH)2-D3 with its specific receptor protein in intestinal mucosal cells regulates a gene coding for vitamin D-dependent calcium-binding protein (CaBP) which is thought to function in the transport process. The recent discovery of putative 1,25-(OH)2-D3 receptors and CaBP in kidney, bone, brain and other tissues prompted the concept that vitamin D may function in these organs through an effector system with common components. The hypothesis, while attractive, has not been rigorously tested. There are significant outstanding questions regarding the exact cellular locations of 1,25-(OH)2-D3 receptors and CaBP and it remains unclear as to whether the synthesis of CaBP in non-intestinal tissue is regulated directly by 1,25-(OH)2-D3. The goal is to gain answers to these questions by bringing to bear new powerful techniques not previously available. First, newly developed immunocytochemical techniques will be used to undertake the first simultaneous mapping of the 1,25-(OH)2-D3 receptor and CaBP in kidney, bone and brain of the adult and developing rat. With such an approach we will identify exactly which cells within each tissue are likely to be responsive to vitamin D. Both immunocytochemical methods and specific radioimmunoassays will then be employed to determine the relative importance of vitamin D deficiency and hypocalcemia in the regulation of CaBP synthesis in these non-intestinal tissues. A final aspect of this project will attempt to probe more directly, the mechanism by which the synthesis of renal CaBP is regulated. In these studies, primary kidney epithelial cells that express CaBP and retain the ability to respond to vitamin D will be used to evaluate the effects of 1,25-(OH)2-D3 and intracellular calcium ion concentration on CaBP synthesis in vitro. The results obtained from these studies will promote a clearer understanding of the relationship between vitamin D and CaBP and thus provide a strong foundation for future studies of possible functions for vitamin D in these tissues.

Parathyroid hormone-related protein was discovered in 1987 and shown to mediate humoral hypercalcemia of malignancy (HHM) a syndrome which had puzzled physicians for decades. This protein is also produced in many normal tissues where it is believed to function in an autocrine or paracrine fashion. Our studies during the last three years have established vascular smooth muscle as an important site of PTHrP production and action. We have characterized the principal mechanisms by which PTHrP is induced by vasoconstrictors and mechanical stimuli. In addition, we have demonstrated novel actions PTHrP on VSMC growth and extracellular matrix production which are mediated through PTH/PTHrP receptors. These observations have upheld our central hypothesis that locally produced PTHrP functions in vascular smooth muscle to restrict both pressor and mitogenic signals induced by vasoconstrictor and mechanical events. In essence we believe we have uncovered a novel compensatory autocrine vasoactive loop not previously appreciated. These advances, together with the discovery that PTHrP is a locally active factor who's production is vital in development, have prompted more sophisticated questions on the processing of PTHrP and the molecular mechanisms responsible for the full range of PTHrP vascular actions. In Aim 1 of the present proposal we will structurally identify the PTHrP forms that are produced in VSMC. In a Aim 2 we will explore in vitro the mechanisms by which PTHrP inhibitors VSMC growth and modulates extracellular matrix production. We will also investigate the involvement of cell cycle related genes in PTHrP growth inhibition of VSMC. In addition, since growth inhibitory activity of PTHrP are associated with an inhibition of osteopontin expression we will determine the effect of PTHrP peptides on VSMC identified in the studies under Aim 1 will be tested for biological activity in our cell models. In the final Aim we will determine the effects of PTHrP in vascular smooth muscle in vivo in transgenic mice. Transgenic mouse models which selectively overexpress PTHrP and its receptor in smooth muscle will be used to examine two distinct functions of PTHrP. First, we will compare the development of thoracic aorta in PTHrP expressing mice, their non- expressing litter mates and int he PTHrP knock out mice using specific smooth muscle cell specific probes (DNA and protein). Secondly we will explore the possibility that overexpression of PTHrP in vascular smooth muscle alters basal and volume stressed cardiovascular hemodynamics. Collectively we believe our studies will greatly expand our understanding of the biology of PTHrP in vascular smooth muscle.

DESCRIPTION (provided by applicant): Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to attract new blood vessels to bone rudiments or regenerates. Other signals, likely emanating from the incoming vasculature, stimulate bone cell specification through interactions with cells within the vascular stem cell niche. The long-term goal of this project is to understand the cellular and molecular mechanisms that control interactions between bone cells and vascular elements. During the past funding period, we have identified the hypoxia inducible factor alpha (HIF-11) pathway as a key component in this process. Using a genetic approach, we have demonstrated that overexpression of HIF1 in mouse osteoblasts through disruption of Vhl profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell non-autonomous mechanisms involving VEGF. Preliminary studies described below suggest that endothelial cells are one source of bone morphogenic signal which couples angiogenesis to bone formation. Angiogenesis also appears to drive osteogenesis when long bones from the mutant and wild type mice are subjected to injury (distraction osteogenesis). Surprisingly however, manipulation of HIF1 levels in mature osteoblasts does not influence the formation of the flat bones of the skull. These results suggest that the mechanisms which couple angiogenesis to osteogenesis are context (skeletal site) specific. In this renewal application, we propose studies to characterize cellular and molecular mechanisms responsible for coupling angiogenesis to osteogenesis in three different skeletal contexts. This renewal grant has three aims: Specific Aim 1: Angiogenic-osteogenic coupling in developing long bones. Specific Aim 2: Angiogenic-osteogenic coupling following bone injury. Specific Aim 3: Angiogenic-osteogenic coupling in the developing cranium. The ultimate goal of this project is to elucidate the functional mechanisms underlying HIF-1/VEGF stimulated angiogenesis and osteogenesis. These findings should lead to better understanding of precise communication between angiogenesis and osteogenesis and aid in the design of new therapies to accelerate bone healing following injury

[unreadable] DESCRIPTION (provided by applicant): Growth hormone (GH) is one of the most important factors-controlling skeletal growth and development, but despite decades of work, the cellular targets and mechanisms of action of GH in bone remain unclear. The principal impediment to the study of GH action is the fact that GH stimulates IGF-1 production making it difficult to distinguish actions due to directly to GH versus those resulting indirectly through IGF-1. Historically, two theories have been developed to explain the biological significance of GH/IGF-1 interactions. The "somatomedin hypothesis" proposed that GH exerts it actions by stimulating peripheral (liver and other tissues) IGF-1 production. Alternatively, the "dual effector theory" postulated that GH directly stimulates target cell differentiation and that GH induced IGF-1 then stimulated clonal expansion of the differentiated cell population. Recent studies suggest a third possibility, namely that GH and IGF-1 can act collaboratively in certain settings through mechanisms involving a physical interaction between their receptors. Advances in technologies to manipulate the mouse genome have for the first time made it possible to distinguish the effects of each of these growth factors in vivo in a normal physiological context. We have used these methods to begin to identify GH actions in osteoblasts of GH that are independent of IGF-1 and are likely to profoundly influence osteoblast function. In this grant proposal, we describe newr in vitro and in vivo studies to define the mechanisms of GH action in osteoblasts and to determine their relationship to IGF-1 action. In Specific Aim 1 we will primary osteoblasts expressing or deficient in the IGF-1 receptor to characterize GHR signaling components and explore the concept of GH/IGH-1 collaboration by comparing the effects of GH on osteoblast proliferation, matrix deposition and differentiated gene expression. We will also examine the effects of GH administration in vivo to mice following conditional disruption of the IGF-1 R and compare the skeletal effects in this model to that observed in mice with an intact IGF-1. In Specific Aim 2, We will analyze the effect of conditional disruption of the GHR on IGF-1 R signaling, osteoblast proliferation, matrix deposition and differentiation in primary calvarial osteoblasts in vitro. Specifically, we will determine how signaling through each receptor and functional activity (e.g. proliferation, apoptosis) is affected by the presence of the other. We will determine the effect of Cre mediated disruption of the GHR from osteoblasts in vivo. Static and dynamic histomorphometric indices of cortical and trabecular bone and CT measurements will be used to compare the bone phenotype in the osteoblast GHR knockout mice to those seen in control mice at selected times during postnatal development. Finally, we will examine further the interaction between GH and IGF-1 in vivo by comparing the bone phenotype of mice lacking both GHR and IGF-1 R in osteoblasts. Completion of these aims should enable us to determine for the first time the mode of GH action in bone. A better understanding of the mechanisms of action of GH will have important implications for the action of this hormone in other tissues and for its use in the clinic. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mammalian skeletal muscle develops and regenerates through a process in which individual myoblasts fuse with one another to create a multinucleated syncytium. Growth hormone (GH) has long been recognized as a critical anabolic factor required for normal muscle development and regeneration. GH treatment improves muscle strength and reduces body fat in humans and animals. Moreover, increases in muscle mass and strength produced with exercise are accompanied by profound increases in the activity of the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis. Conversely, the physiological changes that the human body undergoes with aging including sarcopenia resemble those observed in GH deficiency. However, the mechanisms through which GH produces its effects on skeletal muscle are still poorly understood. In skeletal muscle and other target tissues, GH can function independent of IGF-1 by activating signaling of its cognate receptor (GHR). Alternatively, GH can function indirectly through stimulating production of IGF-1. In this mode, IGF-1 binds to the IGF-1 type I receptor (IGF-1R) causing activation of downstream pathways which also induce anabolic activity in skeletal muscle. The close relationship between GH and IGF-1 has made it virtually impossible to distinguish actions of each of these growth factors in target tissues. Recently, we have devised a genetic approach in mice which enables selective disruption of individual components of the GH/IGF-1 axis. Using this strategy we demonstrated that GHR is absolutely required for normal skeletal muscle development and provide new evidence that GH increases muscle tissue mass by enhancing myoblast fusion. Moreover, in this proposal, we present new data which suggests that GH actions in skeletal muscle may generate additional hormonal signals that enable cross talk between muscle and fat to orchestrate global energy expenditure and regulate body composition. In this project, we will (1) define essential actions of GH in skeletal muscle and (2) identify the mode of GH action in skeletal muscle and determine its relationship to IGF-1. These aims employ complementary genetic mouse models and their myoblasts to identify the mechanisms and of action of GH in skeletal muscle and distinguish effects of GH from those mediated by IGF-1. PUBLIC HEALTH RELEVANCE: The project will investigate how two important growth factor (insulin like growth factor 1 and growth hormone) work to control muscle growth. Distinguishing the actions of GH from those of IGF-1 in muscle cells during postnatal development should ultimately facilitate the development of more specific and effective therapies with these two molecules.

? DESCRIPTION (provided by applicant): In this new project we will explore the role of sensory nerves in bone. In contrast to increasing body of work on to the role of angiogenesis in bone development, few studies have investigated the role of nerves in bone despite a wealth of circumstantial evidence implicating the importance of innervation in skeletal development and repair. This knowledge gap is in part due the difficulty of identifying nerve fibers in calcified tissue and the lack of tractable model systems for study skeletal innervation. To overcome these limitations, our laboratory has validated mouse models for visualizing and disrupting functional signaling of sensory nerves that innervate the skeleton. Using these models we demonstrate that tropomyosin receptor kinase A (TrkA) expressing sensory neurons emanating from the dorsal root ganglion project axons to nerve growth factor (NGF) expressing perichondral cells of developing limbs coincident with incipient mineralization of adjacent cartilage. Elimination of TrkA kinase activity in these sensory nerves during embryogenesis retards axonal ingrowth and attenuates nascent bone mineralization. Disruption of TrkA signaling during embryogenesis reduces postnatal bone acquisition and retards the regeneration of adult bone in response to experimental fracture. These observations, and other studies described below, provide direct evidence for the requirement of TrkA sensory nerves in bone development, acquisition and repair. The studies outlined in this proposal will explore the mechanisms through which sensory nerves function to promote osteogenesis. Our approach will test the hypothesis that nerve growth factor (NGF) produced in the developing and injured limb mesenchyme activates TrkA sensory neurons to promote their survival and guide their axons to primary ossification centers to initiate osteoblast differentiation. This hypothesis will be tested in a setting of bone development (Aim 1) and in response to an established model of experimental bone repair (Aim 2). We believe that our project will yield new insights into the role of sensory nerves in healthy humans and will ultimately inform on the neuropathological manifestations associated with certain bone disorders.

? DESCRIPTION (provided by applicant): The explosion of new knowledge in genomics, cell biology and biomedical engineering technology is ripe for translation into new orthopaedic applications for improving the diagnosis and management of patients with musculoskeletal disorders. Orthopaedic research programs have been slow to capitalize on this opportunity because of inherent structural impediments in this discipline, which have increasingly distanced orthopaedic surgeons and basic scientists in the pursuit of translational research projects. In thi application, we propose a new paradigm for training musculoskeletal scientists in an academic setting, which is modeled after the proven, team science approach used by pharmaceutical companies. The Training in Orthopaedic Team Science (TOTS) program will sponsor the pursuit of well circumscribed, translational research projects by discovery teams composed of orthopaedic residents, post-doctoral Ph.D. fellows, medical students and faculty preceptors. Trainees will complete a Core Curriculum designed to provide a common grounding in musculoskeletal pathobiology and fundamentals in clinical research execution. Both research and didactic phases of the learning experience will occur concurrently by the trainees to ensure constant interaction between basic and clinical team members. An Enrichment Program will catalyze interactions between trainees and faculty and provide valuable career development training. The TOTS Steering Committee will oversee the project and team selection process, and evaluate the program's performance using tools for assessing short and long-term measures. An Internal Oversight Committee will provide independent, ongoing evaluation of the program and will coordinate feedback from trainees, training program faculty, and an External Advisory Committee. Successful implementation of the TOTS program is expected to produce musculoskeletal scientists who will enter the workforce with the requisite experience in translational research and leadership training to move discoveries effectively between the bench and bedside.

PROJECT SUMMARY Our laboratory has demonstrated an essential role for sensory nerves in the ossification of the developing mouse limb. Here, we validated and leveraged several reporter systems to visualize and perturb sensory innervation in mineralized tissues of the long bone. However, unlike the appendicular skeleton, the role of the peripheral nervous system (PNS) in the postnatal craniofacial skeleton remains largely unexplored. In our preliminary work, we demonstrate that the neurotrophin NGF (nerve growth factor) has extensive and specific domains of expression within suture mesenchyme of patent cranial sutures, and that this corresponds with dense innervation of TrkA+ (tropomyosin receptor kinase A) sensory nerves. These observations, and other studies described below, provide the first evidence for the requirement of TrkA sensory nerves in craniofacial skeletal ossification and repair. The studies outlined in this proposal will explore the mechanisms through which sensory nerves function to regulate osteogenesis. Our approach will test the hypothesis that nerve growth factor (NGF) produced in the injured calvarial bone activates TrkA sensory neurons to promote their survival and guide their axons to areas of osteogenesis, vasculogenesis, and bone repair. This hypothesis will be tested in the setting of an established model of experimental bone injury, using a healing circular bone defect adjacent to the patent sagittal suture. We believe that our project will yield new insights into the role of sensory nerves in healthy humans and will ultimately inform on the neuropathological manifestations associated with certain bone disorders.