Luzina Irina G, Highsmith Kendrick, Pochetuhen Kerill, Nacu Natalia, Rao Jaladanki N, Atamas Sergei P
Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Am J Respir Cell Mol Biol. 2006 Sep;35(3):298-305. doi: 10.1165/rcmb.2006-0033OC. Epub 2006 Apr 6.
A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha Gö6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.
一种CC趋化因子CCL18，此前已有报道称其可刺激肺成纤维细胞产生胶原蛋白。本研究聚焦于蛋白激酶C（PKC）在CCL18激活的肺成纤维细胞促纤维化信号传导中的作用。在成纤维细胞中主要表达的三种PKC亚型（PKCalpha、PKCdelta和PKCepsilon）中，有两种亚型（PKCdelta和PKCepsilon）参与了促纤维化的细胞内信号传导。PKCalpha介导的信号传导在胶原蛋白产生调节中的作用尚不清楚。在本研究中，发现PKCalpha主要存在于细胞质中，而PKCdelta和PKCepsilon主要存在于培养的原代肺成纤维细胞的细胞核中。用CCL18刺激后，PKCalpha而非PKCdelta或PKCepsilon发生快速（5 - 10分钟内）的瞬时磷酸化和核转位。用PKCalpha和ERK2的显性负性突变体抑制，而非PKCdelta或PKCepsilon，可消除CCL18刺激的ERK2磷酸化和胶原蛋白产生。PKCalpha的选择性药理抑制剂Gö6976也可消除CCL18对胶原蛋白产生和胶原蛋白启动子报告构建体活性的影响。用CCL18刺激成纤维细胞会导致细胞内钙浓度升高。与已知的PKCalpha信号传导对钙的依赖性一致，用细胞内钙螯合剂BAPTA阻断钙信号会导致PKCalpha核转位、ERK2磷酸化和胶原蛋白产生的消除。这些观察结果表明，在原代肺成纤维细胞中，PKCalpha而非PKCdelta或PKCepsilon介导了CCL18的促纤维化作用。因此，PKCalpha可能成为未来抗纤维化治疗的一个可行靶点。
