Sugita Mizuki, Kuwata Hiroshi, Kudo Ichiro, Hara Shuntaro
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Biochim Biophys Acta. 2010 Jan;1801(1):70-6. doi: 10.1016/j.bbalip.2009.09.013. Epub 2009 Sep 24.
Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A(2) (sPLA(2)-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA(2)-IIA by proinflammatory cytokines was under the control of both classical cPKCalpha and atypical aPKClambda/iota pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1beta/TNFalpha-dependent induction of sPLA(2)-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA(2)-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA(2)-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA(2)-IIA expression was markedly enhanced in cPKCalpha knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKClambda/iota isoform reduced the cytokine-induced expression of sPLA(2)-IIA. These results suggest that the aPKClambda/iota pathway is required for the induction of sPLA(2)-IIA expression and that the cPKCalpha pathway acts as a negative regulator of sPLA(2)-IIA expression in cytokine-stimulated rat fibroblasts.
蛋白激酶C（PKC）是一类参与多种信号转导途径的丝氨酸/苏氨酸激酶。我们研究了PKC在细胞因子刺激的大鼠成纤维细胞3Y1中对IIA组分泌型磷脂酶A2（sPLA(2)-IIA）表达调控中的作用。在此我们表明，通过使用PKC抑制剂、PKC激活剂和PKC基因敲低技术，促炎细胞因子对sPLA(2)-IIA的诱导受经典的cPKCalpha和非典型的aPKClambda/iota途径的控制。用具有广泛特异性的PKC选择性抑制剂（如氯化白屈菜红碱和GF109203X）处理3Y1细胞，以剂量依赖的方式阻断了IL-1β/TNFα依赖性的sPLA(2)-IIA蛋白诱导。用激活cPKC和新型nPKC亚型的PKC激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理，显著减弱了细胞因子依赖性的sPLA(2)-IIA表达诱导。相比之下，用下调这些PKC亚型的PMA进行24小时预处理，显著增强了sPLA(2)-IIA表达。短发夹RNA（shRNA）介导的PKC亚型基因敲低结果显示，与复制对照细胞相比，cPKCalpha基因敲低细胞中细胞因子诱导的sPLA(2)-IIA表达显著增强。相反，aPKClambda/iota亚型的基因敲低降低了细胞因子诱导的sPLA(2)-IIA表达。这些结果表明，aPKClambda/iota途径对于sPLA(2)-IIA表达的诱导是必需的，并且cPKCalpha途径在细胞因子刺激的大鼠成纤维细胞中作为sPLA(2)-IIA表达的负调节因子发挥作用。
