Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway
| Autor: | Delphine Hamelryckx, Charles Lambert, Betty Nusgens, T. T. Giang Ho, Philippe Catroux, Christophe Deroanne, Charles M. Lapière |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
rac1 GTP-Binding Protein
Small interfering RNA Time Factors RHOA Blotting Western Immunoblotting Down-Regulation Enzyme-Linked Immunosorbent Assay RAC1 GTPase CDC42 Transfection p38 Mitogen-Activated Protein Kinases Culture Media Serum-Free GTP Phosphohydrolases Downregulation and upregulation RNA interference Cell Line Tumor Cell Adhesion Humans Gene silencing Microscopy Phase-Contrast Gene Silencing Enzyme Inhibitors Phosphorylation RNA Small Interfering cdc42 GTP-Binding Protein DNA Primers Skin Mitogen-Activated Protein Kinase 1 Mitogen-Activated Protein Kinase 3 biology Reverse Transcriptase Polymerase Chain Reaction Interleukin-8 Cell Biology Fibroblasts Molecular biology Up-Regulation Cell biology biology.protein RNA Interference Matrix Metalloproteinase 1 rhoA GTP-Binding Protein |
| Zdroj: | Journal of Cell Science. 118:1173-1183 |
| ISSN: | 1477-9137 0021-9533 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|