Role of MAP Kinases in the 1,25-Dihydroxyvitamin D3-induced Transactivation of the Rat Cytochrome P450C24 (CYP24) Promoter

Abstract

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...

Global Gene Expression Analysis of ERK5 and ERK1/2 Signaling Reveals a Role for HIF-1 in ERK5-mediated Responses

“…ERK1/2 and ERK5 regulate similar processes within the cell, including chemotaxis, cytoskeletal organization, transcription, and proliferation (7, 13, 20 -22), and have been shown to cooperate in NIH3T3 cell transformation via convergence of p90 RSK and NFB signaling (21). However, mechanisms used by each pathway to elicit specific responses may be distinct and may involve differential localization, substrate preferences, or signal strength (12,13,20,22). For example, although CREB is a substrate for both ERK1/2 and ERK5, the cellular location of ERK5 activation dictates a preferential role for this pathway in mediating CREB-dependent neuronal survival (12).…”

“…Like ERK1/2, ERK5 recognizes phosphorylation sites containing Pro at positions Ϫ2 and ϩ1 to the phosphorylation site. Substrates shared by ERK5 and ERK1/2 include CREB and Ets-1 (12,13), and those specific to ERK5 include members of the myocyte enhancer factor 2 (MEF2) family (2A, 2C, and 2D), Sap1a, MyoD, and Bad (11, 14 -17). Unique to ERK5 is a large C-terminal transcriptional activation domain (18) that physically interacts with MEF2C, further increasing transcriptional activity beyond that achieved by phosphorylation alone (15).…”

The Rapid Effects of 1,25-Dihydroxyvitamin D3 Require the Vitamin D Receptor and Influence 24-Hydroxylase Activity

“…Another open question relates to the physiological relevance of these rapid effects. Their possible role in the genomic control of cell differentiation, cell proliferation, and the regulation of CYP24, a catabolic and activating enzyme in the vitamin D endocrine system (27), only begins to be unveiled (7,31,35). The two current cell models bearing point mutations at key sites of the encoded VDR gene offer unique contributions to these challenging research fields.…”

“…At present, several rapid effects of 1,25-(OH) 2 D 3 have been reported on intracellular calcium entry and distribution, on the opening of Ca 2ϩ and Cl Ϫ channels, and on protein kinase C, MAPK, phospholipase A2, phospholipase C-␤1, and G␣q/11 activation (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). These rapid effects are thought to be involved in the process of cell proliferation/differentiation but also in vitamin D metabolism and catabolism via a MAPK-mediated stimulation of 25-(OH)D 3 -24-hydroxylase gene expression (7,31). The comparison of rapid and long term responses to 1,25-(OH) 2 D 3 in our two cell models strongly support the latter hypothesis as they show the following: (i) a parallelism between rapid calcium and long term 24-hydroxylase responses to low 1,25-(OH) 2 D 3 doses (1 pM to 100 pM) absent in K45E fibroblasts and present in W286R cells; (ii) a parallelism between 1,25-(OH) 2 D 3 effects on 24-hydroxylase activity and CYP24 mRNA expression, no effect in K45E cells, and 2-fold increases in W286R cells; and (iii) the blockage of these effects by PD98059, an inhibitor of the MEK 1/2 kinase.…”

“…These enzyme activity and mRNA expressions are known to be markedly enhanced by 1,25-(OH) 2 D 3 (27)(28)(29). Lastly, to determine precisely the mechanism underlying a possible association between rapid and long term effects, culture experiments have been performed in the presence or absence of verapamil, a blocker of Ca 2ϩ channels, or of PD98059, an inhibitor of the MEK 1/2-kinase (30), which has been shown to inhibit induction of CYP24 gene expression by 1,25-(OH) 2 D 3 (31).…”