Figure 2.
Defective PDGF-B initiated signaling in MT1-MMP-/- VSMCs. (A) Control or MT1-MMP-/- VSMCs were stimulated with PDGF-B for 10 min and cell extracts prepared for analysis. (Upper panel) Total lysates (25 μg/lane) of control or PDGF-B-stimulated VSMCs were analyzed by Western blotting for MT1-MMP and PDGFRβ. (Lower panel) Cell lysates (200 μg/sample) were immunoprecipitated with antibodies against PDGFRβ. For the detection of cell surface PDGFRβ, cells were incubated with Sulfo-NHS-biotin prior to immunoprecipitation. Similar levels of surface-biotinylated and autophosphorylated PDGFRβ were detected using peroxidase-conjugated streptavidin and antibodies against phospho-tyrosine residues (pY), respectively, in precipitates from control or MT1-MMP-null VSMCs. (B) PDGFRβ immunoprecipitates were prepared and separated in SDS-PAGE under nonreducing conditions. Western blotting with antibodies against MT1-MMP revealed its coprecipitation with PDGFRβ from wild-type cells. The total levels of PDGFRβ as detected with goat anti-PDGFRβ antibodies were comparable in control and MT1-MMP-null VSMCs. In a reversed experiment, PDGFRβ was detected from MT1-MMP immunoprecipitates (data not shown). (C) Growth-arrested control or MT1-MMP-/- VSMCs (cultured on type I collagen) were stimulated with PDGF-B (10 ng/mL) for 0-120 min as indicated. (Upper panel) Phosphorylated forms of AKT (pAKT) and ERK (pERK) were detected in VSMC lysates (25 μg/lane). Total ERK1/2 proteins served as loading controls. (Lower panel) Control and MT1-MMP-/- VSMCs were stained with Texas Red-conjugated phalloidin after 2 h PDGF-B treatment. Arrows highlight the areas of cortical actin ruffling in MT1-MMP+/+ VSMCs. (D) Phosphorylated forms of AKT (pAKT) and ERK (pERK) were detected from the whole cell lysates of wild-type and MT1-MMP-/- VSMCs (25 μg/lane) treated with PDGF-B (10 ng/mL), FGF-2 (10 ng/mL), or 10% FBS for 30 min. α-Tubulin served as a protein loading control. (E) Aortic medial explants from wild-type or MT1-MMP-/- mice were stimulated with PDGF-B (10 ng/mL) for 30 min. Protein extracts (50 μg/lane) were subjected to Western blotting with antibodies directed against α-tubulin or the phosphorylated forms of AKT and ERK. (F) Wild-type and MT1-MMP-/- VSMCs were growth-arrested in the absence or presence of synthetic MMP inhibitor BB-94 (5 μM) for 48 h as indicated (similar results were obtained by 24 h incubation; data not shown). Cells were then treated with PDGF-B (10 ng/mL) with or without BB-94 for 30 min as indicated. Phosphorylated forms of AKT and ERK were monitored in cell lysates.