Tumor necrosis factor-α-induced activation of RhoA in airway smooth muscle cells:: Role in the Ca2+ sensitization of myosin light chain20 phosphorylation
Tumor necrosis factor-α-induced activation of RhoA in airway smooth muscle cells:: Role in the Ca2+ sensitization of myosin light chain20 phosphorylation
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DOI：
10.1124/mol.63.3.714
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发表时间：
2003-03-01
影响因子：
3.6
通讯作者：
Nixon, GF
中科院分区：
文献类型：
--
作者：
Hunter, I;Cobban, HJ;Nixon, GF
肿瘤坏死因子-α (TNF) 是一种炎症细胞因子，在支气管哮喘的发病机制中具有潜在的重要作用，并可能导致气道高反应性。最近的证据表明，TNF 可以增加豚鼠气道平滑肌 (ASM) 激动剂刺激的肌球蛋白轻链 (20) (MLC20) 磷酸化的 Ca2+ 敏感性和收缩性。在本研究中，研究了导致 TNF 诱导的 Ca2+ 敏化的潜在细胞内途径。在透化培养的豚鼠 ASM 细胞中，重组人 TNF 在 Ca2+“钳位”条件下刺激 Ca2+ 激活的 MLC20 磷酸化增加。与 Rho 激酶抑制剂 Y27632 预孵育可抑制 MLC20 磷酸化的增加。 TNF 还增加了 GTP 结合的 RhoA 的比例（使用 rhotekin Rho 结合结构域测量），其时间过程与 TNF 诱导的 Ca2+ 敏化作用相一致。在培养的人 ASM 细胞中，重组人 TNF 也以相似的时间过程激活 RhoA。此外，TNF 刺激肌球蛋白磷酸酶调节亚基的磷酸化，而 Y27632 抑制该磷酸化。尽管人 ASM 细胞表达两种受体亚型 TNF-R1 和 TNF-R2，但 RhoA 的激活主要是通过刺激 TNF-R1，尽管 RhoA 不与 TNF-R1 发生免疫沉淀。总之，TNF 诱导的 MLC20 磷酸化的 Ca2+ 敏感性增加是通过刺激 TNF-R1 受体并通过 RhoA/Rho 激酶途径导致肌球蛋白轻链磷酸酶的抑制。这种细胞内机制可能导致 TNF 诱导的气道高反应性。
Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca2+ sensitivity of agonist-stimulated myosin light chain(20) (MLC20) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca2+ sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca2+ activated MLC20 phosphorylation under Ca2+ "clamp" conditions. This increased MLC20 phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca2+ sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca2+ sensitivity of MLC20 phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.