From a study in which we discovered osteoactivin (OA) in bone, we demonstrated that OA mRNA and protein are expressed in osteoblasts and its expression exhibited a temporal pattern being at highest levels during the later stages of matrix maturation and mineralization. Furthermore, the protein is synthesized, processed, glycosylated and secreted by osteoblasts. Using gain-of-function and loss-of-function approaches in osteoblasts, we found that down-regulation of OA decreased osteoblast differentiation and function and over-expression increased osteoblast differentiation and function in vitro. We also demonstrated that the secreted form of OA regulates osteoblast differentiation and function. Treatment with recombinant OA promotes bone formation in vivo. The importance of OA in osteogenesis was confirmed in mice with the null allele for OA and in mice with a natural mutation in the OA gene caused a premature stop codon that results in the generation of a truncated OA protein. Both of these mice exhibit a skeletal phenotype associated with decreased bone mass. During the previous funding period and since the last submission, we established colonies of OA KO and OA mut. mice, and also generated transgenic (Tg) mice that over-express OA in bone. Preliminary data from OA KO, OA mut. and Tg mice support the hypothesis that OA is a novel bone anabolic factor which is synthesized and secreted by osteoblasts, and acts either as an ECM-associated signaling molecule or downstream of BMP2 to regulate osteoblast differentiation and function. In addition to its effects on osteoblasts, we present data showing that OA affects osteoclast differentiation, and we hypothesize that these abnormalities are secondary to altered production of osteoclastogenic factors (e.g. RANKL) by stromal cells/osteoblasts in the bone microenvironment. Studies proposed in aim 1 will evaluate the effects of OA deficiency (OA KO), truncated OA (OA mut.) or OA over-expression (Tg) on bone in vivo, and assess the differentiation and function of bone cells (osteoblasts and osteoclasts) derived from these mice in primary cultures. The presence of various domains in OA might reflect different functions, and evaluation the structure/function relationship and role of the various domains of OA on normal osteoblast differentiation will be investigated in aim 2 of this application. During the previous funding period, we also showed that the secreted isoform of OA can function as an ECM-associated (matricellular) protein and demonstrated that osteoblasts attach to OA via the av[unreadable]1 integrin, resulting in the formation of focal adhesions, cytoskeletal reorganization and the activation of FAK. Studies proposed in aim 3 will test the hypothesis that OA acts as a matricellular protein that binds to specific cell surface integrins on osteoblasts to initiate integrin-activated signaling, cytoskeletal reorganization, and regulate cell function. We recently demonstrated that BMP2 regulates OA expression and that OA is a downstream mediator of BMP2-induced osteoblast function, a response that is mediated by the Smad signaling pathway. We present preliminary data that BMP2 stimulates the recruitment of Smad1, Dlx5 and CBP to the OA promoter and this effect is dependent on the stage of osteoblast differentiation. Studies proposed in aim 4 will investigate the mechanism whereby OA acts as a downstream mediator of BMP2-induced osteoblast differentiation and function, and will evaluate the effects of BMP2 in stimulating the recruitment of Smad1, homeodomain proteins, and CBP co-activators to the OA promoter for transcriptional regulation during osteoblast differentiation. Proposed experiments are expected to generate novel information regarding the effects of OA deficiency or over-expression on bone formation/remodeling in vivo, its mechanisms of action in osteoblasts, and the molecular requirements for OA induction by BMP2 in osteoblasts. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs in the Untied States from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. Osteoactivin is a novel growth factor in bone and the proposed studies will generate new information regarding its effects and mechanisms of action on bone cell development and function. Once we understand its full effects on bone and how it works to promote bone formation, this information will be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with clinically significant bone loss.