Deroanne Christophe F, Hamelryckx Delphine, Ho T T Giang, Lambert Charles A, Catroux Philippe, Lapière Charles M, Nusgens Betty V
Laboratory of Connective Tissues Biology, CBIG/GIGA Research Center, University of Liège, Tour de Pathologie, B23/3, B-4000 Sart Tilman, Belgium.
J Cell Sci. 2005 Mar 15;118(Pt 6):1173-83. doi: 10.1242/jcs.01707. Epub 2005 Feb 22.
The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity.
Rho家族的小GTP酶是由活化的细胞粘附受体触发的细胞信号传导中的关键中间体。在本研究中，我们利用RNA干扰（RNAi），使用小干扰RNA（siRNA）来确定RhoGTP酶家族中最具特征的成员RhoA、Rac1和Cdc42在正常人皮肤成纤维细胞（HSF）中对MMP-1、MMP-2和I型胶原蛋白表达的控制作用。通过特异性siRNA的瞬时转染，在转染后长达7天实现了对三种GTP酶的特异性和持久抑制。Cdc42的沉默而非RhoA或Rac1的沉默导致MMP-1分泌增加15倍。这种上调在mRNA水平得到证实，并且在靶向Cdc42的两种不同siRNA中均观察到。在各种人类细胞系中也观察到了这种调节，并且通过重新表达由携带沉默突变的构建体编码的野生型Cdc42得以挽救，该沉默突变阻止了其被siRNA识别。相比之下，MMP-2和I型胶原蛋白的表达不受每个Rho GTP酶单独沉默的影响。细胞因子蛋白阵列、酶联免疫吸附测定和逆转录PCR测量显示，Cdc42的缺失诱导白细胞介素8和MCP-1的过表达。尽管已知这些细胞因子可诱导MMP-1的表达，但我们表明它们不参与Cdc42介导的MMP-1上调。Cdc42的沉默还诱导ERK1/2和p38丝裂原活化蛋白激酶的磷酸化增加。在Cdc42缺失的细胞上使用化学抑制剂表明，MMP-1的上调依赖于ERK1/2途径，而p38丝裂原活化蛋白激酶途径发挥抑制作用。同时敲低两种或三种Rho GTP酶使我们能够证明RhoA-ROCK途径不参与这种调节，但Rac1的沉默降低了Cdc42抑制的效果。这些数据表明，在体内，当通过整合素的细胞/细胞外基质相互作用诱导细胞骨架组织时，Cdc42通过抑制Rac1和ERK1/2途径将MMP-1表达维持在低水平。因此，Cdc42有助于细胞外基质稳态和结缔组织完整性。
