Osteoporosis, a significant health problem in the military as well as general population in the U.S., poses a significant financial burden. Therefore, successful efforts to reduce the number of osteoporotic fractures would lead to improved health as well as considerable savings in health care costs. To identify candidate genes that may be important in the development of osteoporotic fractures, we are studying a mutant mouse with deletion of gulunolactone oxidase gene, which is involved in the synthesis of ascorbic acid (AA) that develops spontaneous fractures (sfx) at the very early age of 5-7 wks. The importance of AA in skeletal biology is underscored by the fact only a select few genetic abnormalities lead to sfx at a young age and the accumulating epidemiological evidence that individuals with low AA intake have reduced bone mass, greater rate of bone loss and increased fractures. Since genetic defects in any of the molecules involved in AA signaling pathway could increase the risk for osteoprotoic fractures, it is important to fully elucidate the components of AA signaling pathway and their role in bone. In our studies during the current funding period, we have established that AA is essential for increased expression of osterix (Osx), a master osteogenic transcription factor, during differentiation of osteoblasts (OBs). Our proposal will address an unanswered key question of how AA, an antioxidant, modulates transcriptional activity of nuclear factors. To this end, we have new exciting preliminary data for the involvement of prolyl hydroxylase domain protein (PHD)-2 in mediating AA effects in OBs. Based on the published data that p53 blocks OB differentiation, Osx expression and bone development and our preliminary data that AA decreases p53 protein levels via PHD2-dependent mechanism, we will test a model for AA regulation of Osx expression involving PHD2 and p53 by examining 3 hypotheses. To test hypothesis 1, that PHD2 is involved in mediating AA effects on OB differentiation and bone formation (BF) via regulating Osx expression in a HIF-11-independent manner, we will: 1) Establish that OB produced PHD2 is involved in mediating AA effects on OB differentiation by Adenoviral (Ad)-Cre mediated disruption of PHD2 in floxed OBs;2) Establish that AA-induced Osx expression during OB differentiation is mediated by PHD2, independent of HIF11;c) Ascertain in vivo role of PHD2 in the regulation of BF by conditional disruption of PHD2 using Cre/loxP technology;and 4) Confirm that PHD2 is required for AA to rescue BF deficiency in sfx mice by generating PHD2 conditional KO mice that are AA-deficient. To test hypothesis 2, that AA activation of PHD2 results in prolyl hydroxylation of p53 to regulate its degradation by proteosomes, we will: 1) Ascertain that AA regulates prolyl hydroxylation of p53 via PHD2;2) Establish if PHD2 mediated prolyl hydroxylation of p53 is subjected to ubiquitin-mediated proteosomal degradation;and 3) Identify sites of prolyl hydroxylation by mutating potential hydroxylation sites in p53 and determining if mutated p53 is resistant to proteosomal degradation and more potent than WT in inhibiting Osx expression. To test hypothesis 3, that p53 interacts with antioxidant response element (ARE) of Osx promoter and competes with bZIP transcription factors to suppress Osx transctiption, we will: 1) Perform EMSA and ChIP assays using OBs-derived from PHD2 conditional KO and WT mice and confirm that p53 binds to ARE of Osx gene promoter and competes members of bZIP family of transcription factors known to interact with ARE;and 2) Mutate putative AREs within the proximal promoter of Osx and evaluate p53's effect on promoter activity in OBs. Our confirmation of the hypothesis that PHD2 mediates AA effects on Osx expression and OB differentiation via p53-dependent and HIF11-independent mechanism could lead to the development of new drug targets to increase OB differentiation and BF.
PROJECT NARRATIVE Osteoporosis and related bone-wasting diseases are common in the VA population. Therefore, successful efforts to reduce the number of osteoporotic fractures would lead to improved health as well as considerable savings in health care costs. The importance of vitamin C in the pathogenesis of osteoporosis is well established from clinical epidemiological data and studies using genetic mouse models. Studies proposed in this grant application will identify the role of PHD2 in mediating vitamin C's effects on osteoblast differentiation and bone formation. Our confirmation of the hypothesis that PHD2 is an important regulator of osteoblast differentiation could lead to the development of therapeutics to stimulate osteoblast differentiation based on PHD2 targets. Thus, insights into the mechanism of vitamin C action should provide tangible benefits to veterans in the form of improvement in the prognostic and diagnostic clinical tools and ultimately in the discovery of new strategies for osteoporosis prevention and therapy.