Abstract 9842: Bone Morphogenetic Protein-2 Decreases MicroRNA 30b-c to Promote Vascular Smooth Muscle Cell Calcification

Abstract

Bone morphogenetic protein-2 (BMP2) initiates vascular smooth muscle cell (SMC) calcification by upregulating expression of the osteoblast transcription factor Runx2. MicroRNAs (miRs) are negative regulators of gene expression; however, the role of miRs in regulating Runx2 expression remains unknown. Accordingly, we treated human coronary artery SMC with BMP-2 (100 ng/ml), and confirmed a 13-fold (p<0.01) increase in Runx2 mRNA levels at 4 h and a 1.7-fold (p<0.02) increase in protein at 24 h. A miR microarray revealed that BMP2 decreased miR-30 family expression 5.1-fold (p<0.01), and real-time PCR verified this finding: BMP2 decreased expression of miR-30b by 4.4-fold (p<0.01) and miR-30c by 3.4-fold (p<0.01). Downregulation of miR-30b-c by transfection of SMC with anti-miR-30b-c increased Runx2 protein by 1.9-fold (p<0.01) or 3.0-fold (p<0.01), respectively. By contrast, forced expression of miR-30b-c by transfection with pre-miR-30b-c attenuated BMP2-induced expression of Runx2 (1 ± 0 vs. 0.6 ± 0.3 vs. 0.5 ± 0.2 arb units, p <0.02). Next, we cotransfected COS7 cells with a luciferase reporter plasmid containing the Runx2 3' untranslated region (UTR) and pre-miR-30b-c. In this assay system, forced expression of miR-30b-c decreased luminosity (1.0 ± 0 vs. 0.6 ± 0.1 vs. 0.6 ± 0.1 fl. units, p<0.04) indicating that miR-30b-c bind to the Runx2 3'-UTR to modulate expression. Downregulation of miR-30b-c also increased expression of the Runx2-dependent calcification protein osteocalcin (1.0 ± 0 vs. 1.7 ± 0.5 vs. 2.2 ± 0.4 arb. units, p<0.03). Von Kossa staining of SMC transfected with anti-miR-30b-c for 14 d revealed that SMC calcification was increased by 1.7-fold (p<0.03) or 1.8-fold (p<0.03) while forced expression of miR-30b-c prevented calcification (1.2 ± 0 vs. 0.9 ± 0.1 vs. 0.9 ± 0.1 arb units, p<0.01). In BMP2-treated SMC, downregulation of miR-30b or miR-30c increased calcification 1.7± 0.3-fold (p<0.03) or 2.3 ± 0.7-fold (p<0.03) while forced expression of miR-30b-c prevented calcification (0.9 ± 0.1 vs. 1.0 ± 0.1 vs. 1.0 ± 0.1 arb units, p<0.01). These data demonstrate that miR-30b-c regulate Runx2 expression to influence SMC mineralization, and suggest that strategies to regulate expression of these miRNA may limit vascular calcification.