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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

Transient down-regulation of RhoA interferes with CTGF synthesis.(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.
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pone.0121589.g003: Transient down-regulation of RhoA interferes with CTGF synthesis.(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.

Mentions: The missing inhibitory effect of dnRhoA on SRE activation and CTGF expression was unexpected. As a complementary approach to modulate RhoA activity, the GTPase was transiently down-regulated by siRNA. Under these conditions, LPA-induced CTGF secretion was significantly reduced (Fig. 3A). Interestingly basal CTGF secretion was also modulated by siRhoA. This became even more evident, when the promoter activity of CTGF was analyzed (Fig. 3B). A significant reduction of CTGF promoter activity not only in stimulated cells but also in cells cultured without specific stimulation showed that RhoA played an essential role in CTGF regulation. Thus, even though overexpression of dnRhoA clearly altered cell morphology it was not sufficiently effective to reduce CTGF gene expression, while down-regulation of RhoA by siRNA demonstrated regulation of CTGF via this pathway.


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)

Transient down-regulation of RhoA interferes with CTGF synthesis.(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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4376694&req=5

pone.0121589.g003: Transient down-regulation of RhoA interferes with CTGF synthesis.(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.
Mentions: The missing inhibitory effect of dnRhoA on SRE activation and CTGF expression was unexpected. As a complementary approach to modulate RhoA activity, the GTPase was transiently down-regulated by siRNA. Under these conditions, LPA-induced CTGF secretion was significantly reduced (Fig. 3A). Interestingly basal CTGF secretion was also modulated by siRhoA. This became even more evident, when the promoter activity of CTGF was analyzed (Fig. 3B). A significant reduction of CTGF promoter activity not only in stimulated cells but also in cells cultured without specific stimulation showed that RhoA played an essential role in CTGF regulation. Thus, even though overexpression of dnRhoA clearly altered cell morphology it was not sufficiently effective to reduce CTGF gene expression, while down-regulation of RhoA by siRNA demonstrated regulation of CTGF via this pathway.

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