Down-regulation of calponin destabilizes actin cytoskeletal structure in A7r5 cells

Abstract

The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and expression in control and stimulated A7r5 smooth muscle cells were evaluated by Western blotting, laser scanning confocal microscopy (LSCM), and fluorescence resonance energy transfer (FRET) microscopy in cells treated with either 2 × 10−6mol/L TGF-β1 or 2 × 10−5mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and α-actin, localized in fibers in unstimulated control A7r5 smooth muscle cells, were translocated to podosomes following treatment with phorbol-12,13-dibutyrate (PDBu). Confocal colocalization imaging and FRET analysis both indicated substantial association of CaP with α-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKCα, which showed evidence of only slight association with CaP in control cells, exhibited markedly increased (293%) association in PDBu-contracted cells. Platelet-derived growth factor (PDGF)-BB down-regulated CaP to non-detectable levels, whereas transforming growth factor (TGF)-β1 up-regulated (424%) the expression of CaP without affecting the levels of α-actin or PKCα. PDGF-BB resulted in a significant loss in α-actin stress fibers (–47%) and reduced podosome formation (–69%). By comparison, TGF-β1 had no effect on stress fibers in control cells but also reduced (–70%) podosome formation. The results suggest that CaP could play a major role in the stabilization of actin stress fibers in resting cells and may contribute to podosome formation in PDBu-treated cells.