Inactivation of a Novel FGF23 Regulator, FAM20C, Leads to Hypophosphatemic Rickets in Mice

Abstract

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA–mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation “promoter” but not an “inhibitor” in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23.

Functional heterogeneity of osteocytes in FGF23 production: the possible involvement of DMP1 as a direct negative regulator

“…MC3T3-E1 cells regularly do not express an osteocytic phenotype; however, long culture under osteogenic differentiation medium reportedly allows these cells to produce an osteocytic phenotype, such as that involving the DMP1 or FGF23 production (Figure 5a). 47 Treatment of the long-cultured MC3T3-E1 cells with DMP1 (100 ng ml À 1 ) significantly downregulated fgf23 expression, whereas endogenous Dmp1 expression level was significantly upregulated (Figure 5b). These findings were consistent with the observations in UMR106 cells, although other markers that we investigated did not show significant changes in the long-cultured MC3T3-E1 cells (Figure 5b).…”

“…To detect the expression of FGF23 in these cells, we performed long-term culture with osteogenic medium over 4 weeks, modified as previously described. 47 After 4 weeks of culture in the culture dish, the cells were seeded onto well plates for each assay.…”

Family with sequence similarity member 20C is the primary but not the only kinase for the small‐integrin‐binding ligand N‐linked glycoproteins in bone

“…Our MS data confirmed that KO-derived DMP1 had less phosphorylation than that of WT mice. In an earlier study, we reported significant down-regulation of DMP1 in the Fam20C-KO mice (25). It is unclear whether the lower levels of expression and phosphorylation of DMP1 in the KO mice were related to the different MS coverage of the WT-and KO-derived proteins.…”

“…Fam20C-KO mice mediated by Sox2-Cre were generated by a published process (25). The animal procedures were performed in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Texas A&M University Baylor College of Dentistry.…”

“…The animal procedures were performed in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Texas A&M University Baylor College of Dentistry. Genotyping was performed as has been described (25).…”