Regulation of gene expression by the CYP27B1 promoter—study of a transgenic mouse model☆
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
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Cited by (22)
Anabolic effects of vitamin D and magnesium in aging bone
2019, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :Furthermore, the synthesis of 1,25(OH)2D3 was shown to occur in regions other than liver and kidney, such as the brain and testis, as well as other tissues. This was documented using a luciferase reporter linked to the promoter of the rate-limiting enzyme in 1,25(OH)2D3 synthesis [105]. However, despite the widespread expression of enzymes involved in 1,25(OH)2D3 synthesis, most serum 1,25(OH)2D3 results from the processing of vitamin D metabolites in the kidney [106,107], and it is still unclear what role, if any, local generators of vitamin D might have in the body.
The pleiotropic effects of vitamin D in bone
2013, Journal of Steroid Biochemistry and Molecular BiologyCitation Excerpt :We and others have characterised Cyp27b1 promoter activity and Cyp27b1 mRNA expression within trabecular bone and the growth plate [13,37,53–55]. We have demonstrated the regulation of Cyp27b1 mRNA expression in osteoblasts [56,57] which is independent of renal Cyp27b1 expression [13,58–61]. Bone Cyp27b1 level declines with age in rat bone, and increases in the presence of high dietary calcium levels, and is associated with bone mineralization [12,13].
Vitamin D metabolism and biological activities
2011, Molecular and Cellular EndocrinologyBone CYP27B1 gene expression is increased with high dietary calcium and in mineralising osteoblasts
2010, Journal of Steroid Biochemistry and Molecular BiologyRegulation of the 5′-flanking region of the human CYP27B1 gene in osteoblast cells
2009, Molecular and Cellular EndocrinologyCo-expression of CYP27B1 enzyme with the 1.5 kb CYP27B1 promoter-luciferase transgene in the mouse
2008, Molecular and Cellular Endocrinology
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Presented at the 12th Workshop on Vitamin D (Maastricht, The Netherlands, 6–10 July 2003).