Abstract 984: Bone Morphogenetic Protein 2 Induces an Osteoblastic Phenotype in Vascular Smooth Muscle Cells by Activation of Bone Morphogenetic Protein Receptor 2 and Smad Signaling
Bone morphogenetic proteins (BMPs) mediate vascular calcification. In vascular smooth muscle cells (VSMC), BMP2 enhances expression of the osteoblastic transcription factor, core binding factor α1 (Cbfa1). Expression of Cbfa1, in turn, promotes transition to an osteoblastic phenotype. We hypothesized that BMP2 modulates this effect by binding to BMP receptor-2 (BMPR2) to activate Smad signaling, which, in turn, upregulates expression of the osteoblastic transcription factors Cbfa1 and osterix. To examine this hypothesis, VSMCs were transfected with siRNA to BMPR2 to decrease expression of the receptor by 68.8%, and treated with recombinant human BMP2 (20 –200 ng/ml) for up to 7 days. After 3 days of BMP2 treatment, compared to control VSMC, phosphorylation of Smad-1 was diminished significantly in BMPR2-deficient VSMC (201.0 ± 34.9 vs. 54.0 ± 21.6 arb. units, p<0.01), while expression of total Smad1 was unaffected (25.7 ± 0.7 vs. 25.2 ± 1.6 arb. units, p=NS) as determined by Western blotting. Decreased Smad activation was associated with abrogated BMP2-induced Cbfa1 expression in BMPR2-deficient VSMC as compared to control cells (153.0 ± 14.4 vs. 72.5 ± 5.3 arb. units, p<0.001). We next examined the effect of BMP2 on the transcription factor osterix, which may be necessary for Cbfa1-mediated induction of alkaline phosphatase (ALP) expression, a marker of an osteoblastic phenotype. After 5 days of BMP2 treatment, osterix mRNA levels were increased (1.0 ± 0.25 vs. 23.5 ± 3.5 arb. units, p<0.001) compared to untreated cells as determined by quantitative real-time RT-PCR. Compared to untreated VSMC, this effect resulted in increased ALP expression in BMP2-treated cells after 7 days (7.1 ± 0.3 vs.106.0 ± 4.9 arb. units, p<0.0001). To demonstrate the critical role of osterix in ALP expression, we examined ALP at day 3, when expression of Cbfa1 - but not osterix - has been induced; at day 3, there was no increase in ALP expression in BMP2-treated VSMC. These data demonstrate that BMPR2-mediated activation of Smad signaling and osterix expression are crucial for osteoblastic differentiation of VSMC. This novel elucidation of the BMP2 signaling pathway in VSMC may provide new insight into the pathobiological mechanism of vascular calcification.