With DM Gene CYP24A1 (3d) Dkk1 (7d) HHIP (7d) TGFB2 (3d) DSP (7d) Wnt5A (3d) CLEC3B (7d) THBS1 (7d) Gene NLRP1 (3d) GIP3 (7d) COL22A1 (3d) MAPK13 (3d) SLC22A3 (3d) GCLC (3d) TABLE II. Calcitriol and Paricalcitol Signiﬁcantly Modulated Genes Involved in Mouse Cardiovascular and Skeletal Function Compared Calcitriol fold change Paricalcitol fold change R11.1 þ1.4 R1.3 R1.8 R1.3 1.3 1.7 1.6 R5.5 þ1.3 R1.2 R1.5 R1.2 1.3 1.4 1.1 Skeletal disorder Abnormal intramembranous bone formation Fused vertebrae, postaxial polysyndactyly Controls hedgehog proteins in cartilage; Over-expression leads to short skeleton like Ihh knockout Craniofacial chondrogenesis/defects in bones Chondrocyte differentiation defect; lack of limb outgrowth Spinal deformity Cardiovascular disorder Cardiovascular defects Lethal heart defects, ventricular dysfunction, myocyte apoptosis, larger ventricles Congenital heart disease; truncus arteriosus Thrombus in injured vessel Affymetrix probe set numbers can be found in Supplemental data, Tables I and II. CYP24A1, cytochrome P450, family 24, subfamily A; Dkk1, Dickkopf-related protein 1; HHIP, hedgehog interacting protein; TGFB2, transforming growth factor beta 2; DSP, desmoplakin; Wnt5A, wingless-type MMTV site family member 5A; CLEC3B, C-type lectin domain family, member B; THBS1, thombospondin. Mouse phenotypes are described for genes, some of which lack direct human disease data. Values are for the measurement of maximal change (4 h, 1, 3, or 7 days) for calcitriol (10 8 M) þ DM as compared to DM. Bold: P < 0.001: italics: P < 0.05. target tissues in response to vitamin D [Kato et al., 2007]. CYP24A1 mRNA showed an early response (by 4 h), and was the most calcitriol þ DM, paricalcitol þ DM, and R-568 þ DM. The protein expression patterns for HGF and TGFb2 were consistent with those dramatically changed of all the transcripts (Table I and Supple- identiﬁed by the microarray analysis (Tables I and II). mental Table I). Other genes signiﬁcantly regulated by 4 h were the potassium channel subfamily K, member 3 (KCNK3), the antiox- idant, glutamate-cysteine ligase, catalytic subunit (GCLC) and thrombomodulin (THBD). GENE EXPRESSION CONFIRMATION BY BDNA ANALYSIS AND PROTEIN EXPRESSION To validate the gene expression changes detected by in the above analyses, bDNA analysis was performed for a subset of identiﬁed genes to include genes believed to be involved in calciﬁcation that were upregulated, downregulated, and unchanged by exposure to vitamin D sterols in DM. The upregulated gene set included ALPL and tumor necrosis factor (receptor) superfamily member 11B (TNFRSF11B, also known as osteoprotegerin, OPG). The down- regulated gene set included secreted frizzled related protein 3 (FRZB), MGP and ectonucleotide pyrophosphatase/phosphodiester- ase 1 (ENPP1). The unchanged gene set included ﬁbrillin 1 (FBN1) and Runx2. The expression patterns determined by bDNA analysis were similar to those identiﬁed by the microarray analysis (Table IV, VITAMIN D RESPONSE ELEMENT ANALYSIS To identify possible mechanism of transcriptional regulation of genes associated with CASMC calciﬁcation we sought to locate putative VDREs in the promoters of regulated genes and to determine the VDR expression of CASMC. Promoter analyses demonstrated that the promoters of all of the regulated genes identiﬁed contained at least one putative VDRE (Supplemental Tables I and II). RNase protection assays with total RNA isolated from culture expanded primary CASMC conﬁrmed the presence of VDR mRNA in CASMCs (Fig. 4A). In contrast, we were unable to identify CaSR transcripts in CASMC (Fig. 4B). Both VDR and CaSR transcripts were present in RNA isolated from kidney (positive control), but not yeast (negative control) (Fig. 4). The absence of CaSR mRNA was consistent with lack of CASMC response to calcimimetic R-568. DISCUSSION Supplemental Tables I–III). Of note Runx2 expression was not In the present study, we tested the hypothesis that enhanced CASMC altered by AA þ DEX or DM, as seen with the microarray analysis. In addition, protein expression at day 7 was determined for TGFb2 calciﬁcation is associated with altered expression of genes under transcriptional regulation by vitamin D sterols. Consistent with this (upregulated) and HGF (downregulated) (Fig. 3) after exposure to hypothesis, mineral deposition by CASMC in the presence of TABLE III. Other Calcitriol (10 8 M) þ DM and Paricalcitol (10 8 M) þ DM Signiﬁcantly Modulated Genes Compared With DM Calcitriol fold change Paricalcitol fold change Affymetrix probe set numbers can be found in Supplemental data, Tables I and II. NLRP1, NACHT-leucine-rich-repeat and PYD-(pyrin-domain)-containing-1; GIP3, interferon, alpha-inducible protein; COL22A1, collagen type 22, alpha 1; MAPK13, mitogen-activated-protein-kinase 13; SLC22A3, solute-carrier-family-22; GCLC, glutamate-cysteine ligase, catalytic subunit. Values are at the measurement of maximal change (4 h, 1, 3, or 7 days) for calcitriol (10 8 M) þ DM. Bold: P < 0.001. JOURNAL OF CELLULAR BIOCHEMISTRY VITAMIN D STEROLS AFFECT CORONARY ASMC GENES 917 R1.4 1.8 R1.4 R1.3 R2.1 R1.4 R1.2 1.7 R1.2 R1.1 R1.8 R1.2 Gene function family Apoptosis Cytokine Extracellular matrix Cell cycle/signal transduction Cationic channel/transporter Anti-oxidant