Actin-dependent regulation of connective tissue growth factor

Abstract

Expression of connective tissue growth factor (CTGF) in endothelial cells is modulated by shear stress affecting the organization of the cytoskeleton. The molecular connection between alterations of actin and CTGF expression was investigated in human umbilical vein endothelial cells (HUVEC) and a microvascular endothelial cell line. Overexpression of nonpolymerizable monomeric actin R62D interfered with stress fiber formation in HUVEC and concomitantly reduced immunoreactive CTGF. In microvascular endothelial cells, flow-dependent upregulation of CTGF was prevented by this actin mutant. In contrast, overexpression of actin S14C strengthened filamentous actin and increased CTGF expression. These data indicated an inverse relationship between CTGF expression and monomeric actin. Coexpression of the mutant actins and different CTGF promoter constructs revealed an actin-sensitive site between 3 and 4.5 kb of the CTGF promoter. A CArG-like box at -3791 bp was responsible for actin-dependent CTGF induction as shown by mutagenesis. Overexpression of actin S14C activated the nonmutated promoter significantly more strongly than the mutated promoter. Actin polymerization is regulated by the small GTPase RhoA and activation of serum response factor (SRF). Overexpression of constitutively active RhoA or SRF significantly increased CTGF protein synthesis. The 4.5-kb promoter construct, but not the construct with a mutation in the CArG box, was activated by SRF or RhoA, providing evidence for a functional role of this site in CTGF induction. These findings provide novel evidence that monomeric actin is the connecting link between alterations in the cytoskeleton and CTGF gene expression and demonstrate the importance of SRF in regulating CTGF transcription.

Up-Regulation of Connective Tissue Growth Factor in Endothelial Cells by the Microtubule-Destabilizing Agent Combretastatin A-4

“…The effects of CA-4P, the water-soluble form of CA-4, were analyzed in a mouse microvascular endothelial cell line (glEND.2), which has been used previously to characterize the relationship between alterations of the cytoskeleton and CTGF expression (23). Treatment with nanomolar concentrations of CA-4P led to a time-and concentration-dependent disruption of microtubular structures.…”

“…Nonuniform shear stress-induced upregulation of CTGF has been shown to involve activation of Rho proteins (22). In our previous study, G-actin was identified as negative regulator of CTGF expression downstream of RhoA activation, providing evidence for a direct link between alteration of the actin cytoskeleton and CTGF expression (23).…”

The transcriptional coactivators megakaryoblastic leukemia 1/2 mediate the effects of loss of the tumor suppressor deleted in liver cancer 1

“…We therefore sought to determine whether the nuclear localization of MKL1/2 in DLC1-deficient hepatocellular and mammary carcinoma cells results in activation of its target genes. We first tested the expression of CTGF, which has been classified as an SRF-dependent target gene (Muehlich et al, 2007;Medjkane et al, 2009). CTGF also appeared in a recent microarray study by Medjkane et al as a MKL-dependent target gene (Medjkane et al, 2009).…”

“…MKL1 expression was downregulated by 85% at protein and RNA levels ( Figure 5a). Two immediate early genes known to be SRF target genes (Selvaraj and Prywes, 2004;Miano et al, 2007;Muehlich et al, 2007), namely connective tissue growth factor (CTGF) and integrin alpha 5 (Itga5), showed preferential expression in HuH7 versus HepG2 cells, as well as MDA-MB-468 versus MCF7 cells (Figure 5b and Supplementary Figure S4). Both have been reported to have an important role in tumorigenesis (Friedl and Wolf, 2003;Mazzocca et al, 2010).…”

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2

“…Since T 4 is a potent pro-angiogenic factor 21 , an involvement of the T 4-MRTF-SRF axis in vascular growth would require transcriptional activation beyond well-known myogenic proteins 13,14 . However, MRTF coactivaton of SRF induces pro-angiogenic factors such as CCN1 (Cyr61) 22 and maturation factors such as connective tissue growth factor (CTGF) 23,24 . Moreover, SRF controls tip cell behaviour in angiogenesis 25,26 , indicating a role of MRTF-SRF in vascular growth and maintenance.…”