Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

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.

Structural reorganization of the cytoskeleton contributes to the induction of pro-fibrotic connective tissue growth factor in tubular epithelial cells

“…Induction of CTGF by EGTA was dependent on signaling pathways which have earlier been described to regulate CTGF expression in epithelial cells, e.g. ERK and RhoA [19][20][21][22]. Inhibition of ERK1/2 by U0126 (0.5 M) abrogated CTGF induction.…”

“…CTGF expression is regulated via RhoA/Rho kinase signaling [19,20,[23][24][25]. This pathway also played a role in the EGTA-mediated induction of CTGF which was sensitive to treatment with the inhibitor of Rho kinases, H1152 (Fig.…”

“…1C, T, TGF-␤). However, when dense cells were treated with lysophosphatidic acid (L, LPA) as control stimulus [19], the secretion of CTGF was strongly induced. After treatment with LPA for 4 h (Fig.…”

G Protein-Coupled Estrogen Receptor Regulates Actin Cytoskeleton Dynamics to Impair Cell Polarization

“…Analysis of actin polymerization rate and expression revealed that both are concomitantly reduced by GPER/RhoA signaling in fibroblasts, pointing toward an unidentified negative feedback pathway. Regulation of gene expression by RhoAmediated actin polymerization has been previously described in proximal tubular epithelial cells through the myocardin-related transcription factor A/serum response factor (MRTF-A/SRF) axis (Giehl et al, 2015). MRTF-A is normally inactive in the cytoplasm through binding to G-actin monomers.…”

The cytoskeleton as a novel target for treatment of renal fibrosis

“…Upregulation of CTGF is dependent, in part, on the actin cytoskeleton; this has been in shown in primary human tubular epithelial cells (Kroening et al, 2009) and other cell types (Sakai et al, 2013;Chowdhury and Chagour, 2004). More specifically, CTGF regulation in a human proximal tubular cell line (HKC-8) is dependent upon the MAL/myocardin related transcription factor A (MRTF; megakaryoblastic leukemia 1)/serum response factor pathway downstream of RhoA (Giehl, et al, 2015). This pathway is perhaps the prototypic pathway regulated by the cytoskeleton as monomeric actin regulates gene expression through serum response factor by inhibition of MAL/MRTF (Leitner et al, 2011).…”