Transient down-regulation of Rho kinases is not sufficient to impair CTGF expression.

<p>HKC-8 cells were treated with siRNA directed against GFP, ROCK1 (R1) or ROCK2 (R2), or a combination of both (R1/2) 3 h after seeding. (A) One day after siRNA transfection, HKC-8 cells were transfected with SRE constructs. Relative luciferase activity was determined in control cells and cells stimulated with LPA for 4 h. The graph summarizes means ± SD of 3 to 5 independent experiments. SRE activity in cells stimulated with LPA was set to 1 in each experiment. Statistics were calculated for LPA-stimulated samples. *** p< 0.001, ANOVA with Tukey’s multiple comparison. (B) Phosphorylated MYPT (pMYPT) was detected by Western blot analysis in control cells and in cells stimulated with LPA for 3–5 min 48 h after siRNA treatment. Detection of total MYPT was used as control. The graph summarizes data of 3 experiments (mean ± SD). Expression of pMYPT/MYPT in LPA-stimulated siGFP-transfected cells was set to 1 in each experiment; * p<0.05 compared to LPA-stimulated siGFP-transfected cells. (C) One day after siRNA transfection, HKC-8 cells were transfected with CTGF promoter constructs. Stimulation with LPA was 4 h. The graph summarizes means ± SD of 3–4 independent experiments. Promoter activity in LPA-stimulated siGFP-transfected cells was set to 1 in each experiment. (D) CTGF protein was detected in cellular homogenates. Data are means ± SD of 3–5 independent experiments. CTGF protein detected in LPA-stimulated cells was set to one in each experiment. * p<0.05 compared to LPA-stimulated siGFP-transfected cells. (E) CTGF protein was detected in cell culture supernatants. Data are means ± SD of 3–4 independent experiments.</p

Actin mutants alter CTGF expression.

<p>(A) HKC-8 cells were transfected with the flag-tagged polymerization-defective actin mutant R62D. 24 h after transfection, cells were stimulated with LPA (10 μM) for 2 h. Mutated actin (anti-flag, red) and CTGF (green) were visualized by indirect immunofluorescence; nuclei were stained with Hoechst (blue). Open arrow indicates CTGF expression in a LPA-stimulated cell; closed arrows indicate low CTGF expression in actin R62D transfected cells. Scale bar: 20 μm. (B) HKC-8 cells were transfected with the flag-tagged polymerization favoring actin mutant S14C. Cells were fixed after transfection without further stimulation. Mutated actin (anti-flag, red) and CTGF (green) were visualized by indirect immunofluorescence; nuclei were stained with Hoechst (blue). Open arrows indicate CTGF expression in transfected cells. Scale bar: 20 μm. (C): HKC-8 cells were transfected with actin expression plasmids (actin R62D and S14C) or eGFP as control and with a luciferase-coupled promoter construct comprising three SRE elements. Expression of cotransfected beta galactosidase was used as reference. Cells were stimulated with LPA (L, 10 μM) for 4 h and compared to control cells (C). Data are means ± SD of 2–4 experiments with biological duplicates. SRE activity of LPA-stimulated actin R62D-transfected cells was set to 1. # p < 0.01 compared to the respective R62D actin- or eGFP-transfected cells; * p < 0.001 analyzed separately compared to eGFP-treated cells. ANOVA with Tukey’s multiple comparison test; ++ p<0.001, + p<0.05, analysis of eGFP and R62D treated cells, LPA-stimulated cells compared to control cells. (D) Cells were treated as in (C), but co-transfected with a 4.5 kb CTGF promoter construct. # p < 0.01 compared to the respective R62D actin- or eGFP-transfected cells; * p < 0.01 analyzed separately compared to eGFP-treated cells. ANOVA with Tukey’s multiple comparison test; ++ p<0.001, analysis of eGFP and R62D treated cells, LPA-stimulated cells compared to control cells.</p

CTGF expression is dependent on Rho kinase activity.

<p>(A) HKC-8 cells were transfected with luciferase-coupled SRE constructs. Expression of transfected beta galactosidase was used as reference. Cells were preincubated for 30 min with Y27632 (Y, 10 μM) or H1152 (0.75 μM) and were then stimulated with LPA (L, 10 μM, grey bars) for 4 h. Data are means ± SD of 3 experiments with duplicate samples. In each experiment, means of control values were set to 1. *** p < 0.001 compared to LPA-stimulated control cells; ANOVA with Tukey’s multiple comparison test. (B) HKC-8 cells were treated as in A, but transfected with a 4.5 kb CTGF promoter construct. ## p < 0.01 compared to non-stimulated control cells, *** p < 0.001, compared to LPA-stimulated cells, ANOVA with Tukey’s multiple comparison test. (C) HKC-8 cells were preincubated with Y27632 (10 μM) for 30 min and then incubated with LPA (10 μM) for 2 h. CTGF was detected by Western blotting in the cell culture supernatants (secreted CTGF) and homogenates (cellular CTGF). Samples were detected on one blot which had to be rearranged (dotted line). The graphs summarize quantification of multiple experiments with LPA-stimulated cells set to 1 in each experiment: cellular CTGF: n = 2 ± half range; secreted CTGF control: n = 3, secreted CTGF LPA-stimulated: n = 6; *** p<0.001 compared to LPA-stimulated cells, ANOVA with Tukey’s multiple comparison test. (D) HKC-8 cells were transfected with siGFP or siROCK1/ROCK2 at day 1, incubated with Y27632 (10 μM) at day 2 and stimulated with LPA (10 μM) for 1 h at day 3. F-actin was visualized with PromoFluor phalloidin. Scale bar: 20 μm.</p

Differential regulation of CTGF by EHT1864 in epithelial cells of proximal and distal origin.

<p>Primary proximal and distal tubular epithelial cells were pre-incubated with EHT1864 (10 μM) for 30 min and then incubated with LPA (10 μM) for 2 and 5 h. Secreted CTGF was detected in the cell culture supernatants. Data are means of 3 (2 h) and 4 (5 h) preparations of cells obtained from different patients. Secretion of control cells was set to one in each experiment. ** p < 0.01, two-sided Student’s t-test.</p

Overexpression of activated or dominant negative Rac1 alters cell morphology but not CTGF expression.

<p>(A) HKC-8 cells were transfected with dominant negative Rac1 (T17N) or constitutively active Rac1 (G12V) coupled to eGFP. 24 h after transfection, cells were stimulated with LPA (10 μM) for 1 h. F-actin fibers were visualized with PromoFluor phalloidin. Arrows indicate transfected cells. Scale bar: 20 μm. (B) HKC-8 cells were transfected with eGFP-coupled dominant negative (dn) or constitutively active (ca) Rac1, or eGFP (GFP) as control together with luciferase-coupled SRE constructs. Expression of cotransfected beta galactosidase was used as reference. Cells were stimulated with LPA (L, 10 μM) for 4 h. Data shown are means ± SD of 3–5 independent experiments performed with duplicate transfections. In each experiment the mean of the unstimulated control values was set to 1. (C) Cells were treated as in (B) but transfected with a 4.5 kb CTGF promoter construct. Data are means of 3 independent experiments with duplicate transfections. In each experiment the mean of the unstimulated control values was set to 1.</p

Transient down-regulation of RhoA interferes with CTGF synthesis.

<p>(A) HKC-8 cells were transfected with siRNA against GFP or RhoA. After 48 h, cells were stimulated with LPA (L, 10 μM) for 2 h. Secreted CTGF was precipitated from the cell culture supernatants and analyzed by Western blotting. The graph summarizes data of 4 independent experiments. CTGF expression in controls cells was set to 1 in each experiment. Means ± SD, * p < 0.05, ANOVA with Tukey’s multiple comparison test. (B) HKC-8 cells were treated with siRNA directed against GFP or RhoA 3 h after seeding. One day after siRNA transfection, HKC-8 cells were transfected with the 4.5 kb CTGF promoter construct. Stimulation with LPA was 4 h. The graph summarizes means ± SD of 3 independent experiments. Promoter activity in LPA-stimulated siGFP-transfected cells was set to 1 in each experiment. * *p < 0.001, ANOVA with Tukey’s multiple comparison test.</p

Overexpression of activated RhoA activates SRE activity and enhances CTGF promoter activity.

<p>(A) HKC-8 cells were transfected with constitutively active RhoA (G14V) or dominant negative RhoA (T19N) coupled to eGFP. 24 h after transfection, cells were stimulated with LPA (10 μM) for 1 h. Actin fibers were visualized with PromoFluor phalloidin. Arrows indicate transfected cells. Scale bar: 20 μm. (B) HKC-8 cells were transfected with eGFP-coupled dominant negative (dn) or constitutively active (ca) Rho GTPases, or eGFP (-) as control together with luciferase-coupled SRE constructs. Expression of cotransfected beta galactosidase was used as reference. Cells were stimulated with LPA (L, 10 μM) for 4 h. Data shown are means ± SD of 3 independent experiments performed with duplicate transfections. In each experiment the mean of the unstimulated control values was set to 1. SRE activity in caRhoA in control cells (C) was significantly increased (p < 0.001); ANOVA with Tukey’s multiple comparison test. (C) HKC-8 cells were transfected with eGFP-coupled constitutively active RhoA (caRhoA) or eGFP (Co) and luciferase-coupled 4.5 kb CTGF promoter constructs. Cotransfected Beta galactosidase was used as reference. Data are means ± SD of 3 experiments performed in duplicates. The mean values of control cells were set to 1. CTGF promoter activity was significantly higher in RhoA transfected cells (p < 0.05, ANOVA with Tukey’s multiple comparison test.) (D) HKC-8 cells were transfected with eGFP. 24 h after transfection the cells were stimulated with LPA for 1 h. MKL1 was detected by indirect immunofluorescence. Scale bar: 20 μm. (E) HKC-8 cells were transfected with eGFP-tagged dominant negative RhoA (T19N) constitutively active RhoA (G14V) (green) and stimulated with LPA for 1 h. Expression of MKL1 was detected by immunocytochemistry (red). Scale bar: 20 μm.</p

Time-dependent effects of Rac1 inhibition by EHT1864 on CTGF regulation.

<p>(A) HKC-8 cells were transfected with SRE promoter constructs. After pre-incubation with EHT1864 (10 μM) for 30 min, the cells were stimulated with LPA (10 μM) for the times indicated. Data are means ± SD from one experiment with triplicate transfections. (B) HKC-8 cells were transfected with SRE or 4.5 kb CTGF promoter constructs. Relative luciferase activity was determined in control cells and cells pre-treated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for 3–4 h. Data are means ± SD of 7 (SRE) and 3 (CTGF promoter) experiments. Activity in LPA-stimulated cells was set to 1 in each experiment. *** p<0.001, compared to cells stimulated with LPA, ANOVA with Dunnett’s multiple comparison test. (C) HKC-8 cells were pre-incubated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for the times indicated. Cellular CTGF was determined in preparations of cellular homogenates by Western blotting. Tubulin was used to control for equal blotting and detection. The graph summarizes data of n = 3 experiments, stimulated with LPA for 1 h (CTGF/tubulin, means ± SD). Secreted CTGF was determined in precipitates of cell culture supernatants by Western blotting. The graph summarizes data of n = 4 experiments, stimulated with LPA for 2 h. Data were normalized to LPA-stimulated CTGF synthesis. *** p<0.001, ** p<0.01, ANOVA with Tukey’s multiple comparison test. (D) HKC-8 cells were pre-incubated with EHT1864 (10 μM) for 30 min and then stimulated with LPA (10 μM) for 4 h. Secreted CTGF (sCTGF) was determined in precipitates of cell culture supernatants and cellular CTGF (cCTGF) in cellular homogenates by Western blotting. Vinculin (vinc) served as control. Samples were detected on one blot which had to be rearranged. Data of 4–5 experiments (cellular CTGF, 4 h) and 5–7 experiments (secreted CTGF, 4–6 h) are summarized in the graphs. CTGF expression in LPA-stimulated cells was set to 1 in each experiment. * p<0.05, ***p<0.001 compared to LPA-stimulated cells, ANOVA with Tukey’s multiple comparison test. # p<0.05, paired 2-sided t-test, EHT1864-treated cells compared to control cells.</p

Cellular effects of Rac1 inhibition by EHT1864.

<p>(A) HKC-8 cells were incubated with EHT1864 (10 μM) for the times indicated. Rac1 activity was determined by pull down experiments. The blot is representative of 3 experiments with comparable results. Samples were run on one blot which had to be rearranged. pERK1/2, ERK1/2 and vinculin were detected by immunoblot procedure. The blot shows duplicate biological samples. The graph summarizes data of n = 3–6 experiments (pERK1/2 / ERK1/2 or pERK1/2 / vinculin), means ± SD, *** p<0.001, compared to control cells, ANOVA with Dunnett’s multiple comparison test. pCofilin and tubulin were detected on one blot which had to be rearranged. The graph summarizes data of n = 4 experiments (pCofilin/Tubulin), means ± SD, *** p<0.001, compared to control cells, ANOVA with Dunnett’s multiple comparison test. (B) HKC-8 cells were treated with EHT1864 (10 μM) for 30 min. F-actin was visualized with PromoFluor phalloidin. Scale bar: 20 μm. (C) HKC-8 cells were seeded around barriers. After removal of the barriers (t = 0 h) the cells were treated with EHT1864 (10 μM) for 24 h. Scale bar: 200 μm. The graph summarizes the relative migration velocity of cells treated with 5 or 10 μM EHT1864 or 10 μM Y27632. Data are means ± SEM of 3 experiments with 4 determinations each. ** p< 0.01, * p<0.05 compared to control cells.</p

Isoform-specific changes in F-actin fiber formation by ROCK1 and ROCK2 siRNA.

<p>(A) HKC-8 cells were transfected with siRNAs as indicated. After 48 h the cells were stimulated with LPA (10 µM) for 2 h. Cells were stained for F-actin (red) and paxillin (green). Scale bar: 50 µm. (B) HKC-8 cells were treated as in A. N-cadherin was visualized by immunocytochemistry. Scale bar: 20 µm. (C) HKC-8 cells or hPTEC were transfected with siRNAs against ROCK1 (R1), ROCK2 (R2) or GFP as indicated. After 72 h, expression of N-cadherin, E-cadherin, and tubulin was detected by Western blotting. (D) hPTECs were treated as in A. Cells were stained for F-actin and for N-cadherin to distinguish between proximal and distal cells.</p