AbstractPHosphate‐regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X‐linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH‐related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP‐mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR‐106 osteoblastic cells with PTHrP(1‐34), and used RT‐PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1‐34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1‐34)‐mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull‐down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1‐34). J. Cell. Physiol. 227: 2378–2387, 2012. © 2011 Wiley Periodicals, Inc.