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.
Highlights
The small GTPases RhoA and Rac1 are major regulators of cell morphology by modulating fibrous actin (F-actin) structures
The actin S14C polymerization favoring mutant was incorporated into F-actin fibers and induced spreading of HKC-8 cells (Fig. 1B and S2 Fig.)
To induce connective tissue growth factor (CTGF) synthesis, cells were stimulated with lysophosphatidic acid (LPA), which is a known activator of RhoA-Rho kinase signaling [22]
Summary
The small GTPases RhoA and Rac are major regulators of cell morphology by modulating fibrous actin (F-actin) structures. RhoA-induced actin polymerization has been shown to reduce monomeric actin which allows MKL1 to interact with serum response factor (SRF) and leads to upregulation of a subset of SRF-responsive genes [2]. The binding site of the MKL1-SRF complex, the CArG box element, closely resembles the SRE element, which mediates growth factor dependent activation of SRF, but does not contain the flanking Ets binding sites [3]. A CArG box-like element is enclosed in the promoter of connective tissue growth factor (CTGF, CCN2) [4]. Expression of this matricellular protein has been proven to be sensitive to all types of changes in actin cytoskeletal organization [5, 6]. Examples are upregulation of CTGF in endothelial cells upon shear stress [7] or in cardiomyocytes upon stretching [8]
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