Regulation of gene expression by the CYP27B1 promoter—study of a transgenic mouse model

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Abstract

The enzyme 25-hydroxyvitamin D 1-hydroxylase (CYP27B1) is the rate limiting enzyme in the two-step activation process of Vitamin D to its active form 1,25-dihydroxyvitamin D (1,25D) and is located in the mitochondrial fraction of the proximal tubular cells of the kidney. More recently CYP27B1 activity and expression have also been identified in a number of non-renal cells, which is suggestive of new, previously unidentified roles for Vitamin D in the human body. Although the regulation of CYP27B1 activity and expression has been a major focus of interest over the past decades, the exact molecular mechanism behind the regulation of CYP27B1 activity and expression and the role of the CYP27B1 promoter, herein, are still poorly understood. In this study, we created a transgenic mouse model that expresses the luciferase reporter gene under the control of the full-length, 1.5 kb, human CYP27B1 promoter. This animal model allows us to study in vivo the tissue-specific, CYP27B1 promoter-controlled, regulation of the expression of the CYP27B1 gene.

Introduction

The enzyme 25-hydroxyvitamin D 1-hydroxylase or CYP27B1, is the key enzyme in the two-step activation process of Vitamin D to its biologically active form 1,25-dihydroxyvitamin D (1,25D) [1]. It is located in the proximal tubular cells of the kidney. The activity of the CYP27B1 enzyme is apparently tightly regulated by a number of physiological and dietary factors such as parathyroid hormone (PTH), calcium, phosphate, 1,25D, and factors involved in growth and differentiation [2], [3], [4].

Although the kidney was originally believed to be the sole producer of the active form of 1,25D, more recently, CYP27B1 enzyme activity and mRNA expression have been identified in a number of extra-renal tissues. These include the intestine, brain, and bone [5], [6], [7]. These findings raise the possibility that, besides its classical roles in mineral homeostasis and bone metabolism, 1,25D has other, so far unidentified roles in the human body. Evidence from dietary and knock-out animal studies has lead to the suggestion of a potential role for 1,25D in a wide range of physiologocal and patho-physiological processes, including autoimmune diseases, psoriasis, and cancer.

Although the metabolism of 1,25D has been the focus of intensive research for a number of decades, the molecular mechanisms underlying the regulation of CYP27B1 expression and activity in the kidney and in extra-renal cells are still poorly understood. In vitro studies, using promoter constructs of the CYP27B1 promoter, have suggested regulatory regions in this promoter for factors known to affect 1,25D synthesis such as PTH and 1,25D; the precise sequences through which these factors regulate gene transcription are, however, unknown.

To fully understand the regulation of CYP27B1 gene expression in vivo, we established a transgenic mouse model that expresses the luciferase reporter gene under the control of the full-length, 1.5 kb, human CYP27B1 promoter. This animal model allows us to identify factors that modulate CYP27B1 expression through response elements located within the 1.5 kb CYP27B1 promoter.

Section snippets

DNA construct

The pGL3-pCYP27B1-luciferase (pGL3-pCYP27B1-luc) construct contained the luciferase reporter gene flanked by the 1541 bp (−1497 to +44 relative to the transcriptional start site) promoter region of the human CYP27B1 gene. The complete 1541 KpnI–XhoI fragment was inserted upstream of the luciferase reporter gene in the pGL3-Basic vector (Promega Corp., NSW, Australia). The 3513 bp pGL3-pCYP27B1-luc construct was excised by KpnI and BamHI restriction enzyme digestion.

Generation of transgenic mice and genomic DNA analysis

Transgenic mice were generated

Transgene expression

To investigate the tissue-specific transcriptional activity of the 1.5 kb CYP27B1 promoter in vivo, three independent transgenic mouse lines (2992, 3039, and 3042) were generated expressing the luciferase reporter gene under the control of the 1.5 kb CYP27B1 promoter. In all three lines, a very similar pattern of transgene expression was detected, suggesting that the 1.5 kb CYP27B1 promoter controls gene expression in vivo in a tissue-specific manner. The absolute levels of luciferase activity

Discussion

The 1.5 kb human CYP27B1 promoter directed reporter gene expression in a tissue-specific manner in three independent transgenic mice lines. Although the tissue-specific expression pattern of the transgene did not differ significantly from one line to the other, the absolute levels of luciferase activity varied greatly among the three lines. The variation in transgene expression between independent lines, is a common observation in studies utilizing transgenic animals [8], [9]. The introduction

References (12)

  • K. Dobie et al.

    Variegated gene expression in mice

    Trends Genet.

    (1997)
  • G. Jones et al.

    Current understanding of the molecular actions of Vitamin D

    Physiol. Rev.

    (1998)
  • J.L. Omdahl et al.

    Hydroxylase enzymes of the Vitamin D pathway: expression, function, and regulation

    Annu. Rev. Nutr.

    (2002)
  • M. Ishida et al.

    Hydroxylation of 25-hydroxyvitamin D3 by renal mitochondria from rats of different ages

    Endocrinology

    (1987)
  • T. Nesbitt et al.

    Insulin-like growth factor-I regulation of renal 25-hydroxyvitamin D-1-hydroxylase activity

    Endocrinology

    (1993)
  • D.K. Panda et al.

    25-Hydroxyvitamin D 1 alpha-hydroxylase: structure of the mouse gene, chromosomal assignment, and developmental expression

    J. Bone Miner. Res.

    (2001)
There are more references available in the full text version of this article.

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Presented at the 12th Workshop on Vitamin D (Maastricht, The Netherlands, 6–10 July 2003).

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