Vitamin D is a secosteroid that is metabolically activated and degraded through the actions of three cytochrome P450 hydroxylase enzymes. Bioactivation occurs through the sequential actions of cytochromes P450C25 and P450C1, resulting in synthesis of the pleiotropic hormone 1,25-dihydroxyvitamin D (1,25VD), which regulates over 60 genes whose actions include those associated with calcium homeostasis and immune responses as well as cellular growth, differentiation, and apoptosis. Inactivation of 1,25VD occurs by C23/C24 oxidation pathways that are catalyzed by the multifunctional cytochrome P450C24 enzyme. Both P450C1 and P450C24 are highly regulated enzymes whose differential expression is controlled in response to numerous cellular modulatory agents such as parathyroid hormone (PTH), calcitonin, interferon gamma, calcium, phosphorus, and pituitary hormones as well as the secosteroid hormone 1,25VD. Most thoroughly studied at the molecular level are the actions of PTH to upregulate P450C1 gene expression and 1,25VD to induce the expression of P450C24. The regulatory action of PTH is mediated through the protein kinase A pathway and involves the phosphorylation of transcription factors that function at the proximal promoter of the P450C1 gene. The upregulation of P450C24 by 1,25VD has both a rapid nongenomic and a slower genomic component that are functionally linked. The rapid response involves protein kinase C and mitogen-activated protein kinase (MAPK) pathways that direct the phosphorylation of nuclear transcription factors. The slower genomic actions are linked to the binding of 1,25VD to the vitamin D receptor (VDR) and the interaction of the VDR-1,25VD complex with its heterodimer partner retinoid-X-receptor and associated coactivators. The regulatory complex is assembled on vitamin D response elements in the proximal promoter of the P450C24 gene and functions to increase the transcription rate.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired

We previously demonstrated augmented endothelium-derived nitric oxide (EDNO)-dependent pulmonary arterial dilation and increased arterial endothelial nitric oxide synthase (eNOS) levels in chronic hypoxic (CH) and monocrotaline (nonhypoxic) models of pulmonary arterial hypertension. Therefore, we hypothesized that the long-term elevation of arterial eNOS levels associated with CH is related to pulmonary hypertension or some factor(s) associated with hypertension and not directly to hypoxia. To test this hypothesis, we examined responses to the EDNO-dependent dilator ionomycin in U-46619-constricted, isolated, saline-perfused lungs from control rats, CH (4 wk at 380 mmHg) rats, and rats previously exposed to CH but returned to normoxia for 4 days or 2 wk. Microvascular pressure was assessed by double-occlusion technique, allowing calculation of segmental resistances. In addition, vascular eNOS immunoreactivity was assessed by quantitative immunohistochemistry, and eNOS mRNA abundance was determined by RT-PCR assays. Our findings indicate that 4-day and 2-wk posthypoxic rats exhibit persistent pulmonary hypertension, likely due to maintained arterial remodeling and polycythemia associated with prior exposure to CH. Furthermore, arterial dilation to ionomycin was augmented in lungs from each experimental group compared with controls. Finally, arterial eNOS immunoreactivity and whole lung eNOS mRNA levels remained elevated in posthypoxic animals. These findings suggest that altered vascular mechanical forces or vascular remodeling contributes to enhanced EDNO-dependent arterial dilation and upregulation of arterial eNOS in various models of established pulmonary hypertension.

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...

The hormone 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 1 or calcitriol) is a pleiotropic secosteroid that functions in the regulation of calcium homeostasis, cellular differentiation and proliferation, and immune function (1-5). Nuclear actions of 1,25-(OH) 2 D 3 involve the transcriptional regulation of gene expression, which is mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor that belongs to the nuclear receptor superfamily (1, 6 -10). Activated VDR can bind as either a homodimer or a heterodimeric complex to a DNA sequence known as the vitamin D-responsive element (VDRE) present in the promoter of target genes (11-13). Heterodimers consisting of VDR and retinoid X receptor (RXR) are widely documented (12), although VDR heterodimeric complexes have also been demonstrated for both the retinoic acid receptor (14, 15) and the thyroid hormone receptor (16). Vitamin D-responsive elements generally display a binding motif that consists of two imperfect, direct repeat hexameric sequences (i.e. halfsites) that are separated by 3 bp or, more rarely, by 6 bp; these VDREs are referred to as DR-3 and DR-6, respectively (12).Metabolic inactivation of 1,25-(OH) 2 D 3 and conversion to water-soluble calcitroic acid occurs through the C-24 oxidation pathway. The initial step in this pathway involves the 24-hydroxylation of 1,25-(OH) 2 D 3 by the mitochondrial enzyme 25-hydroxyvitamin D 3 24-hydroxylase (CYP24) (17, 18). Rats fed a normal diet express a low level of CYP24, predominantly in the kidney. However, the enzyme can be substantially induced in kidney and intestine (19 -23) and various other cells (24 -29) by 1,25-(OH) 2 D 3 treatment. Up-regulation of CYP24 expression (19 -23) increases the metabolic clearance of 1,25-(OH) 2 D 3 , and, thereby, feedback regulates the hormone's ambient and cellular levels (17, 18). The mechanism whereby 1,25-(OH) 2 D 3 acts to modulate cellular CYP24 expression is of fundamental importance to understanding the hormone's role in health and disease.Molecular regulatory studies of CYP24 gene expression by 1,25-(OH) 2 D 3 are in progress and promoter analysis data for rat (14, 30 -33) and human (34) have been reported. In the rat CYP24 gene promoter, three VDREs on the antisense strand have been identified. We have previously defined the proximal VDRE (30) in its native promoter context, while the two more distal VDREs have been tested by fusing to a heterologous promoter (14, 31). To date, however, the functionality of the VDREs has not been verified in the context of the native CYP24 promoter, and there is no direct information available regarding the contribution of each VDRE to vitamin D induction or whether there is a cooperative interaction between the response elements. These issues are addressed in the current investigation, in which mutagenic constructs of the rat CYP24 promoter have been used in transient gene expression and gel mobility shift analysis. Construction of Mutant Clones-A 365-bp PvuII/StuI fragment containing 298 bp of 5Ј-flanking se...