Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation.
Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation.
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DOI：
10.1152/ajpcell.00188.2002
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发表时间：
2003-03
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影响因子：
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通讯作者：
H. Nobe;K. Nobe;F. Fazal;P. de Lanerolle;R. Paul
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作者：
H. Nobe;K. Nobe;F. Fazal;P. de Lanerolle;R. Paul
成纤维细胞在含有天然1型胶原的培养基中生长时形成纤维。所产生的收缩力可以被精确地量化并用于分析调节成纤维细胞收缩的信号转导途径。小牛血清（30%）诱导持续收缩，伴随细胞内钙（[Ca（2+）]（i））的瞬时增加。钙调蛋白抑制剂W-7、钙/钙调蛋白依赖性蛋白激酶抑制剂KN-62和肌球蛋白轻链激酶抑制剂ML-7对收缩或[Ca（2+）]（i）反应均无影响。酪氨酸激酶抑制剂染料木黄酮和蛋白激酶C抑制剂calphostin C均对力或[Ca（2+）]（i）无显著影响。相反，Rho激酶抑制剂（R）-（+）-trans-N-（4-吡啶基）-4-（1-氨乙基）-环己烷甲酰胺（Y-27632）和HA 1077以剂量依赖性方式抑制收缩，而不影响[Ca（2+）]（i）反应。应力纤维的形成也被Y-27632抑制。令人惊讶的是，小牛血清，Y-27632，和小牛血清加Y-27632没有改变单或二磷酸化的肌球蛋白调节轻链（MRLC）相比，对照未处理的纤维。这些结果表明，小牛血清诱导的NIH 3 T3成纤维细胞纤维的持续收缩是由Rho激酶介导的，但不依赖于[Ca（2+）]（i）、钙/钙调蛋白或蛋白激酶C依赖性途径的持续增加或MRLC磷酸化的增加。
Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca(2+)](i)). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca(2+)](i) responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca(2+)](i). In contrast, the Rho kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca(2+)](i) response. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca(2+)](i), calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.