Dykes Ava C, Wright Gary L
The Joan C. Edwards School of Medicine, Marshall University, 1542 Spring Valley Drive, Huntington, WV 25704, USA.
Can J Physiol Pharmacol. 2007 Feb;85(2):225-32. doi: 10.1139/y07-005.
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 x 10(-6 ) mol/L TGF-beta 1 or 2 x 10(-)5 mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and alpha-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 alpha-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKC alpha, 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)-beta 1 up-regulated (424%) the expression of CaP without affecting the levels of alpha-actin or PKC alpha. PDGF-BB resulted in a significant loss in alpha-actin stress fibers (-47%) and reduced podosome formation (-69%). By comparison, TGF-beta 1 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.
在对照和佛波酯处理的A7r5平滑肌细胞中，研究了h1钙调蛋白（CaP）表达水平变化对肌动蛋白细胞骨架组织的影响。通过蛋白质印迹法、激光扫描共聚焦显微镜（LSCM）和荧光共振能量转移（FRET）显微镜，对用2×10⁻⁶ mol/L转化生长因子-β1（TGF-β1）或2×10⁻⁵ mol/L血小板衍生生长因子-BB（PDGF-BB）处理以改变h1钙调蛋白表达的对照和刺激的A7r5平滑肌细胞中的蛋白质结合和表达进行了评估。单免疫染色样本显示，在未刺激的对照A7r5平滑肌细胞中定位于纤维的CaP和α-肌动蛋白，在用佛波醇-12,13-二丁酸酯（PDBu）处理后转移至足体。共聚焦共定位成像和FRET分析均表明，CaP与对照细胞应力纤维和PDBu处理细胞足体中的α-肌动蛋白有大量结合。蛋白激酶Cα（PKCα）在对照细胞中仅显示与CaP有轻微结合的证据，而在PDBu收缩的细胞中结合明显增加（293%）。血小板衍生生长因子（PDGF）-BB将CaP下调至不可检测水平，而转化生长因子（TGF）-β1上调（424%）CaP的表达，而不影响α-肌动蛋白或PKCα的水平。PDGF-BB导致α-肌动蛋白应力纤维显著减少（-47%），足体形成减少（-69%）。相比之下，TGF-β1对对照细胞中的应力纤维没有影响，但也减少了（-70%）足体形成。结果表明，CaP可能在静息细胞中肌动蛋白应力纤维的稳定中起主要作用，并可能有助于PDBu处理细胞中足体的形成。
