Rombouts Krista, Lottini Benedetta, Caligiuri Alessandra, Liotta Francesco, Mello Tommaso, Carloni Vinicio, Marra Fabio, Pinzani Massimo
Department of Internal Medicine, University of Florence, Italy.
Exp Cell Res. 2008 Apr 15;314(7):1444-54. doi: 10.1016/j.yexcr.2008.01.029. Epub 2008 Feb 12.
Myristoylated alanine-rich protein kinase c substrate (MARCKS) has been suggested to be implicated in cell adhesion, secretion, motility and mitogenesis through regulation of the actin cytoskeletal structure. In the present study, a possible link between MARCKS and the platelet-derived growth factor (PDGF) signaling pathway was investigated in activated human hepatic stellate cells (hHSC), critical regulators of hepatic fibrogenesis. PDGF-BB stimulation resulted in a bi-directional movement of MARCKS that coincided with the phosphorylation of MARCKS and the activation of both PKCepsilon and PKCalpha. Biochemical inhibition of PKC kinase activity and small interfering RNA (siRNA) against PKCepsilon demonstrated that PKCepsilon is indispensable for PDGF-BB-induced MARCKS phosphorylation and cell migration. Immunoprecipitation studies revealed an association between MARCKS and the PDGFbeta-receptor, while the PDGFbeta-receptor and PKCalpha associated with focal adhesion kinase (FAK). Transient transfection with MARCKS DNA plasmid remarkably reduced PDGF-BB stimulated cell motility. In contrast, siRNA against MARCKS increased cell migration in RNAi treated cells in comparison to the scrambled control cells. In conclusion, the present study indicates that MARCKS play a major key role in PDGF-BB-induced chemotaxis in activated hHSC.
豆蔻酰化富含丙氨酸的蛋白激酶C底物(MARCKS)已被认为通过调节肌动蛋白细胞骨架结构参与细胞黏附、分泌、运动和有丝分裂。在本研究中,在活化的人肝星状细胞(hHSC)(肝纤维化的关键调节因子)中研究了MARCKS与血小板衍生生长因子(PDGF)信号通路之间的可能联系。PDGF-BB刺激导致MARCKS的双向移动,这与MARCKS的磷酸化以及PKCε和PKCα的激活同时发生。PKC激酶活性的生化抑制和针对PKCε的小干扰RNA(siRNA)表明,PKCε对于PDGF-BB诱导的MARCKS磷酸化和细胞迁移是必不可少的。免疫沉淀研究揭示了MARCKS与PDGFβ受体之间的关联,而PDGFβ受体和PKCα与粘着斑激酶(FAK)相关。用MARCKS DNA质粒进行瞬时转染显著降低了PDGF-BB刺激的细胞运动性。相反,与乱序对照细胞相比,针对MARCKS的siRNA增加了RNAi处理细胞中的细胞迁移。总之,本研究表明MARCKS在活化的hHSC中PDGF-BB诱导的趋化作用中起主要关键作用。