(Verbatim from the Applicant): Parathyroid hormone related protein (PTHrP) is required for endochondral development and critically regulates growth plate cartilage organization and mineralization. We have discovered that articular cartilage PTHrP expression becomes robust in osteoarthritis (OA) but that chondrocytes preferentially express the least widely expressed PTHrP isoform (PTHrP 1-1 73). PTHrP isoforms are endoproteolytically cleaved to generate biologically active peptides. Moreover, the unique C-terminal 140-173 domain peptide of PTHrP 1-173 determines marked effects on chondrocyte matrix synthetic function and the elaboration of PPi (a major regulator of chondrocyte mineralization) by an intracrine mechanism. We will test the hypotheses that the 147-150 KKKK motif of PTHrP 1-173 modulates both endoproteolytic processing and the subcellular and nuclear localization of PTHrP 1-173. We also hypothesize that PTHrP 1-173 processing at the 147-150 motif critically regulates chondrocyte synthetic and mineralizing functions. To test these hypotheses, we will identify the peptides derived from PTHrP 1-173 by endoproteolysis in cultured articular chondrocytes and chondrocytic TC28 cells. We also will identify the molecular mechanism by which chondrocytes process proPTHrP 1-173 at the N-terminus to derive active PTHrP, and process the 147-150 KKKK motif to affect the biologic activity of PTHrP 1-173. In doing so, we will test the hypothesis that furin-like subtilisin family proprotein convertases (PCs) are at least partially responsible. Methods employed in these studies will include physical and immunochemical approaches to characterize PTHrP 1-173 derived peptides in cultured chondrocytes and a genetic screening approach for cleavage site-specific endoproteases. Having defined how chondrocytes process PTI-IrP 1-173, and the peptides generated, we will determine how the C-terminal processing products affect chondrocyte functions. We will focus on growth, collagen synthesis, PPi elaboration and mineralization. We also will test the hypothesis that a nuclear localization signal (NLS) at the 147-150 domain of PTHrP 1-173 is critical for certain regulatory effects of PTHrP 1-173 on chondrocyte functions. To do so, we will permeabilize chondrocytes and treat with labeled wild-type and mutant forms of PTHrP 1-173, and PTHrP 140-173. Last, we will directly test the hypothesis that PTHrP 1-173 modulates hydroxyapatite and CPPD crystal deposition by cultured chondrocytes. Completion of these studies will indicate not only how PTHrP 1-173 can modulate OA, but also how functional properties of the PTHrP 1-173 C-terminal 140-173 domain can be harnessed to promote cartilage repair.
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