Cloning of porcine 25-hydroxyvitamin D3 1alpha-hydroxylase and its regulation by cAMP in LLC-PK1 cells.
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.
Yoshida
T
T
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Yoshida
N
N
Nakamura
A
A
Monkawa
T
T
Hayashi
M
M
Saruta
T
T
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002762:Cholecalciferol / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004338:Drug Combinations; D018374:LLC-PK1 Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine
D018076:DNA, Complementary; D004338:Drug Combinations; D012333:RNA, Messenger; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase