Komalavilas Padmini, Penn Raymond B, Flynn Charles R, Thresher Jeffrey, Lopes Luciana B, Furnish Elizabeth J, Guo Manhong, Pallero Manuel A, Murphy-Ullrich Joanne E, Brophy Colleen M
Center for Metabolic Biology, College of Liberal Arts and Sciences, Arizona State University, PO Box 873704, Tempe, AZ 85287-3704, USA.
Am J Physiol Lung Cell Mol Physiol. 2008 Jan;294(1):L69-78. doi: 10.1152/ajplung.00235.2007. Epub 2007 Nov 9.
Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.
环磷酸腺苷（cAMP）/cAMP依赖性蛋白激酶A（PKA）信号通路的激活可导致气道平滑肌（ASM）舒张。本研究旨在探讨小热休克相关蛋白HSP20在介导PKA依赖性ASM舒张中的作用。对人ASM细胞进行基因工程改造，使其组成性表达绿色荧光蛋白-PKA抑制融合蛋白（PKI-GFP）或仅表达GFP。用异丙肾上腺素（ISO）或福斯高林激活cAMP依赖性信号通路，可导致GFP细胞中HSP20磷酸化增加，但PKI-GFP细胞中未出现这种情况。福斯高林处理GFP细胞而非PKI-GFP细胞，导致中央肌动蛋白应力纤维消失，粘着斑复合物数量减少。这种应力纤维的消失与GFP细胞中肌动蛋白解聚蛋白丝切蛋白的去磷酸化有关，但PKI-GFP细胞中未出现这种情况。为证实磷酸化的HSP20在PKA诱导的ASM舒张中起作用，将完整的牛ASM条带先用5-羟色胺预收缩，然后用ISO处理。PKA信号通路的激活导致牛ASM舒张，这与HSP20的磷酸化和丝切蛋白的去磷酸化有关。最后，用具有蛋白转导结构域 的HSP20磷酸肽模拟物处理，可使预收缩的牛ASM条带部分舒张。总之，ISO诱导的HSP20磷酸化或HSP20的合成磷酸肽类似物可降低丝切蛋白的磷酸化并破坏ASM中的肌动蛋白，这表明HSP20介导ASM舒张的一种可能机制是通过调节肌动蛋白丝动力学。
