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Biochem. J. (2005) 389 (753–762) (Printed in Great Britain)

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Molecular action of 1,25-dihydroxyvitamin D₃ and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site

Barbara K. NUTCHEY*, Josef S. KAPLAN*, Prem P. DWIVEDI*, John L. OMDAHL†, Antonio FERRANTE‡§, Brian K. MAY* and Charles S. T. HII‡§¹

*School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5000, Australia, †Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM 87131-5221, U.S.A., ‡Department of Paediatrics, University of Adelaide, Adelaide, SA 5006, Australia, §Department of Immunopathology, Women's and Children's Hospital, 72 King William Road, SA 5006, North Adelaide, Australia, and School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D₃ 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D₃) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys⁷¹Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at -171/-163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5´-TGTCGGTCA-3´, is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.

Key words: cytochrome P450C24, 1,25-dihydroxyvitamin D₃, extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), phorbol ester, transcriptional regulation.

Abbreviations used: AP-1, activator protein-1; CYP24, 25-hydroxyvitamin D₃ 24-hydroxylase; 1,25D, 1,25-dihydroxyvitamin D₃; EBS, Ets-1 binding site; EMSA, electrophoretic mobility-shift assay; ERK, extracellular-signal-regulated kinase; GST, glutathione S-transferase; HEK-293T cell, human embryonic kidney 293T cell; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MKK, MAP kinase kinase; oFSHR, ovine follicle-stimulating hormone receptor; PKC, protein kinase C; RasGRP, Ras guanine-nucleotide-releasing protein; RXR, retinoid-X receptor; VDR, vitamin D receptor; VDRE, vitamin D response element; VSE, vitamin D stimulatory element; WT, wild-type.

¹To whom correspondence should be addressed (email charles.hii@adelaide.edu.au).

Received 23 November 2004/12 April 2005; accepted 19 April 2005

Published as BJ Immediate Publication 19 April 2005, doi: 10.1042/BJ20041947

The Biochemical Society, London ©2005