6443 The Role of GDF5 in Regulating Enthesopathy Development in the Hyp mouse Model of XLH

Abstract Disclosure: A. Hughes: None. M. Sorsby: None. S. Almardini: None. S. Venkat: None. A. Alayyat: None. M. Rahman: None. J. Baker: None. R. Rana: None. V. Rosen: None. E.S. Liu: Advisory Board Member; Self; Ascendis Pharma. Other; Self; Inozyme - DSMB. X-linked hypophosphatemia (XLH) is characterized by high serum FGF23 levels, impaired production of 1,25 dihydroxyvitamin D, and hypophosphatemia. Up to 60% of adults with XLH develop enthesopathy, which is a mineralization of the bone-tendon attachment site, that causes significant pain and impaired mobility. We previously showed that entheses from mice with XLH (Hyp) have an expansion of hypertrophic appearing enthesopathy cells (HECs) that stain positive for cartilage proteoglycans (Safranin O) and alkaline phosphatase activity (ALP+). The HECs have enhanced bone morphogenetic protein (BMP) and Indian hedgehog (IHH) signaling, implicating these pathways which regulate chondrogenesis in XLH enthesopathy development. Therefore, Hyp mice were treated daily with palovarotene, a retinoid acid gamma receptor agonist that blocks BMP signaling, from postnatal day (P) 7 (prior to enthesopathy development) to P30. Treated Hyp entheses normalized BMP/IHH signaling, indicating that BMP signaling plays a pathogenic role in XLH enthesopathy development and that IHH signaling is activated by BMP signaling in entheses.We previously showed that mRNA expression of BMP/GDF factor Gdf5 is increased in Hyp entheses P14. In the current studies, we performed RNAscope on WT and Hyp entheses, demonstrating that Gdf5 expression is similarly robust in embryonic entheses but decreases dramatically postnatally by P14. While Gdf5 expression is similar between WT and Hyp entheses e16.5 and P2, it is enhanced in P14 Hyp entheses compared to WT. Since Gdf5 expression is increased in Hyp entheses and GDF5 promotes chondrocyte maturation and hypertrophy, we undertook studies to determine the role of GDF5 in Hyp enthesopathy development by deleting Gdf5 globally in Hyp mice (Gdf5KO/Hyp) and conditionally in cells that express Scx (markers of enthesis/tendon) in Hyp mice (Gdf5f/f;ScxCre+/Hyp). In both murine models, Gdf5KO/Hyp and Gdf5f/f;ScxCre+/Hyp entheses had similar significant decreases in immunoreactivity for BMP/IHH signaling markers and ALP activity compared to Hyp and Gdff/f/Hyp controls, respectively. Consistent with these results, mRNA expression of BMP/IHH signaling target genes were significantly decreased in Gdf5f/f;ScxCre+/Hyp entheses compared with Gdff/f/Hyp control entheses. Deletion of Gdf5 in WT mice or in the Scx+ cells of WT mice did not alter BMP /IHH signaling in entheses. Taken together, these data suggest that although GDF5 action is not essential for regulating BMP/IHH signaling in WT entheses, the enhanced Gdf5 expression in Hyp entheses plays a pathogenic role in XLH enthesopathy development. Moreover, GDF5 action in Scx+ cells in Hyp mice regulates the enhanced BMP/IHH signaling seen in Hyp entheses. Blocking BMP/GDF5 signaling may be important in the treatment of XLH enthesopathy. Presentation: 6/1/2024