Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase

The subcellular location and some properties of the rat kidney 25-hydroxyvitamin D3-1 alpha-hydroxylase are described. Enzyme activity can be measured as previously discussed (Tanaka, Y., and DeLuca, H.F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 196-199) using saturating substrate (25-hydroxyvitamin D3) concentrations. The reaction is linear with time for up to 30 min at a substrate concentration of 80 microM and 9-11 mg/ml mitochondrial protein. The enzyme, located in the mitochondria, requires molecular oxygen and a source of NADPH. Succinate supplies NADPH for 1 alpha-hydroxylation through reversal of electron transport and transhydrogenation as shown by inhibition with antimycin A and dinitrophenol. Malate supplies NADPH for the reaction via the mitochondrial malic enzyme or malate dehydrogenase and transhydrogenase as indicated by the lack of inhibition by antimycin A but inhibition with dinitrophenol. Metyrapone and carbon monoxide both inhibit 1 alpha-hydroxylation indicating the involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, has no effect on 1 alpha-hydroxylation.     

25-hydroxyvitamin D3 1alpha-hydroxylase and vitamin D receptor expression in papillary thyroid carcinoma.

Vitamin D receptor (VDR) and 25-hydroxyvitamin D3 1-alpha-hydroxylase expression have recently been shown to be upregulated in several tumors and thought to represent an important endogenous response to tumor progression. Little is known about the expression of these proteins in thyroid carcinoma, although previous reports have documented evidence of the biological effect of vitamin D in thyroid cells. Using paraffin-embedded and frozen sections of papillary thyroid carcinoma, we utilized real-time quantitative RT-PCR and immunohistochemistry to characterize the expression of VDR and 1-alpha-hydroxylase in thyroid follicular cells, with special emphasis on papillary thyroid carcinoma (PTC). VDR and 1-alpha-hydroxylase expression were increased in PTC compared with normal thyroid tissue and especially high in areas of lymphocyte infiltration. Expression of VDR and 1-alpha-hydroxylase in PTC may be compatible with an overall favorable prognosis for this tumor type and may constitute important prerequisites for using vitamin D and/or vitamin D analogs in the treatment of PTC.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

25-hydroxyvitamin D3-1alpha-hydroxylase expression in normal and malignant human colon.

1,25-dihydroxyvitamin D(3) has anti-mitotic, pro-differentiating, and pro-apoptotic activity in tumor cells. We demonstrated that the secosteroid can be synthesized and degraded not only in the kidney but also extrarenally in intestinal cells. Evaluation of 1,25-dihydroxyvitamin D(3)-synthesizing CYP27B1 hydroxylase mRNA (real-time PCR) and protein (immunoblotting, immunofluorescence) showed enhanced expression in high- to medium-differentiated human colon tumors compared with tumor-adjacent normal mucosa or with colon mucosa from non-cancer patients. In high-grade undifferentiated tumor areas expression was lost. Many cells co-expressed CYP27B1 and the vitamin D receptor. We suggest that autocrine/paracrine antimitotic activity of 1,25-dihydroxyvitamin D(3) could prevent intestinal tumor formation and progression.     

The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism.     

Localization of 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the mammalian kidney

The tRNA from Ehrlich ascites tumor cells is deficient in the modified nucleoside Q (queuosine). Continuous infusion of Q base (queuine) to tumor-bearing mice reverses the deficiency of Q in Ehrlich ascites tRNA, and coincidently, causes an inhibition of tumor growth.     

Analysis of basal regulatory elements in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene promoter

5'-Deletion analysis of the 1.7-kb mouse 1alpha-hydroxylase gene promoter reveals that the minimal promoter region for basal activity is -85/+22 and requires a functional CCAAT element. Mutational analysis also demonstrates that deletion of the internal promoter region from nucleotides -1125 to -86 leads to a 25- to 30-fold increase in basal promoter activity. The increased activity is not the result of positional effects, but is caused in part by the removal of an AC repeat. Further analysis of the promoter revealed an enhancer element localizes to an upstream region -1385 to -1125, which contains three consensus AP-1 sites. Deletion of the most proximal AP-1 site causes a 60% loss of enhancer activity. The data suggest the presence of the AC repeat prevents the full potential activation of the 1alpha-hydroxylase promoter by factors binding to AP-1 sites.     

Parathyroid hormone stimulates dephosphorylation of the renoredoxin component of the 25-hydroxyvitamin D3-1 alpha-hydroxylase from rat renal cortex

Parathyroid hormone (PTH) stimulates the renal conversion of 25-OH-vitamin D3 to 1,25-(OH)2-vitamin D3 in young animals. There is evidence that PTH acts via cAMP and cAMP-dependent protein kinase, but the identity of the phosphorylated protein(s) is unknown. The present study investigates the possibility that phosphorylation modification of specific components of the renal mitochondrial, cytochrome P-450-linked 25-OH-vitamin D3-1 alpha-hydroxylase is involved in the regulation of 1,25-(OH)2-vitamin D3 production. Mitochondria were isolated from [32P]phosphate-labeled renal cortical slices which had been divided into control and agonist-treated groups. The hydroxylase protein components from the solubilized mitochondria were partially purified using p-chloroamphetamine-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. Phosphorylation was observed only in a protein with an Mr = 12,000 and a pI of 4.2 by autoradiography of the gels. This radiolabeled protein was immunoprecipitated with adrenodoxin antibody. Additionally, the protein in the same Mr region of the polyacrylamide gel reacted with adrenodoxin antibody and co-migrated with bovine adrenodoxin. PTH and forskolin treatment resulted in decreased phosphate incorporation into the protein, whereas A23187 treatment increased the phosphorylation. In parallel experiments, affinity-isolated hydroxylase from control and PTH-treated slices was used to assess in vitro hydroxylase activity using [3H]25-hydroxyvitamin D3 as substrate. The hydroxylase activity derived from PTH-treated tissue was significantly higher than that of control. From these data, it is proposed that renal response to PTH in terms of 25-hydroxyvitamin D3 hydroxylase stimulation involves dephosphorylation of renoredoxin, the ferrodoxin component of this hydroxylase complex.     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.     

Vestibular dysfunction in vitamin D receptor mutant mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. Vitamin D deficits are associated with muscle weakness and osteoporosis, whereas vitamin D supplementation may improve muscle function, body sway and frequency of falls, growth and mineral homeostasis of bones. The loss of muscle strength and mass, as well as deficits in bone formation, lead to poor balance. Poor balance is one of the main causes of falls, and may lead to dangerous injuries. Here we examine balance functions in vitamin D receptor deficient (VDR−/−) mice, an animal model of vitamin D-dependent rickets type II, and in 1α-hydroxylase deficient (1α-OHase−/−) mice, an animal model of pseudovitamin D-deficiency rickets. Recently developed methods (tilting box, rotating tube test), swim test, and modified accelerating rotarod protocol were used to examine whether the absence of functional VDR, or the lack of a key vitamin D-activating enzyme, could lead to mouse vestibular dysfunctions. Overall, VDR−/− mice, but not 1α-OHase−/− mice, showed shorter latency to fall from the rotarod, smaller fall angle in the tilting box test, and aberrant poor swimming. These data suggest that VDR deficiency in mice is associated with decreased balance function, and may be relevant to poorer balance/posture control in humans with low levels of vitamin D.