A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice.

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca(++), PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis.

Ethanol extract of Fructus Ligustri Lucidi increases circulating 1,25-dihydroxyvitamin D3 by inducing renal 25-hydroxyvitamin D-1α hydroxylase activity.

OBJECTIVE: The present study was designed to determine whether Fructus Ligustri Lucidi (FLL) ethanol extract can directly regulate vitamin D metabolism both in vivo and in vitro. METHODS: Eleven-month-old, aged Sprague-Dawley sham-operated and ovariectomized (OVX) female rats were fed a normal-calcium (Ca) diet (0.6% Ca, 0.65% phosphorus) and received either FLL (700 mg/kg) or vehicle daily for 12 weeks. The in vitro effects of FLL on vitamin D metabolism were studied using primary cultures of the rat renal proximal tubules. mRNA and protein expressions of 25-hydroxyvitamin D-1α hydroxylase (1-OHase) and vitamin D receptor (VDR) in the kidney and proximal tubule were measured using real-time polymerase chain reaction and Western blotting, respectively. The concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesized by renal 1-OHase were measured by a competitive enzyme immunoassay. RESULTS: FLL treatment significantly increased serum 1,25(OH)2D3 levels in both sham (P < 0.01) and OVX (P < 0.05) rats. FLL increased renal 1-OHase and VDR protein and mRNA expressions in sham rats. Protein expression of renal 1-OHase, but not VDR, was also up-regulated in OVX rats during FLL treatment. 1-OHase mRNA and 1-OHase activity were increased by FLL treatment in primary cultures of renal proximal tubule cells. CONCLUSIONS: FLL could increase the circulating levels of 1,25(OH)2D3 in vivo in aged female rats by directly stimulating 1-OHase activity. Thus, it might be an ideal oral agent that can help to improve the ability to induce 1,25(OH)2D3 synthesis and Ca balance in postmenopausal women who are of high risk of developing osteoporosis.

Chemoprevention of colorectal neoplasia by estrogen: potential role of vitamin D activity.

Postmenopausal hormone replacement therapy lowers colon cancer incidence. In humans, the mechanism is unknown, but animal models suggest that it may involve activation of the vitamin D receptor (VDR) pathway. The aims of our study were to determine whether estrogen intervention affects global gene expression in rectal mucosal biopsies and whether vitamin D-related genes are affected. Estradiol was given to raise serum estradiol to premenopausal levels in 10 postmenopausal women under close nutritional control. Primary end points were expression of VDR, CYP24A1, CYP27B1, and E-cadherin in rectal mucosa by reverse transcription-PCR and examining response to estradiol by genome-wide arrays. Responses in gene expression in rectal biopsies to estrogen were determined in each subject individually and compared with a human estrogen response gene array database and a custom array in vitro-generated database. Cluster analysis showed that subjects maintained their overall gene expression profile and that interindividual differences were greater than intraindividual differences after intervention. Eight of 10 subjects showed significant enrichment in estrogen-responsive genes. Gene array group analysis showed activation of the VDR pathway and down-regulation of inflammatory and immune signaling pathways. Reverse transcription-PCR analysis showed significant up-regulation of VDR and E-cadherin, a downstream target of vitamin D action. These data suggest that the chemopreventive action of hormone replacement therapy on colon neoplasia results, at least in part, from changes in vitamin D activity. Evaluation of gene arrays is useful in chemopreventive intervention studies in small groups of subjects.

[The influence of isoflavonoids on the antitumor activity of vitamin D3]. / Wplywu izoflawonoidów na aktywnosc przeciwnowotworowa witaminy D3.

Isoflavonoids exert a regulatory function on the expression of cytochrome P450 enzymes and also up-regulate the vitamin D(3) receptor (VDR) on cancer cells, which increase their sensitivity to 1,25-dihydroxyvitamin D(3) , the hormonally active form of vitamin D(3) . Isoflavonoids are also able to raise the serum level of the active form of vitamin D(3) due to their inhibitory activity on CYP24, the enzyme involved in the degradation of 1,25-dihydroxyvitamin D(3) and its precursor 25-OH-D(3) to inactive compounds. Another enzyme, CYP27B1, involved in the synthesis of 1,25-dihydroxyvitamin D(3) , is stimulated by isoflavonoids, and this may result in a similar effect of increasing in the serum level of 1,25-dihydroxyvitamin D3. CYP27B1 and CYP24 were found in kidneys (the main location of 1,25-(OH) (2)D(3) synthesis) and also in brain cells, osteoclasts, keratinocytes, macrophages, intestine epithelial cells, and in some cancer cells. The expression of VDR was detected not only in the cells primarily targeted by 1,25-dihydroxyvitamin D3, but also in epithelial and mesenchymal cells. Therefore, combined treatment with isoflavonoids and 1,25-dihydroxyvitamin D3 might be effective in both cancer prevention and treatment.

Leptin attenuates gene expression for renal 25-hydroxyvitamin D3-1alpha-hydroxylase in mice via the long form of the leptin receptor.

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We previously showed that leptin administration to leptin-deficient obese (ob/ob) mice suppressed mRNA expression and activity of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). In leptin receptor-deficient (db/db) mice, we presently examined whether leptin affects 1alpha-hydroxylase expression in renal tubules through the active form of the leptin receptor (ObRb). Elevated serum concentrations of calcium and 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in untreated ob/ob mice showed sharp reduction with leptin administration (4 mg/kg, i.p. every 12h for 2 days); no such reduction of elevation occurred in db/db mice. ObRb mRNA was expressed in kidney, brain, fat, lung, and bone in wild-type and ob/ob mice, but not db/db mice. The ob/ob and db/db mice showed large increases in renal 1alpha-hydroxylase mRNA expression and activity. Leptin administration (4 mg/kg) completely abrogated these increases in ob/ob but not db/db mice. Renal 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA synthesis also was greatly elevated in ob/ob and db/db mice; excesses decreased significantly with leptin administration in ob/ob mice, but increased in db/db mice. Renal tubular cells in primary culture expressed mRNAs including proximal tubules markers (1alpha-hydroxylase and megalin), parathyroid hormone receptor, and vitamin D receptor. Calcitonin receptor mRNA, synthesized mainly in distal tubules, was scant, indicating that most cultured cells were from proximal tubules. Cells did not express ObRb mRNA. Forskolin exposure at 10(-6)M for 3 or 6h significantly increased 1alpha-hydroxylase mRNA. Leptin at 10(-6)M did not change mRNA expression in either presence or absence of forskolin. Accordingly, leptin attenuates renal 1alpha-hydroxylase gene expression through ObRb. Furthermore, leptin appears to act indirectly on renal proximal tubules to regulate 1alpha-hydroxylase gene expression.

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.