Fig. 2. MT1-MMP promotes PDGFRβ- and PDGF-BB-dependent suppression of VSMC contractile proteins. (A) Quantitative assessment of SMA and calponin mRNA expression by real-time PCR. mRNA was isolated from subconfluent tenth-passage VSMCs cultured with the wide-spectrum MMP inhibitor BB-94 (10 µM), PDGFRβ kinase inhibitor AG1296 (10 µM) and EGFR tyrosine-kinase inhibitor AG1478 (10 µM) as indicated. The expression data are normalized against mouse TBP and presented relative to the mRNA expression in mock (0.1% DMSO)-treated wild-type cells (mean ±1 s.d., n=3). (B) Early-passage wild-type (MT1^+/+) and MT1-MMP^–/– VSMCs were cultured on polymerized collagen I in the presence of TIMP2 (4 µg/ml) for 48 hours as indicated, and exposed to PDGF-BB (25 ng/ml) under serum-free conditions. After 48 hours, the cells were lysed and the lysates analyzed by immunoblotting for the relative levels of calponin, SMA and vimentin. β-tubulin served as a loading control. Mean values of the ratios between normalized SMA and vimentin protein levels are presented below each lane (n=3). Relative mobilities of the molecular-mass markers are indicated in kDa. (C) Calponin expression was assessed by immunofluorescence staining of cells exposed to PDGF-BB as above. Filamentous actin was visualized with phalloidin (F-actin) and nuclei with DAPI. (D) MT1-MMP^–/– VSMCs were transfected with EGFP expression-vector alone (Mock), or with either expression construct for wild-type MT1-MMP (MT1-MMP) or the catalytically inactive mutant (MT1-E240A) as indicated. Calponin expression (red) was assessed by immunofluorescence after 48 hours of treatment with PDGF-BB.