Cloning of porcine 25-hydroxyvitamin D3 1α-hydroxylase and its regulation by cAMP in LLC-PK1 cells

Tadashi Yoshida, Noriko Yoshida, Akira Nakamura, Toshiaki Monkawa, Matsuhiko Hayashi, Takao Saruta

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The 25-hydroxyvitamin D3 1α-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1α, 25-dihydroxyvitamin D3 (1α,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, 1α,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 1α,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.

Original languageEnglish
Pages (from-to)963-970
Number of pages8
JournalJournal of the American Society of Nephrology
Volume10
Issue number5
Publication statusPublished - 1999 May

Fingerprint

25-Hydroxyvitamin D3 1-alpha-Hydroxylase
LLC-PK1 Cells
Calcifediol
Mixed Function Oxygenases
Organism Cloning
Swine
Messenger RNA
Complementary DNA
Cytochrome P-450 Enzyme System
Hand
8-Bromo Cyclic Adenosine Monophosphate
Polymerase Chain Reaction
Calcitriol
Cholecalciferol
Colforsin
Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases
Adenylyl Cyclases
Genes
Reverse Transcription

ASJC Scopus subject areas

  • Nephrology

Cite this

Cloning of porcine 25-hydroxyvitamin D3 1α-hydroxylase and its regulation by cAMP in LLC-PK1 cells. / Yoshida, Tadashi; Yoshida, Noriko; Nakamura, Akira; Monkawa, Toshiaki; Hayashi, Matsuhiko; Saruta, Takao.

In: Journal of the American Society of Nephrology, Vol. 10, No. 5, 05.1999, p. 963-970.

Research output: Contribution to journalArticle

Yoshida, Tadashi ; Yoshida, Noriko ; Nakamura, Akira ; Monkawa, Toshiaki ; Hayashi, Matsuhiko ; Saruta, Takao. / Cloning of porcine 25-hydroxyvitamin D3 1α-hydroxylase and its regulation by cAMP in LLC-PK1 cells. In: Journal of the American Society of Nephrology. 1999 ; Vol. 10, No. 5. pp. 963-970.
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abstract = "The 25-hydroxyvitamin D3 1α-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1α, 25-dihydroxyvitamin D3 (1α,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, 1α,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 1α,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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