Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway

Abstract

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 longlasting 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 wildtype 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.

RhoA-GDP Regulates RhoB Protein Stability

“…For the 28 S rRNA, the efficiency of the RT-PCR was controlled by a synthetic RNA co-transcribed and co-amplified with the same primers as the endogenous RNA to yield an amplification product of slightly larger size. The RT-PCR conditions were described elsewhere (18). Briefly, 10 ng of total RNA and a known copy number of the standard synthetic RNA were reverse transcribed (70°C for 15 min).…”

“…This technology that recently allowed us to highlight the regulation operated by Cdc42 on matrix metalloprotease-1 (18) has several advantages as compared with classical methods. Beside a higher specificity, siRNA suppresses both GDP-and GTP-bound forms allowing to better evaluate the contribution of either form in the global function of individual RhoGTPase.…”

Pituicyte Stellation is Prevented by RhoA-or Cdc42-Dependent Actin Polymerization

“…RhoA and Cdc42 siRNAs (for sequences see Deroanne et al 2005, the "first" siCdc42 sequence was used in the present study) were purchased from Eurogentec (Seraing, Belgium) and transfected into cells grown in regular (10%) serum medium using a Ca 2+ phosphate protocol as described previously (Vouret-Craviari et al 2004). Cells were analyzed 48 h after the second transfection.…”

Anti-tumor activity of Gastrodia elata Blume is closely associated with a GTP-Ras-dependent pathway

“…RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR) were carried out, as described previously, with slight modifications (12)(13)(14)(15). Briefly, the total cellular RNA was extracted from B16 cells using the TRIzol reagent (Gibco BRL, Grand Island, NY).…”