The role of GDF5 in regulating enthesopathy development in the Hyp mouse model of XLH.

X-linked hypophosphatemia (XLH) is caused by mutations in PHEX, leading to rickets and osteomalacia. Adults affected with XLH develop a mineralization of the bone-tendon attachment site (enthesis), called enthesopathy, which causes significant pain and impaired movement. Entheses in mice with XLH (Hyp) have enhanced bone morphogenetic protein (BMP) and Indian hedgehog (IHH) signaling. Treatment of Hyp mice with the BMP signaling blocker palovarotene attenuated BMP/IHH signaling in Hyp entheses, thus indicating that BMP signaling plays a pathogenic role in enthesopathy development and that IHH signaling is activated by BMP signaling in entheses. It was previously shown that mRNA expression of growth/differentiation factor 5 (Gdf5) is enhanced in Hyp entheses at P14. Thus, to determine a role for GDF5 in enthesopathy development, Gdf5 was deleted globally in Hyp mice and conditionally in Scx + cells of Hyp mice. In both murine models, BMP/IHH signaling was similarly decreased in Hyp entheses, leading to decreased enthesopathy. BMP/IHH signaling remained unaffected in WT entheses with decreased Gdf5 expression. Moreover, deletion of Gdf5 in Hyp entheses starting at P30, after enthesopathy has developed, partially reversed enthesopathy. Taken together, these results demonstrate that while GDF5 is not essential for modulating BMP/IHH signaling in WT entheses, inappropriate GDF5 activity in Scx + cells contributes to XLH enthesopathy development. As such, inhibition of GDF5 signaling may be beneficial for the treatment of XLH enthesopathy.

X-linked hypophosphatemia (XLH) is a rare bone disorder that leads to short stature and poorly mineralized bones. As adults, patients with XLH often develop a mineralization of the bone-tendon attachment site, called enthesopathy, which results in significant pain. We previously showed that Achilles bone-tendon attachment sites (entheses) in mice with XLH (Hyp) have an enthesopathy characterized by increased bone morphogenetic protein (BMP) signaling. In the current studies, we show that treating Hyp mice with the BMP signaling inhibitor palovarotene prevents enthesopathy, demonstrating that the increased BMP signaling in Hyp entheses leads to enthesopathy development. We also reported that gene expression of Gdf5, which activates BMP signaling, is enhanced in Hyp entheses. Therefore, to determine if the enhanced Gdf5 expression leads to the increased BMP signaling seen Hyp entheses, Gdf5 was deleted from Hyp mice and also deleted specifically in the entheses of Hyp mice. In both mouse models, enthesopathy development was attenuated, demonstrating that the increased Gdf5 expression in Hyp entheses plays a role in enthesopathy development. These data indicate that blocking GDF5 and BMP signaling may prevent enthesopathy in patients with XLH.

GDF5 deficiency prevents cardiac rupture following acute myocardial infarction in mice.

BACKGROUND: We previously showed that growth differentiation factor 5 (GDF5) limits infarct expansion post-myocardial infarction (MI). We now examine the acute post-MI role of GDF5 in cardiac rupture. METHODS AND RESULTS: Following permanent ligation of the left anterior descending artery, GDF5 deficiency (i.e., GDF5 knockout mice) reduced the incidence of cardiac rupture (4/24 vs. 17/24; P < .05), and improved survival over 28-d compared to wild-type (WT) mice (79% vs. 25%; P < .0001). Moreover, at 3-d post-MI, GDF5-deficient mice manifest: (a) reduced heart weight/body weight ratio (P < .0001) without differences in infarct size or cardiomyocyte size; (b) increased infarct zone expression of Col1a1 (P < .05) and Col3a1 (P < .01), suggesting increased myocardial fibrosis; and (c) reduced aortic and left ventricular peak systolic pressures (P ≤ .05), suggesting reduced afterload. Despite dysregulated inflammatory markers and reduced circulating monocytes in GDF5-deficient mice at 3-d post-MI, reciprocal bone marrow transplantation (BMT) failed to implicate GDF5 in BM-derived cells, suggesting the involvement of tissue-resident GDF5 expression in cardiac rupture. CONCLUSIONS: Loss of GDF5 reduces cardiac rupture post-MI with increased myocardial fibrosis and lower afterload, albeit at the cost of chronic adverse remodeling.

HOXA10 promotes Gdf5 expression in articular chondrocytes.

Growth differentiation factor 5 (GDF5), a BMP family member, is highly expressed in the surface layer of articular cartilage. The GDF5 gene is a key risk locus for osteoarthritis and Gdf5-deficient mice show abnormal joint development, indicating that GDF5 is essential in joint development and homeostasis. In this study, we aimed to identify transcription factors involved in Gdf5 expression by performing two-step screening. We first performed microarray analyses to find transcription factors specifically and highly expressed in the superficial zone (SFZ) cells of articular cartilage, and isolated 11 transcription factors highly expressed in SFZ cells but not in costal chondrocytes. To further proceed with the identification, we generated Gdf5-HiBiT knock-in (Gdf5-HiBiT KI) mice, by which we can easily and reproducibly monitor Gdf5 expression, using CRISPR/Cas9 genome editing. Among the 11 transcription factors, Hoxa10 clearly upregulated HiBiT activity in the SFZ cells isolated from Gdf5-HiBiT KI mice. Hoxa10 overexpression increased Gdf5 expression while Hoxa10 knockdown decreased it in the SFZ cells. Moreover, ChIP and promoter assays proved the direct regulation of Gdf5 expression by HOXA10. Thus, our results indicate the important role played by HOXA10 in Gdf5 regulation and the usefulness of Gdf5-HiBiT KI mice for monitoring Gdf5 expression.