Abstract 3724: Reactive Oxygen Species Mediate Bone Morphogenetic Protein 2 Induced Calcification of Human Coronary Artery Smooth Muscle Cells by Increasing RUNX2 Expression
Bone morphogenetic proteins (BMP) mediate vascular calcification. In vascular smooth muscle cells (SMC), BMP2 induces expression of RUNX2, a key transcription factor that promotes transition to an osteoblastic phenotype; however, the mechanism by which BMP2 increases RUNX2 remains unknown. We hypothesized that BMP2 stimulates reactive oxygen species (ROS) accumulation to modulate RUNX2 expression. To examine this hypothesis, we first treated human coronary artery SMC with BMP2 (100 ng/ml) or vehicle and confirmed transition to an osteoblastic phenotype. Compared to control SMC, BMP2-treated cells demonstrated increased RUNX2 protein levels (198% control, p<0.01) after 3 days; increased alkaline phosphatase activity (253.6 ± 12.8 vs. 759.4 ± 15.7 ng/ml/mg protein, p<0.01) after 10 days; and enhanced von Kossa staining for calcification after 21 days. To demonstrate that BMP2-mediated ROS generation increased RUNX2 expression, we measured ROS by 2, 7-dichlorofluorescein fluorescence. After 24 hr, BMP2-treated SMC demonstrated elevated ROS levels (198.3 ± 12.8 vs. 866.4 ± 22.6 units, p<0.01) owing to an increase in NADPH oxidase activity (22.9 ± 8.4 vs. 108.2 ± 11.0 nmol O2−/mg protein/min, p<0.01) compared to control SMC. In BMP2-treated SMC, this was associated with increased expression of the ROS-related transcription factors Sp1 (264% control, p<0.01) and NF-KB subunits p50 (356% control, p<0.01) and p65 (288% control, p<0.01). Next, we examined a 1 kb segment of the RUNX2 promoter and identified a region that bound both Sp1 and NF-KB (−561 – −512). A mobility shift assay revealed that Sp1 and NF-KB protein-DNA binding occurred at this site in BMP2-treated cells. To confirm that increased RUNX2 expression is ROS-dependent, SMC were treated with apocynin (30 μmol/L) to inhibit NADPH oxidase activity. Compared to BMP2-treated SMC, cells treated with apocynin and exposed to BMP2 demonstrated diminished ROS formation (35% control p<0.01) and decreased expression of RUNX2 mRNA (98% control, p=NS) and protein (102% control, p=NS). These findings demonstrate that BMP2-mediated calcification of SMC is dependent upon increased ROS levels to induce RUNX2 expression and suggest a novel therapeutic target to limit this pathophysiological process.