1,25-(OH)2D3 Down-Regulates Expression of Phex, a Marker of the Mature Osteoblast

Abstract

Mutations in the PHEX/Phex gene, which encodes for a protein with homology to neutral endopeptidases, are responsible for human and murine X-linked hypophosphatemia. The present study examined Phex messenger RNA (mRNA) and protein expression in cultured osteoblasts and its regulation by 1,25-(OH) 2 D 3 . Phex mRNA levels were quantitated on Northern blots by densitometric analysis relatively to GAPDH mRNA levels. Immunoreactive Phex protein levels were evaluated by immunoprecipitation using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide.␤-Glycerophosphate-induced matrix mineralization in primary osteoblast cultures was associated with significant increases in Phex mRNA and protein. Phex mRNA and protein levels were low or undetectable in proliferating preosteoblastic MC3T3-E1 cells and dramatically increased concomitantly with initiation of matrix mineralization. The pattern of Phex expression, however, was similar in nonmineralizing cultures grown in the absence of ␤-glycerophosphate, indicating that the induction of Phex expression in MC3T3-E1 cells was related to cell differentiation rather than matrix mineralization. 1,25-(OH) 2 D 3 inhibited mineral deposition and down-regulated Phex mRNA and protein expression in a time-and dose-dependent manner.These results indicate that Phex is a marker of the fully differentiated osteoblast and that its expression is stimulated during ␤-glycerophosphate-induced mineralization in primary osteoblast cultures and down-regulated by 1,25-(OH) 2 D 3 , an inhibitor of matrix mineralization. These findings add support for Phex having an important role in bone mineralization. (Endocrinology 140: 1192(Endocrinology 140: -1199(Endocrinology 140: , 1999 X -LINKED hypophosphatemia is associated with hypophosphatemia that results from impaired renal phosphate reabsorption, rickets, and osteomalacia that lead to stunted growth and skeletal deformities (1). Despite extensive investigations of the murine homologue, the Hyp mouse (2), the underlying mechanism for the bone and renal abnormalities is not understood. The defective renal phosphate transport in Hyp mice (3) has been attributed to decreased renal expression of the Na-Pi cotransporter gene and immunoreactive protein (4) and is thought to result from the effect of a humoral factor (5, 6). The failure of phosphate therapy and the need for concurrent administration of supraphysiological doses of 1,25-(OH) 2 D 3 to correct the bone osteomalacic lesions in affected patients (7,8) and mice (9, 10) have indicated that hypophosphatemia is not the sole cause of the defective bone mineralization. Evidence has been provided for an associated osteoblast dysfunction in this disorder. They include the presence of characteristic hypomineralized periosteocytic lesions in cortical bone of XLH patients (11) that do not completely disappear in treated patients despite normalization of the mineralization process (12) and the failure of Hyp bone cells to produce normal bone when transplanted into norma...

MEPE has the properties of an osteoblastic phosphatonin and minhibin

“…Serum levels of 1,25-dihydroxy vitamin-D 3 were elevated in the MEPE-treated animals and as expected in PTH-treated animals after 31 h. This is consistent with the fact that 1,25-dihydroxy vitamin-D 3 suppresses MEPE and PHEX mRNA expression and markedly elevated levels of MEPE and PHEX also occur in vitamin D receptor (VDR) knock-out mice [2,19,60]. Also, we have reported an increase in 1,25-dihydroxy vitamin-D 3 synthesis in proximal tubule renal cells (Hu-CL8 cell line) exposed to OHO tumor conditioned media in vitro [73].…”

Osteocyte-Specific Monoclonal Antibody MAb OB7.3 Is Directed Against Phex Protein

“…The protein was therefore designated chicken Phex. The fact that we isolated an 89-kDa protein, while others have isolated a 97-100-kDa protein from mice, (24,25) probably indicates differences between species; for instance, in glycosylation patterns or in amino acid sequence. This idea is supported by the fact that the expected molecular weight of human PHEX is calculated to be 86 kDa (ExPaSy Accession No.…”

Fibroblast Growth Factor 23 Is a Counter-Regulatory Phosphaturic Hormone for Vitamin D

“…In addition, expression of a dominant negative VDR inhibited 1,25(OH) 2 D 3 stimulation of FGF23 promoter activity ( Figure 5C), indicating that FGF23 is regulated directly by 1,25(OH) 2 D 3 in osteoblasts. It is interesting that 1,25(OH) 2 D 3 also is reported to suppress PHEX mRNA levels in osteoblasts and bone (30,31) and reductions in PHEX can lead to increased FGF23 expression in osteoblasts (32,33). Therefore, there seem to be both direct and indirect mechanisms for 1,25(OH) 2 D 3 to regulate FGF23 production by osteoblasts.…”