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First published online 22 February 2005
doi: 10.1242/jcs.01707

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

¹ Laboratory of Connective Tissues Biology, CBIG/GIGA Research Center, University of Liège, Tour de Pathologie, B23/3, B-4000 Sart Tilman, Belgium
² L'Oréal Research and Development, 188 rue Paul Hochart, 94152 Chevilly-Larue, France

View larger version (42K): [in a new window]   Fig. 1. Specific silencing of RhoA, Rac1 and Cdc42 mediated by transient transfection of siRNA. (A) Western-blot analysis of whole-cell lysates of HSFs transfected with calcium phosphate alone (CaP), with 20 nM siRNAs targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42), or with 20 nM irrelevant siRNA (siScr). 72 hours after transfection, the cells were lysed in SDS-PAGE loading buffer and analysed by immunoblotting with specific antibodies to RhoA, Rac1, Cdc42 and ERK1/2. (B) Western-blot analysis of whole-cell lysates of HSFs transfected with 20 nM siScr or with 20 nM siCdc42. Cells were lysed in SDS-PAGE loading buffer between day 1 and day 9 after transfection and analysed by immunoblotting with specific antibodies to Cdc42 and ERK1/2.

View larger version (77K): [in a new window]   Fig. 2. Morphological effects of RhoA, Rac1 or Cdc42 silencing in HSFs. Representative phase-contrast microscopy of HSFs 72 hours after transfection with calcium phosphate alone (CaP), 20 nM irrelevant siRNA (siScr) or with 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42). Bar, 20 µm.

View larger version (36K): [in a new window]   Fig. 3. Cdc42 silencing upregulates MMP-1 protein level. (A) Representative western-blot analysis of serum-free medium conditioned by HSFs for 16 hours between days 2 and 3 after transfection with calcium phosphate alone (CaP), with 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42), or with 20 nM irrelevant siRNA (siScr). (B) Densitometric analysis of A. Results are the means±s.d. of three independent experiments. (C) Representative western-blot analysis of cell lysates of HSFs 72 hours after transfection (cell lysates) or serum-free medium conditioned by HSFs for 16 hours between days 2 and 3 after transfection (C.M.). Cells were transfected with the indicated concentrations of either an irrelevant siRNA (siScr) or the siRNA targeting Cdc42 (siCdc42).

View larger version (22K): [in a new window]   Fig. 4. Cdc42 silencing upregulates MMP-1 but not MMP-2 or 1I mRNA steady-state levels. (A) Representative quantitative RT-PCR analysis of MMP-1, MMP-2 and 1I mRNA levels, and of the 28S rRNA in total RNA extracted from HSFs cultured in DMEM containing 10% FCS 72 hours after transfection with calcium phosphate alone (CaP), an irrelevant siRNA (siScr) or an siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42). Sample-to-sample variations in RT-PCR efficiency are controlled by adding a known copy number of synthetic RNA co-transcribed and co-amplified with the same primers, to generate a product of larger size (*). (B) Densitometric analysis of A. Results are the means±s.d. of three independent experiments. (C) Western-blot analysis of whole-cell lysates of HSFs transfected with calcium phosphate alone (CaP), with 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42), or with 20 nM irrelevant siRNA (siScr). 72 hours after transfection, the cells were lysed in SDS-PAGE loading buffer and analysed by immunoblotting with specific antibodies to Stat1 and ERK1/2.

View larger version (42K): [in a new window]   Fig. 5. A second siRNA targeting another region of the Cdc42 mRNA also upregulates MMP-1. Representative western-blot analysis of whole-cell lysates and of serum-free medium conditioned by HSFs 72 hours after transfection with 20 nM irrelevant siRNA (siScr), the first siRNA targeting Cdc42 (1st siCdc42) or the second siRNA targeting Cdc42 (2nd siCdc42), using specific antibodies to Cdc42, ERK1/2 and MMP-1. RT-PCR analysis of MMP-1 mRNA level and 28S rRNA was performed with total RNA extracted from HSFs cultured in DMEM containing 10% FCS 72 hours after transfection with 20 nM irrelevant siRNA (siScr), the first siRNA targeting Cdc42 (1st siCdc42) or the second siRNA targeting Cdc42 (2nd siCdc42). Sample-to-sample variations in RT-PCR efficiency are controlled using a known copy number of synthetic RNA co-transcribed and co-amplified with the same primers to generate a product of larger size (*).

View larger version (59K): [in a new window]   Fig. 6. Multiple knock down of Rho GTPases revealed a role for Rac1, but not RhoA, in MMP-1 overexpression following Cdc42 silencing. HSFs were transfected with the indicated siRNAs at a concentration of 10 nM. To reach the same final concentration of 30 nM siRNA in each condition, transfection mix were supplemented with the irrelevant siScr siRNA. Representative western-blot analysis of whole-cell lysates 72 hours after transfection (cell lysates) and of serum-free medium conditioned for 16 hours between days 2 and 3 after transfection. Cell lysates and conditioned medium (C.M.) were analysed by immunoblotting with specific antibodies to RhoA, Rac1, Cdc42, ERK1/2 and MMP-1. (bottom) Densitometric analysis of MMP-1 measurements by western-blot analysis of C.M. Results are expressed as the means±s.d. of three independent experiments. The concentrations of the siRNA in the table below the figures are in nM.

View larger version (26K): [in a new window]   Fig. 7. MCP-1 and IL-8 overexpression following Cdc42 silencing. (A) Analysis of serum-free medium conditioned for 16 hours between days 2 and 3 after transfection by cytokine proteoarray. HSFs were transfected with 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42), or with 20 nM irrelevant siRNA (siScr). White arrows indicate the positions of the IL-8, IL-6 and MCP-1 signals, respectively, from top to bottom (B) Representative semiquantitative RT-PCR analysis of MCP-1 and IL-8 mRNA level and of 28S rRNA in total RNA extracted from HSF 72 hours after transfection with calcium phosphate alone (CaP), an irrelevant siRNA (siScr) or an siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42). The efficiency of the 28S RT-PCR is controlled using a known copy number of synthetic RNA co-transcribed and co-amplified with the same primers to generate a product of larger size (*). (C) ELISA measurements of MCP-1 and IL-8 in serum-free medium conditioned by HSFs for 16 hours between days 2 and 3 after transfection.

View larger version (50K): [in a new window]   Fig. 8. Overexpression of MMP-1 following Cdc42 silencing is mediated through the ERK1/2 pathway. (A) Western-blot analysis of whole-cell lysates from starved HSFs 72 hours after transfection with calcium phosphate alone (CaP), 20 nM irrelevant siRNA (siScr) or 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42). Cell lysates were analysed by immunoblotting with specific antibodies to phospho-ERK1/2, phospho-p38 and total ERK1/2 (ERK1,2). (B) Representative western-blot analysis of whole-cell lysates 72 hours after transfection (cell lysates) and of serum-free medium conditioned for 16 hours between days 2 and 3 after transfection (C.M.). HSFs were transfected with 20 nM irrelevant siRNA (siScr) or with 20 nM siRNA targeting Cdc42 (siCdc42) and cultured between days 1 and 3 after transfection with the indicated concentrations of the MEK-kinase inhibitor U0126, of the PI3K inhibitor LY294 and of the p38-MAP-kinase inhibitor SB203580. Cell lysates and C.M. were analysed by immunoblotting with specific antibodies to Cdc42, ERK1/2 and MMP-1.

View larger version (44K): [in a new window]   Fig. 9. The ERK1/2 pathway is also required for MMP-1 overexpression following Cdc42 silencing with a second siRNA. (A) Representative western-blot analysis of whole-cell lysates of starved HSFs 72 hours after transfection with calcium phosphate alone (CaP) or with the indicated concentrations of an irrelevant siRNA (siScr) or the second siRNA targeting Cdc42 (2nd siCdc42). Cell lysates were analysed by immunoblotting with specific antibodies to phospho-ERK1/2 and ERK1/2. (B,C) Representative western-blot analysis of serum-free medium conditioned by HSFs for 16 hours between days 2 and 3 after transfection with 20 nM irrelevant siRNA (siScr), with 20 nM first siRNA targeting Cdc42 (1st siCdc42) or with 20 nM second siRNA targeting Cdc42 (2nd siCdc42) and cultured between days 1 and 3 after transfection with the indicated concentrations of the MEK-kinase inhibitor U0126 (B) or the p38-MAP-kinase inhibitor SB203580 (C).

View larger version (51K): [in a new window]   Fig. 10. MMP-1 overexpression following Cdc42 silencing is observed in various human cell lines. Representative western-blot analysis of cell lysates of human breast adenocarcinoma cell line HS578T, fibrosarcoma cell line HT1080 and melanoma cell line A2058 72 hours after transfection, and of serum-free medium conditioned by the same cells for 16 hours between days 2 and 3 after transfection (C.M.). Cells were transfected with 20 nM irrelevant siRNA (siScr) or 20 nM first siRNA targeting Cdc42 (siCdc42). Cell lysates and C.M. were analysed by immunoblotting with specific antibodies to Cdc42, ERK1/2 and MMP-1.

View larger version (35K): [in a new window]   Fig. 11. The rescue of Cdc42 knock down represses MMP-1 overexpression. RT-PCR analysis of Cdc42 and mutated Cdc42 (mCdc42) mRNA, and 28S rRNA levels was performed with total RNA extracted from HS578T cells transfected with 20 nM irrelevant siRNA (siScr) or 20 nM first siRNA targeting Cdc42 (siCdc42) and 1 µg either empty pShuttle (vector) or pShuttle-mCdc42 (mCdc42). Total RNA was extracted 72 hours after transfection with the siRNA. (top left) The amplification of mCdc42 mRNA performed with total RNA extracted from HS578T cells 48 hours after transfection with pShuttle-mCdc42, with (+RT) or without (–RT) the reverse transcription step. Representative western-blot analysis with specific antibodies to Cdc42, ERK1/2 and MMP-1 of whole-cell lysates and of serum-free medium conditioned (C.M.) by HS578T cells transfected with 20 nM irrelevant siRNA (siScr) or 20 nM first siRNA targeting Cdc42 (siCdc42) and 1 µg empty pShuttle (vector) or pShuttle-mCdc42 (mCdc42). (bottom) Densitometric analysis of MMP-1 measurements by western-blot analysis of C.M. Results are the means±s.d. of three independent experiments.