The long-term goals of this Merit project are focused on characterizing the roles and mechanisms of actions of vitamin C in skeletal biology since its deficiency induces skeletal fractures. During the last funding period, we identified novel roles fo prolyl hydroxylase domain proteins (PHDs) in regulating articular cartilage (AC) development besides mediating vitamin C effects on osteoblasts (OBs). By using mice with a conditional knockout (cKO) of the Phd2 gene in OBs and chondrocytes, we determined that loss of Phd2 in OBs resulted in osteopenia while chondrocyte-specific KO of Phd2 lead to a dramatic increase in trabecular bone mass caused by increased endochondral bone formation (EBF), thus suggesting distinct roles and mechanisms for PHD2 in the two cell types. New preliminary data described in this proposal show that Phd2 and Phd3 are differentially expressed in the superficial zone (SZ) and middle zone (MZ) of AC, respectively, and that transition of AC chondrocyte progenitors in the SZ into differentiating chondrocytes in the MZ capable of EBF is tightly regulated by relative levels of Phd2 and Phd3 expression. Furthermore, our data show that PHD2 and PHD3 regulate AC chondrocytes by precise control of hypoxia-inducible factor (HIF)1? and HIF2? levels and by an epigenetic mechanism involving hydroxylation of 5- methyl cytosine (5-mC). Based on these data, our major goal in this competitive renewal application is to elucidate the role of Phd2 and Phd3 in AC development and in the pathogenesis of osteoarthritis (OA), since OA is a significant public health problem in the U.S., posing a substantial financial burden with no available therapies for treatment. We will use new genetic mouse models to test the prediction that dysregulation of Phd2/Phd3 expression in AC contributes to the pathogenesis of OA by involving HIF signaling and epigenetic modification of genome activity.
In Specific Aim 1, we will test the hypothesis that loss of Phd2 expression in AC progenitors impairs development and maintenance of AC by promoting HIF1? signaling. We will generate mice in which expression of Phd2 is disrupted in Prg4 expressing AC progenitors and examine the AC phenotype as a function of age and in response to a destabilization of medial meniscus (DMM) injury and determine if the AC phenotype in Phd2 cKO mice is abolished by disruption of Hif1? expression in AC progenitors.
In Specific Aim 2, we will test the hypothesis that loss of Phd3 expression in differentiating chondrocytes regulates the AC phenotype by promoting HIF2? signaling. We will generate mice in which expression of Phd3 is disrupted in aggrecan expressing differentiating chondrocytes and determine if the AC phenotype in Phd3 cKO mice is abolished by disruption of HIF2? signaling.
In Specific Aim 3, we will test the hypothesis that Phd2 and Phd3 regulate transition of AC progenitors into differentiating chondrocytes in part via epigenetic control of genome activity via regulating hydroxylation of 5-mC. Successful completion of our proposed studies will provide important information on the pathway by which Phd2 and Phd3 regulate AC and could provide novel PHD-based drug targets for treatment of OA prevalent in aging veterans.
There is an urgent need for development of novel anabolic drugs for the treatment of osteoarthritis, a debilitating degenerative joint disease of enormous economic burden in the U.S. and worldwide. Based on exciting preliminary data that prolyl hydroxylase domain protein (PHD)2 and PHD3 regulate proliferation and differentiation of articular cartilage progenitors, studies proposed in this application will examine the role and mechanism of action of these two enzymes in articular cartilage development, maintenance and pathogenesis. Because Phd2 and Phd3 sequences are conserved between mice and humans, future confirmation of a role for these proteins in humans could provide a basis for the future development of PHD- based novel drugs to promote articular cartilage development for treatment of osteoarthritis, the most common joint disorder in aging veterans and in the general population.
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