Eric R. Hines

Molecular cloning of the murine PHEX gene promoter.

We report the novel cloning of the murine PHEX promoter, the gene that is mutated in X-linked hypophosphatemic rickets (XLH). Four promoter/reporter gene constructs, -133/+104, -542/+104, -1061/+104, and -2866/+104, showed significant luciferase activity (4.9-13.2-fold over background) when transfected into rat osteogenic sarcoma (UMR-106) cells.

Molecular cloning of murine sodium-phosphate cotransporter type IIb (Na/P(i)-IIb) gene promoter and characterization of gene structure.

We report the cloning of the murine Na/P(i)-IIb cotransporter gene, which spans more than 18 kilobases and consists of 12 introns and 13 exons. Three promoter/reporter gene constructs, -159/+73, -429/+73 and -954/+73, showed significant luciferase activity (22-82-fold over background) when transfected into in rat intestinal epithelial (RIE-1) cells.

Regulation of the murine PHEX gene by vitamin D3

Introduction: The mechanism for normal bone mineralization is complex and has not been fully elucidated, however it is known to be influenced by factors such as stress, systemic factors (e.g. estrogen) and local factors such as bone morphogenic proteins and interleukins. Osteomalacia is characterized by defective bone mineralization, with increased unmineralized matrix. The PHEX gene (Phosphate encoding gene with Homologies to Endopeptidases on the X chromosome) is abnormal in X-linked hypophosphatemic rickets (XLH) aka “Phosphate Diabetes.” This manifests as hypophosphatemia, low or inappropriately normal 1,25 (OH)2 vitamin D3 (D3) levels, high serum alkaline phosphatase and osteomalacia. PHEX is mainly expressed in osteoblasts, and is theorized to regulate an osteoblast derived factor termed “phosphatonin” that modulates mineralization of the extracellular matrix. D3 has been shown to downregulate PHEX in primary and cultured mouse osteoblasts. PHEX deficient osteoblasts have an altered response to D3 treatment ; further, D3 decreases PHEX mRNA levels and protein synthesis in normal osteoblast culture, with preliminary evidence indicating that this decrease is at the transcriptional level. The murine PHEX promoter has been previously cloned by our laboratory and its functionality was demonstrated in rat osteoblast-like (UMR-106) cells . Examination of this sequence for the presence of putative cis-acting elements identified multiple binding sites, including those for estrogen and glucocorticoid receptors; a putative binding site for vitamin D receptor was not identified. The present study was undertaken to confirm that D3 regulates PHEX at a transcriptional level both in vivo and in vitro, and to determine the promoter region responsible for this regulation.