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Cloning of Porcine 25-Hydroxyvitamin D₃ 1-Hydroxylase and Its Regulation by cAMP in LLC-PK₁ Cells

Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.

Correspondence to Dr. Matsuhiko Hayashi, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Phone and Fax: 81-3-3359-2745; E-mail: matuhiko{at}mc.med.keio.ac.jp

Abstract. The 25-hydroxyvitamin D₃ 1-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1, 25-dihydroxyvitamin D₃ (1,25(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 CYP27B1 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 µmol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 µmol/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, 1,25(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 1,25(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.

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