Synergistic induction of CTGF by cytochalasin D and TGFß-1 in primary human renal epithelial cells: Role of transcriptional regulators MKL1, YAP/TAZ and Smad2/3.

ABSTRACT

Changes in cell morphology that involve alterations of the actin cytoskeleton are a hallmark of diseased renal tubular epithelial cells. While the impact of actin remodeling on gene expression has been analyzed in many model systems based on cell lines, this study investigated human primary tubular epithelial cells isolated from healthy parts of tumor nephrectomies. Latrunculin B (LatB) and cytochalasin D (CytoD) were used to modulate G-actin levels in a receptor-independent manner. Both compounds (at 0.5µM) profoundly altered F-actin structures in a Rho kinase-dependent manner, but only CytoD strongly induced the pro-fibrotic factor CTGF (connective tissue growth factor). CTGF induction was dependent on YAP as shown by transient downregulation experiments. However, CytoD did not alter the nuclear localization of either YAP or TAZ, whereas LatB reduced nuclear levels particularly of TAZ. CytoD modified MKL1, a coactivator of serum response factor (SRF) regulating CTGF induction, and promoted its nuclear localization. TGFß-1 is one of the major factors involved in tubulointerstitial disease and an inducer of CTGF. Preincubation with CytoD but not LatB synergistically enhanced the TGFß-1-stimulated synthesis of CTGF, both in cells cultured on plastic dishes as well as in polarized epithelial cells. CytoD had no direct effect on the phosphorylation of Smad2/3, but facilitated their phosphorylation and thus activation by TGFß-1. Our present findings provide evidence that morphological alterations have a strong impact on cellular signaling of one of the major pro-fibrotic factors, TGFß-1. However, our data also indicate that changes in cell morphology per se cannot predict those interactions which are critically dependent on molecular fine tuning of actin reorganization.