Development of enthesopathy in the mouse model of X-linked hypophosphatemia - X-linked hypophosphatemia (XLH) is the most common form of inheritable rickets characterized by mutations in phex

These mutations result in elevated serum levels of FGF23, which leads to hypophosphatemia, rickets, and osteomalacia. FGF23 also inhibits vitamin D 1-a-hydroxylase (CYP27B1), thus blocking 1,25 dihydroxyvitamin D (1,25D) production. Pathologic mineralization of the enthesis (tendon-bone attachment site), referred to as enthesopathy, is a debilitating complication of XLH that causes significant pain and impaired mobility in affected individuals. Common sites affected include the patellar and achilles entheses. The pathogenesis of XLH enthesopathy is poorly understood. We previously demonstrated that achilles entheses from mice with XLH (HYP) have an expansion of hypertrophic appearing cells (HECS) that exhibit an aberrant chondrogenic phenotype with enhanced BMP/IHH signaling by P14. Treatment of HYP mice with 1,25D or a FGF23 blocking antibody (FGF23AB) early in development (P2) similarly prevented enthesopathy despite the dramatic increase in FGF23 expression in bone, suggesting impaired 1,25D action underlies the enhanced BMP/IHH signaling observed in HYP enthesopathy. In both mice and humans with XLH, 1,25D therapy cannot reverse enthesopathy, supporting the hypothesis that early restoration of 1,25D is needed to prevent enthesopathy. The increase in serum 1,25D levels wane post-initiation of FGF23AB in both mice and humans with XLH, suggesting FGF23AB may not be effective in preventing enthesopathy in XLH patients. There is no data on the effects of optimized 1,25D monotherapy or FGF23AB on enthesopathy in XLH patients. Therefore, given the similar responses of mice and humans to 1,25D and FGF23AB, studies on the hormonal regulation of XLH enthesopathy are essential to guiding future clinical studies on enthesopathy prevention. Preliminary data show that XLH enthesopathy results from impaired 1,25D action, not actions specific to FGF23 or consequences of the HYP mutation. Studies in aim I will examine mice with global deletions of CYP27B1, FGF23, or both with or without the HYP mutation to address the hypothesis that impaired 1,25D action leads to enhanced BMP/IHH signaling and enthesopathy. Studies will also elucidate if decreased local 1,25D action leads to enthesopathy. Since our data demonstrates increased BMP signaling is accompanied by enhanced GDF5 expression in HYP entheses, studies in aim II will identify a pathogenic role for GDF5/BMP signaling in HYP enthesopathy development. Studies will determine the time course of GDF5 expression in WT and HYP entheses. Ablation of GDF5 in HYP entheses will define the role of GDF5 in the activation of BMP/IHH signaling in XLH enthesopathy. Inhibition of BMP signaling in HYP mice will show that IHH signaling is activated by BMP signaling in entheses and enhanced BMP/IHH signaling directly leads to enthesopathy. Taken together, these studies will identify novel hormonal and molecular regulators of XLH enthesopathy and normal enthesis development. They will also identify targets for the design of new therapies to prevent enthesopathy.