Abstract 12655: Smad Signaling Activates NADPH Oxidase to Increase Endoplasmic Reticulum Stress and Promote Calcification of Bone Morphogenetic Protein-2-Stimulated Human Coronary Artery Smooth Muscle Cells
Vascular smooth muscle cells exposed to bone morphogenetic protein-2 (BMP-2) or oxidant stress demonstrate increased Runx2 expression, transition to an osteoblast-like phenotype, and calcification; however, the mechanistic link between BMP-2 and oxidant stress remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with BMP-2 (0 – 200 ng/ml) and after 21 d confirmed calcification by Von Kossa staining, with a maximal effect observed with 100 ng/ml BMP−2. BMP-2 (100 ng/ml) increased the expression of Runx2, osterix, and alkaline phosphatase, consistent with HCSMC phenotype transition. In BMP-2-treated HCSMC, there was an increase in p47phox phosphorylation, NADPH oxidase activity, and reactive oxygen species levels after 15 min (1,316.2 ± 89.9 vs. 2,191.2 ± 182.1 arb. fluor. units, p< 0.01). This response was mediated by the bone morphogenetic protein receptor-2 (BMPR2) and Smad 1/5/8-Smad 4 signaling as cells transfected with an siRNA to BMPR2 or Smad 4 demonstrated decreased NADPH oxidase activity and Runx2 expression compared to control-transfected cells. To determine how Smad signaling activated NADPH oxidase, we examined the role of protein kinase G (PKG) in p47phox phosphorylation as PKG is a known binding partner of the activated Smad complex. In BMP-2-treated HCSMC, the PKG inhibitor KT5823 decreased phosphorylated p47phox by 90% (p<0.01) and NADPH oxidase activity by 82% (p<0.01). BMP-2-mediated oxidant stress increased Runx2 expression through a mechanism involving endoplasmic reticulum (ER) stress. BMP-2 increased the expression of GRP78, phospho-IRE1α, and the transcription factor XBP1; these effects were abrogated in cells transfected with siRNA to BMPR2 or p47phox. Analysis of a 1 kb segment of the Runx2 promoter revealed a single XBP1 binding site (−596 – −591); electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that XBP1 bound to the Runx2 promoter at this site. These data demonstrate that BMP-2 activates Smad signaling leading to PKG-mediated phosphorylation of p47phox, NADPH oxidase activation, and increased oxidant stress. This mechanism, in turn, increases ER stress, activates XBP1, and increases Runx2 expression to promote vascular calcification.
- © 2010 by American Heart Association, Inc.