The current study investigated the action of 1,25-dihydroxyvitamin D 3 (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogenactivated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction of CYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor ␣ (RXR␣) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR␣ and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR␣ and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXR␣ (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXR␣ phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.The hormonally active form of vitamin D 3 is 1,25-dihydroxyvitamin D 3 (1,25D). 1 This secosteroid hormone plays a central role in calcium homeostasis and bone metabolism and participates in a diverse range of cellular actions including inhibition of tumor cell growth, induction of cell differentiation, and modulation of the immune response (1-4). The transcriptional actions of 1,25D are mediated by the nuclear vitamin D receptor (VDR), which heterodimerizes with retinoid X receptor (RXR) and binds to specific vitamin D response element (VDRE) sites in the promoter region of vitamin D-responsive genes (5). Transactivation by the liganded VDR/RXR is dependent upon the binding of one or more coactivator complexes that permit bridging to the RNA polymerase II machinery (6, 7). In the unliganded state, some nuclear receptors including VDR (8) can bind a co-repressor that inhibits transactivation (9, 10), but this repressor dissociates upon ligand binding.Maintenance of cellular 1,25D levels is critical to regulation of the function of the hormone in which high levels of 1,25D are to...