Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C19 and PC19
Expression of 25-hydroxyvitamin D3-1??-hydroxylase and functional vitamin D signalling in human vascular smooth muscle cells
M. A. Lubczanska1, D. Zehnder1, R. Bland1
1. The Clinical Science Research Laboratories, Warwick Medical School, The University of Warwick, Coventry, United Kingdom.
Progressive medial arterial calcification is a major cause of cardiovascular mortality in patients with chronic kidney disease. It is known that vascular smooth muscle cells (VSMC) play a key role in this process. They are capable of phenotypic transformation into osteoblast-like cells and they promote the deposition of calcium. Recent studies have suggested a protective role for vitamin D. The exact mechanism for this process is unclear and vitamin D may have several actions. Some of these may be due to circulating levels of 1,25-dihydroxvitamin D3 (1,25D3), but it is also apparent that local synthesis of 1,25D3 is important, where it may act in a paracrine/autocrine manner. Synthesis of 1,25D3 from the major circulating form, 25 hydroxyvitamin D3 (25D3), is catalysed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase, CYP27B1). 1,25D3 is metabolised by 24-hydroxylase (24-OHase, CYP24A1). This study investigated the expression and regulation of 1α-OHase, 24-OHase and the vitamin D receptor (VDR) in arterial tissue from healthy patients and primary cultures of human aortic smooth muscle cells (HAoSMC). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison tests; data are presented as mean ± S.E.M. (n=3). RT-PCR analyses demonstrated that HAoSMC expressed 1α-OHase and 24-OHase mRNA. The transcripts were similar to that seen in HKC-8 cells (a kidney cell line, which expresses both hydroxylases). VDR mRNA is also expressed. Western blot analyses identified single protein bands in HAoSMC and normal arteries demonstrating expression of both 1α-OHase and 24-OHase. The bands correspond to the size of the 1α-OHase and 24-OHase proteins (approximately 56kDa) and were identical to those seen in HKC-8 cells and human kidney tissue. VDR protein was detected as a double band of approximately 54 and 56kDa in all samples. These results were confirmed by immunohistochemistry of arteries. 24-OHase mRNA was significantly increased by 1,25D3 (10nM; 6 hours; p<0.05. Conversely, 1α-OHase protein was significantly decreased by 1,25D3 (1nM; 6 hours; p<0.05). Importantly 24-OHase mRNA was also increased and 1α-OHase protein decreased by 25D3 (100nM & 10nM respectively; 6 hours; p<0.05) indicating 1α-OHase activity in the HAoSMC. This data clearly demonstrates that arteries and VSMC express 1α-OHase and functional vitamin D signalling. We believe we are the first to demonstrate the presence and localization of 24-OHase in human arteries and VSMC. We have also shown that VSMC are able to synthesize sufficient 1,25D3 to stimulate a local response. Therefore we would postulate that alterations in the local production and metabolism of 1,25D3 modulates the function of VSMC and may play a role in the development and progression of the vascular calcification seen in kidney disease.
Where applicable, experiments conform with Society ethical requirements