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Actin-mediated gene expression depends on RhoA and Rac1 signaling in proximal tubular epithelial cells.

Giehl K, Keller C, Muehlich S, Goppelt-Struebe M - PLoS ONE (2015)

Bottom Line: Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants.Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression.Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction of Cellular Motility, Internal Medicine V, Justus-Liebig-University Giessen, Giessen, Germany.

ABSTRACT
Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses.

No MeSH data available.


Related in: MedlinePlus

Cellular effects of Rac1 inhibition by EHT1864.(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.
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pone.0121589.g007: Cellular effects of Rac1 inhibition by EHT1864.(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.

Mentions: To further address Rac1 signaling in HKC-8 cells the activity of Rac1 was inhibited by the specific low molecular weight inhibitor EHT1864, which blocks the GTP binding site [24]. Treatment of the cells with 10 μM EHT1864 reduced Rac1 activity as determined by pull down assays (Fig. 7A). Furthermore, it reduced phosphorylation of known effector proteins of Rac1, namely phosphorylation of ERK1/2 and Cofilin (Fig. 7A). The morphology of the cells was reminiscent of the appearance of cells transfected with dnRac1, characterized by F-actin spikes (Fig. 7B). Functionally, EHT1864 prevented cell migration (Fig. 7C) which was stimulated in control cells in the presence of the Rho kinase inhibitor Y27632 in line with published results [25].


Actin-mediated gene expression depends on RhoA and Rac1 signaling in proximal tubular epithelial cells.

Giehl K, Keller C, Muehlich S, Goppelt-Struebe M - PLoS ONE (2015)

Cellular effects of Rac1 inhibition by EHT1864.(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.
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pone.0121589.g007: Cellular effects of Rac1 inhibition by EHT1864.(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.
Mentions: To further address Rac1 signaling in HKC-8 cells the activity of Rac1 was inhibited by the specific low molecular weight inhibitor EHT1864, which blocks the GTP binding site [24]. Treatment of the cells with 10 μM EHT1864 reduced Rac1 activity as determined by pull down assays (Fig. 7A). Furthermore, it reduced phosphorylation of known effector proteins of Rac1, namely phosphorylation of ERK1/2 and Cofilin (Fig. 7A). The morphology of the cells was reminiscent of the appearance of cells transfected with dnRac1, characterized by F-actin spikes (Fig. 7B). Functionally, EHT1864 prevented cell migration (Fig. 7C) which was stimulated in control cells in the presence of the Rho kinase inhibitor Y27632 in line with published results [25].

Bottom Line: Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants.Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression.Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction of Cellular Motility, Internal Medicine V, Justus-Liebig-University Giessen, Giessen, Germany.

ABSTRACT
Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF-mediated CTGF expression in proximal tubular cells, whereas Rac1 signaling is more complex with adaptive cellular responses.

No MeSH data available.


Related in: MedlinePlus