Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.
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.
Deroanne
Christophe F
CF
Laboratory of Connective Tissues Biology, CBIG/GIGA Research Center, University of Liège, Tour de Pathologie, B23/3, B-4000 Sart Tilman, Belgium. c.deroanne@ulg.ac.be
Hamelryckx
Delphine
D
Ho
T T Giang
TT
Lambert
Charles A
CA
Catroux
Philippe
P
Lapière
Charles M
CM
Nusgens
Betty V
BV
D015153:Blotting, Western; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000378:metabolism; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis*; D008858:Microscopy, Phase-Contrast; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D004791:Enzyme Inhibitors; D016209:Interleukin-8; D034741:RNA, Small Interfering; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D020781:Matrix Metalloproteinase 1; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein