Fig. 4. Human CASMCs express vitamin D receptor (VDR), but not calcium sensing receptor (CaSR), mRNA by RNase protection assays. A: The 282 nt VDR probe protected a 184 nucleotide (nt) fragment. B: The 540 nt CaR probe protected a 448 nt fragment. The 138 nt cyclophilin (cyclo) probe protected a 103 nt fragment. Lane 1: Probe; lanes 2 and 3: CASMC lot number 3F0246; lane 4: CASMC lot number 00077; lane 5: yeast (negative control); lane 6: kidney (positive control). expression. Other studies reported either no effect of calcitriol or paricalcitol on phosphate induced bovine [Wolisi and Moe, 2005] and human SMC calciﬁcation [Wu-Wong et al., 2006], or calcitriol-, but not paricalcitol-, induced calciﬁcation in rat SMC [Cardus et al., 2007]. Future studies are needed to clarify if differences in experimental design or model systems underlie these conﬂicting reports. The lack of effect by R-568 observed in the current study is consistent with the lack of CaSR expression in these CASMCs and with previous reports in bovine ASMC. Although CaSR mRNA was not detected in this study, CaSR mRNA and/or protein and calcimimetic responses were demonstrated in rat and human SMC by others [Farzaneh-Far et al., 2000; Smajilovic et al., 2006; Molostvov et al., 2007; Ivanovski et al., 2009; Alam et al., 2009]. Farzaneh-Far et al. showed that calcium and CaSR agonists induced MGP/promoter reporter expression in rat aorta SMCs. However, CaSR transcripts were not detected and this response was attributed to a separate CaSR-like receptor. Smajilovic et al. demonstrated CaSR mRNA (RT-PCR) and protein (immunocytochemical) in rat aortic SMC, and showed that calcium and neomycin induced proliferation, which was not inhibited by dominant-negative CaSR silencing. This suggested that other receptors/pathways for calcium and neomycin were operative to induce proliferation. Molostvov et al. demonstrated CaSR mRNA (RT-PCR) in human aortic SMCs; and, that responses to CaSR agonists were inhibited by knock-down CaSR-siRNA methods. In addition, Molostov et al. showed that CaSR expression declined with severity of kidney disease in patient vessels. Alam et al. demonstrated CaSR protein (immunohistochem- istry) in VSMCs on human arterial sections and in human and bovine SMCs (immunoﬂuorescence or Western blot) in vitro. Alam et al. also showed that the calcimimetic R-568 caused a delay in mineralization of human SMCs cultures, and upregulated CaSR. These experiments supported an anti-mineralization protective role for SMC CaSR. Ivanovski et al. demonstrated that the calcimimetic R-568 inhibited phosphate-induced human aortic SMC mineraliza- tion after 10 days in culture medium that contained 1% serum; and, silencing with CaSR-siRNA abrogated this calcimimetic effect. In the present study, we cannot explain why the human CASMCs and, previously, bovine aorta SMCs did not respond to calcimimetic exposure, other than absence of the CaSR. It is likely that different species, donors, culture systems, and assay methods account for these divergent results for the effects of calcimimetics on SMCs. It is widely believed that VC is a highly regulated, cell-mediated, multi-factorial process associated with the expression of chondro/ osseous genes and their protein products [Jono et al., 2000; Tyson et al., 2003]. Two commonly studied osteo/chondrogenic genes, Runx2 and osteocalcin (OC), have been shown to be upregulated in human fetal SMC upon exposure to elevated phosphorus concen- trations [Jono et al., 2000]. In contrast, normal adult human SMCs often express osteo/chondrogenic genes, including osteocalcin and Runx2, spontaneously in vitro [Shanahan and Weissberg, 1999; Tyson et al., 2003]. Thus, Runx2 may be regulated differently in fetal and adult SMC [Jono et al., 2000]. In this study, we demonstrate that Runx2 and osteocalcin mRNA are expressed in our hCASMCs and their mRNA expression levels were not changed by vitamin D sterol þ DM. Further, the expression of Runx2 protein was not changed by DM or vitamin D sterols (data not shown), conﬁrming the ﬁndings in a previous report [Wu-Wong et al., 2007a]. Although the expression of these two osteo/chondrogenic genes was not changed, many other genes related to mineralization were altered in vitamin D sterol-treated CASMC resulting in a gene expression pattern indicative of a shift to a mineralizing osteoblast-like cell phenotype [Favus, 2006]. Such a shift is represented by concurrent increases in pro-mineralization gene expression (e.g., ALPL, TGFB2, BMP4 and 6) and decreases in anti-mineralization gene expression (ENPP1) along with changes in IBSP, OPG and the chondrocyte genes, ANXA3, CILP, TNC, ITGA8, and cytokine IL-6. Changes in the expression of pro- and anti-apoptotic genes are consistent with the proposed role of apoptosis as a calciﬁcation initiator in human CASMC [Reynolds et al., 2004]. In addition to these genes implicitly involved in mineralization, we also observed altered expression of genes normally expressed in heart or skeleton that are associated with cardiovascular or joint diseases including, DSP, THBS1, THBD and HGF, HGF binding protein (CLEC3B), ASPN, S100A4, SLC22A3, KCNK3, HHIP, Dkk1. This study is limited by the model system used. This study was designed to determine the gene expression changes associated with vitamin D sterol enhancement of VC induced by uremic-like conditions found in secondary HPT. This precluded the use of cells from the second donor that lacked a calciﬁcation response to high P, even though they may have calciﬁed in the presence of high P and vitamin D sterols. Therefore, these data can be considered JOURNAL OF CELLULAR BIOCHEMISTRY VITAMIN D STEROLS AFFECT CORONARY ASMC GENES 919