HYDROGEN-PEROXIDE STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE IN BOVINE TRACHEAL MYOCYTES - IMPLICATIONS FOR HUMAN AIRWAY DISEASE
HYDROGEN-PEROXIDE STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE IN BOVINE TRACHEAL MYOCYTES - IMPLICATIONS FOR HUMAN AIRWAY DISEASE
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DOI：
10.1165/ajrcmb.11.5.7946386
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发表时间：
1994-11-01
影响因子：
6.4
通讯作者：
HERSHENSON, MB
中科院分区：
文献类型：
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作者：
ABE, MK;CHAO, TSO;HERSHENSON, MB
我们已经表明，高氧暴露的未成年大鼠诱导气道平滑肌层增厚和细胞周转平行于发现在气道支气管肺发育不良和慢性，严重哮喘患者。我们假设活性氧通过直接刺激调节细胞生长的信号转导通路促进气道重塑。为了在培养的细胞中验证这一假设，我们评估了过氧化氢（H2 O2）对牛气管肌细胞中丝裂原活化蛋白（MAP）激酶激活的影响。MAP激酶是一个40至46 kD的胞质丝氨酸/苏氨酸激酶家族，其参与将促有丝分裂信号转导至细胞核。将静止细胞暴露于H2 O2（25至200 μ m; 2至60分钟），之后进行细胞提取物的SDS-PAGE。使用抗MAP激酶抗血清的Western分析显示，H2 O2暴露5至30分钟后，42和44 kD MAP激酶条带的流动性降低，反映了酶活性所需的苏氨酸和酪氨酸残基的磷酸化。MAP激酶激活，激酶复性试验表明，这表明几乎4倍增加42和44 kD MAP激酶活性。下调蛋白激酶C（PKC）与佛波醇12，13-二丁酸酯（PDBu）部分降低H2 O2刺激的MAP激酶活性，表明H2 O2诱导MAP激酶激活通过PKC依赖和PKC非依赖途径。Western分析表明，H2 O2暴露后，分子量约为72和125 kD的蛋白质的酪氨酸磷酸化增加，表明H2 O2可以刺激多种胞质蛋白的酪氨酸磷酸化，包括MAP激酶。H2 O2对MAP激酶的激活表明活性氧中间体可以激活调节细胞增殖的信号转导途径中不可或缺的组分。这些发现可能对人类气道疾病具有重要意义，其中异常气道平滑肌增殖可能起作用。
We have shown that hyperoxic exposure of immature rats induces airway smooth muscle layer thickening and cell turnover parallel to that found in the airways of patients with bronchopulmonary dysplasia and chronic, severe asthma. We hypothesized that reactive oxygen species could promote the observed airway remodeling by directly stimulating signal transduction pathways that regulate cell growth. To test this hypothesis in cultured cells, we assessed the effects of hydrogen peroxide (H2O2) On mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes. The MAP kinases are a family of 40 to 46 kD cytosolic serine/threonine kinases that participate in the transduction of mitogenic signals to the cell nucleus. Quiescent cells were exposed to H2O2 (25 to 200 mu m; 2 to 60 min), after which SDS-PAGE of cell extracts was performed. Western analysis using an anti-MAP kinase antiserum revealed a decrease in the mobility of the 42 and 44 kD MAP kinase bands after H2O2 exposures of 5 to 30 min, reflecting the phosphorylation at threonine and tyrosine residues required for enzymatic activity. MAP kinase activation was demonstrated by kinase renaturation assays, which showed an almost 4-fold increase in 42 and 44 kD MAP kinase activity. Down-regulation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) partially reduced H2O2-stimulated MAP kinase activity, suggesting that H2O2 induces MAP kinase activation via both PKC-dependent and PKC-independent pathways. Western analysis using a phos- photyrosine monoclonal antibody revealed increased tyrosine phosphorylation of proteins with approximate molecular weights of 72 and 125 kD after H2O2 exposure, demonstrating that H2O2 can stimulate the tyrosine phosphorylation of multiple cytosolic proteins, including MAP kinase. The activation of MAP kinase by H2O2 indicates that reactive oxygen intermediates can activate components integral to a signal transduction pathway that regulates cell proliferation. These findings may hold significance for human airway diseases in which abnormal airway smooth muscle proliferation may play a role.