[Show abstract][Hide abstract] ABSTRACT: Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.
[Show abstract][Hide abstract] ABSTRACT: The biosynthesis of 1alpha, 25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) in renal proximal tubules. It was recently demonstrated that LLC-PK1 cells express CYP27B1 mRNA, which is regulated by intracellular cAMP but not vitamin D3. To clarify the effect of calcitonin on vitamin D3 metabolism in vitro, LLC-PK1 cells were incubated with hormonal factors, and expression of CYP27B1 mRNA was measured by quantitative reverse transcription-PCR. Calcitonin at 100 nmol/L significantly increased CYP27B1 mRNA expression by 24 h (271 +/- 21% of control). Incubation with calcitonin over a range of 1 micromol/L to 1 pmol/L resulted in a concentration-dependent increase in CYP27B1 mRNA levels. It is known that the calcitonin receptor has dual intracellular signaling pathways, via protein kinases A and C. Both 500 micromol/L 8-bromo-cAMP, a protein kinase A activator, and 100 nmol/L phorbol 12-myristate 13-acetate, a protein kinase C activator, increased CYP27B1 mRNA levels at 24 h (207 +/- 54 and 246 +/- 58% of control, respectively). However, calcitonin-induced CYP27B1 mRNA expression was only inhibited by the protein kinase C inhibitors staurosporine and calphostin C. The protein kinase A inhibitors Rp-cAMPS at 10 and 100 micromol/L and H-89 at 10 micromol/L had no effect on the action of calcitonin, in spite of cAMP-activation by calcitonin. The present data suggest that calcitonin upregulates CYP27B1 mRNA expression via the protein kinase C pathway in LLC-PK1 cells.
No preview · Article · Jan 2000 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.
No preview · Article · Jun 1999 · Journal of the American Society of Nephrology