Mauro Annunziata, Ciccarelli Carmela, De Cesaris Paola, Scoglio Arianna, Bouché Marina, Molinaro Mario, Aquino Angelo, Zani Bianca Maria
Department of Experimental Medicine, University of L'Aquila, Via Vetoio, Coppito II, 67100 L'Aquila, Italy.
J Cell Sci. 2002 Sep 15;115(Pt 18):3587-99. doi: 10.1242/jcs.00037.
We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.
我们之前曾提出，蛋白激酶Cα（PKCα）在12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）介导的人胚胎横纹肌肉瘤细胞（RD）生长停滞和肌源性分化中发挥作用。在此，通过监测TPA触发的信号通路，我们证明PKCα通过诱导c - Jun氨基末端蛋白激酶（JNKs）的瞬时激活以及p38激酶和细胞外信号调节激酶（ERKs）（均称为丝裂原活化蛋白激酶，MAPKs）的持续激活来介导这些效应。组成型活性PKCα异位表达后MAPKs的激活也得到了证实，但其显性负性形式则未激活。我们通过监测MAPK通路的激活，以及使用MEK1/2抑制剂（UO126）、p38抑制剂（SB203580）和JNK及p38激动剂（茴香霉素）处理来剖析MAPK通路，研究了MAPKs对生长停滞和肌源性分化的选择性贡献。生长停滞信号要么由JNK的瞬时和持续激活（分别由TPA和茴香霉素触发）引发，要么通过同时抑制ERK和JNK的激活（UO126）引发，并且由p38维持而非诱导。因此，我们认为JNK在控制ERK介导的有丝分裂活性中起关键作用。值得注意的是，肌节肌球蛋白的表达由TPA和UO126诱导，但被p38抑制剂消除。这一发现表明p38在控制肌源性程序中起关键作用。茴香霉素持续激活p38和JNKs，但阻止TPA诱导的肌球蛋白表达。与此负性作用一致，在UO126预处理的细胞中，茴香霉素对JNKs的再激活也阻止了肌球蛋白表达。这表明，与TPA介导的肌源性过程中发生的瞬时JNK激活不同，持久的JNK激活支持生长停滞状态，但拮抗p38介导的肌球蛋白表达。最后，我们使用MEK抑制剂的结果表明ERK通路在调节分化的RD细胞中与肌源性相关的形态方面起关键作用。
