Co-expression of CYP27B1 enzyme with the 1.5 kb CYP27B1 promoter-luciferase transgene in the mouse
Introduction
The hydroxylation of 25-hydroxyvitamin D3 (25D) to form the biologically active 1,25 dihydroxyvitamin D3 (1,25D) is mediated by the mitochondrial enzyme 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). CYP27B1, located to the proximal convoluted tubule cells of the kidney, is stimulated by parathyroid hormone (PTH) in response to hypocalcemia (Booth and Tsai, 1977). Circulatory levels of 1,25D have been demonstrated to arise almost exclusively from renal CYP27B1 activity (Anderson and O’Loughlin, 2004) and an increase in circulating levels of 1,25D, as result of increased renal CYP27B1 activity, directly stimulates the absorption of calcium in the duodenal intestine and is capable of restoring circulating calcium to homeostatic levels (Anderson and May, 2003). 1,25D has also been demonstrated, mainly by in vitro methods, to mediate a variety of physiological processes including cellular proliferation and differentiation. Although there has been some conjecture as to the importance of extra-renal synthesis of vitamin D, we have previously shown that the CYP27B1 substrate, 25 hydroxyvitamin D (25D), regulates bone cell activity in a CYP27B1 enzyme activity-dependent manner (Atkins and Anderson, 2007). Therefore the expression of CYP27B1 in a variety of tissues such as skin, bone, brain, mammary, colon and prostate suggests that 25D is converted to 1,25D and presumably acts in either a paracrine or autocrine fashion to regulate various physiological processes (Fu and Portale, 1997, Jones and Strugnell, 1998, Hewison and Zehnder, 2000, Panda and Al Kawas, 2001, Welsh, 2004, Kemmis and Salvador, 2006). A reduction in the locally synthesised 1,25D has been suggested to be involved in the progression of prostate, breast and colon cancers (Ma and Nonn, 2004, Matusiak and Murillo, 2005, Townsend and Banwell, 2005).
While factors such as parathyroid hormone (PTH), calcium, phosphate and 1,25D have been demonstrated to regulate the expression of CYP27B1 in the kidney (Garabedian and Holick, 1972, Fraser and Kodicek, 1973, Bland and Walker, 1999, Murayama and Takeyama, 1999, Brenza and DeLuca, 2000, Gao and Dwivedi, 2002, Armbrecht and Boltz, 2003, Armbrecht and Hodam, 2003, Hendrix and Anderson, 2005), current evidence suggest that the regulation of CYP27B1 in other tissues is independent from that in the kidney. For example in keratinocytes, PTH does not induce the expression of CYP27B1 despite its potent stimulation of CYP27B1 expression in the kidney (Flanagan and Wang, 2003). We have shown that the age-related regulation of CYP27B1 mRNA in bone cells is independent of serum PTH levels and is distinct from the CYP27B1 expression in the kidney (Anderson and O’Loughlin, 2005). While locally produced 1,25D may play a significant role in physiological and patho-physiological processes in a number of tissues, little is known about the regulatory factors that control 1,25D synthesis at these extra-renal sites.
The brain has been identified as an important extra-renal site for the expression of CYP27B1. We and others have demonstrated that the expression of CYP27B1 mRNA occurs in the rat and human brain (Fu and Lin, 1997, Panda and Al Kawas, 2001, Hendrix and Anderson, 2004). Despite this information, little is known of the regulation of CYP27B1 expression in the brain. Immunohistochemical techniques have been used to describe the cellular distribution of this enzyme and the vitamin D receptor in human Purkinje cells of the cerebella as well as within neuronal cells of the cerebral cortex (Zehnder and Bland, 2001, Eyles and Smith, 2005). In cell cultures, CYP27B1 is present within glial cells and has been demonstrated to catalyse the final hydroxylation of 25D to 1,25D (Neveu and Naveilhan, 1994). These findings are of considerable interest considering the growing body of evidence that vitamin D is involved in mammalian brain function (McGrath and Feron, 2001, Garcion and Wion-Barbot, 2002, Eyles and Brown, 2003, Becker and Eyles, 2005, Feron and Burne, 2005, McGrath and Barnett, 2005, McGrath and Burne, 2005).
In order to examine the regulation of the CYP27B1 gene expression in the kidney, brain and other extra-renal tissues in vivo, we established a transgenic mouse model to directly measure the regulation of promoter activity for the CYP27B1 gene (Hendrix and Anderson, 2004). The relatively short 5′-flanking region of the human CYP27B1 gene is 1501 bp in length and defined by the presence of the methyltransferase-like 1 (METTL1) gene located immediately upstream of this region (Bahr and Hankeln, 1999). Using this mouse model, we have demonstrated that the 1501 bp 5′-flanking region of the human CYP27B1 gene contains many if not all the regulatory elements required for the control of endogenous CYP27B1 gene expression in the kidney (Hendrix and Anderson, 2005). We now report on the specific regulation of the CYP27B1 transgene within the brain, testes and other non-renal tissues and identify novel sites for expression.
Section snippets
Transgenic animals
The pCYP27B1(-1501bp)-Luc transgenic mice were offspring of founder 2992 as described previously (Hendrix and Anderson, 2004) and were bred at the Institute of Medical and Veterinary Science (Adelaide, Australia). Three 12-week-old male mice were used to study the tissue distribution of the luciferase transgene and endogenous CYP27B1 protein expression by immunohistochemistry. The effect of age on the expression of the pCYP27B1(-1501 bp)-Luc transgene was studied by killing mice at 2, 4, 6, 8,
Results
The cellular locations of expressed luciferase protein and endogenous CYP27B1 protein were investigated by immunohistochemistry and confocal microscopy using whole tissue slices from a pCYP27B1(−1501 bp)-Luc transgenic mouse. In the kidney, the presence of both luciferase and CYP27B1 protein were detected primarily in the proximal convoluted tubular cells of the kidney (Fig. 1). When confocal images of the sections stained for luciferase and CYP27B1 enzyme were merged, the change in colour
Discussion
The co-localisation of endogenous CYP27B1 expression and the luciferase transgene in the kidney is largely restricted to proximal convoluted tubules of the kidney confirming our and other previous findings (Anderson and O’Loughlin, 2004, Hendrix and Anderson, 2004, Hendrix and Anderson, 2005). The co-localisation of the endogenous CYP27B1 with the transgene also occurred in all other tissues investigated including the specific sites within the brain and testes. This indicates that the
Acknowledgements
The authors thank Dr Martin Hewison of the Cedars-Sinai Medical Centre, Los Angeles, CA, for providing the sheep anti-mouse CYP27B1 polyclonal antibody.
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These authors are joint first authors.