Cloning and characterization of the proximal murine Phex promoter

Phex is an endopetidase that regulates systemic phosphate homeostasis. We i nvestigated Phex gene transcription by cloning and performing functional an alysis of the 2736 bp of the 5 ' flanking region of the mouse Phex gene con taining its promoter. We identified a transcription start site, a consensus TATA-box, and multiple potential cis-acting regulator elements. To determi ne whether the promoter directs cell-type restricted Phex expression, we tr ansfected full-length and 5 ' -deleted Phex luciferase reporter constructs into various cell lines. Phex-expressing C5.18 chondrocytes displayed the h ighest activity of the transfected Phex promoter constructs compared with n on-Phex-expressing COS-7 cells, whereas promoter activity was intermediate in ROS 17/2.8 osteoblasts and maturation stage-dependent in MC3T3-E1 osteob lasts. Analysis of sequential 5 ' -deletion mutants of the Phex promoter in ROS 17/2.8 cells revealed bimodal activity, suggesting that both positive and negative cis-acting regions may be present. The chondrogenic factor SOX 9 markedly stimulated Phex promoter activity, whereas Cbfa1, PTH, and 1,25( OH)(2)D-3 had no effect. Our findings are consistent with the predominant e xpression of Phex in bone and cartilage. Additional studies will be needed to confirm the regulatory regions in the Phex promoter that function in a c ell-restricted manner.