The long-term goal of this project is to identify the molecular mechanisms responsible fur the pathogenesis of senile osteoporosis. Because impaired bone formation (BF) is a major contributor to this disease, and because of the importance of the IGF system in the regulation of BF, our studies have focused towards identifying the role of individual IGF system components in the regulation of BE. In this regard, studies during the past grant period have revealed that: (1) IGF binding protein-5 (BP-5) could increase BE via a mechanism independent of IGFs (i.e. BP-5 is a growth factor); and (2) BP-5 could function as a physiologic regulator of BF, and that low serum BP-5 could play a pathogenic role in hip fracture patients. In order to define the role of BP-5 as a BE regulator, we need to understand the molecular mechanisms controlling the level of BP-5 and the molecular pathways involved in BP-5 action in bone. To this end, we have made two exciting and pivotal discoveries which form the basis for the continuation studies proposed in this application: (1) ADAM-9 (A Disintegrin And Metalloprotease) is an BP-5 protease in osteoblasts (OBs); and (2) FHL2 (Four and a Half LIM Domain) interacts with BP-5 in OBs. ADAM-9 and FHL2 are newly discovered proteins and relatively little is known about any aspects of these proteins in bone. Studies proposed in Specific Aim 1 deal with the role of ADAM-9 in OBs and these studies are based on the hypothesis that degradation of BP-5 by ADAM-9 is a key control mechanism in regulating BP-5 concentrations in the conditioned medium (and perhaps serum) and, thereby, osteoblast proliferation/differentiation. To this end, we will express recombinant ADAM-9 and test whether purified recombinant ADAM-9 cleaves BP-5 but not other IGFBPs. To evaluate if ADAM-9 is the predominant BP-5 protease, we will perform immunodepletion experiments with antibodies specific to ADAM-9 in the conditioned medium of human OBs. To determine if ADAM-9 is regulated, we will test the effect of osteoregulatory agents, known to modulate BP-5 proteolysis, on synthesis and activity of ADAM-9. To evaluate the functional role of ADAM-9, we will modulate expression of ADAM-9 in human OBs by transgenic approaches using MLV vectors and determine the consequence of changes in ADAM-9 expression on BP-5 proteolysis and BF parameters in vitro. Studies proposed in Specific Aim 2 deal with the role of FHL2 in OBs, and these studies are based on the hypothesis that FHL2 interacts with BP-5 and mediates IGF-independent actions of BP-5 in OBs. To test if BP-5 binds to FHL2 and shuttles it to the nucleus, we will over-express wild-type/nuclear localization sequence mutated BP-5 along with FHL2 fused to different fluorescent proteins and use a fluorescent energy transfer technique to monitor interactions between FHL2 and BP-5. To evaluate the significance of BP-5 interaction with FHL2, we will test the effects of BP-5 on bone formation parameters in mice lacking functional FHL2. We will also evaluate the BP-5 functional pathway by applying the ProteinChip Arrayer to determine if the complex of BP-5/FHL2 binds to other proteins in the nucleus of OBs. Successful completion of the proposed studies will provide information on the molecular pathways involved in the regulation of the BF process, which will eventually lead to better understanding the pathogenesis of why some people have impaired BF during aging, and suggest treatment options to correct BF deficiency in those patients.
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