Profibrotic epithelial phenotype: a central role for MRTF and TAZ.

Epithelial injury is a key initiator of fibrosis but - in contrast to the previous paradigm - the epithelium in situ does not undergo wide-spread epithelial-mesenchymal/myofibroblast transition (EMT/EMyT). Instead, it assumes a Profibrotic Epithelial Phenotype (PEP) characterized by fibrogenic cytokine production. The transcriptional mechanisms underlying PEP are undefined. As we have shown that two RhoA/cytoskeleton-regulated transcriptional coactivators, Myocardin-related transcription factor (MRTF) and TAZ, are indispensable for EMyT, we asked if they might mediate PEP as well. Here we show that mechanical stress (cyclic stretch) increased the expression of transforming growth factor-ß1 (TGFß1), connective tissue growth factor (CTGF), platelet-derived growth factor and Indian Hedgehog mRNA in LLC-PK1 tubular cells. These responses were mitigated by siRNA-mediated silencing or pharmacological inhibition of MRTF (CCG-1423) or TAZ (verteporfin). RhoA inhibition exerted similar effects. Unilateral ureteral obstruction, a murine model of mechanically-triggered kidney fibrosis, induced tubular RhoA activation along with overexpression/nuclear accumulation of MRTF and TAZ, and increased transcription of the above-mentioned cytokines. Laser capture microdissection revealed TAZ, TGFß1 and CTGF induction specifically in the tubular epithelium. CCG-1423 suppressed total renal and tubular expression of these proteins. Thus, MRTF regulates epithelial TAZ expression, and both MRTF and TAZ are critical mediators of PEP-related epithelial cytokine production.

TGF-ß1 regulates the expression and transcriptional activity of TAZ protein via a Smad3-independent, myocardin-related transcription factor-mediated mechanism.

Hippo pathway transcriptional coactivators TAZ and YAP and the TGF-ß1 (TGFß) effector Smad3 regulate a common set of genes, can physically interact, and exhibit multilevel cross-talk regulating cell fate-determining and fibrogenic pathways. However, a key aspect of this cross-talk, TGFß-mediated regulation of TAZ or YAP expression, remains uncharacterized. Here, we show that TGFß induces robust TAZ but not YAP protein expression in both mesenchymal and epithelial cells. TAZ levels, and to a lesser extent YAP levels, also increased during experimental kidney fibrosis. Pharmacological or genetic inhibition of Smad3 did not prevent the TGFß-induced TAZ up-regulation, indicating that this canonical pathway is dispensable. In contrast, inhibition of p38 MAPK, its downstream effector MK2 (e.g. by the clinically approved antifibrotic pirferidone), or Akt suppressed the TGFß-induced TAZ expression. Moreover, TGFß elevated TAZ mRNA in a p38-dependent manner. Myocardin-related transcription factor (MRTF) was a central mediator of this effect, as MRTF silencing/inhibition abolished the TGFß-induced TAZ expression. MRTF overexpression drove the TAZ promoter in a CC(A/T-rich)6GG (CArG) box-dependent manner and induced TAZ protein expression. TGFß did not act by promoting nuclear MRTF translocation; instead, it triggered p38- and MK2-mediated, Nox4-promoted MRTF phosphorylation and activation. Functionally, higher TAZ levels increased TAZ/TEAD-dependent transcription and primed cells for enhanced TAZ activity upon a second stimulus (i.e. sphingosine 1-phosphate) that induced nuclear TAZ translocation. In conclusion, our results uncover an important aspect of the cross-talk between TGFß and Hippo signaling, showing that TGFß induces TAZ via a Smad3-independent, p38- and MRTF-mediated and yet MRTF translocation-independent mechanism.

YAP/TAZ Are Mechanoregulators of TGF-ß-Smad Signaling and Renal Fibrogenesis.

Like many organs, the kidney stiffens after injury, a process that is increasingly recognized as an important driver of fibrogenesis. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are related mechanosensory proteins that bind to Smad transcription factors, the canonical mediators of profibrotic TGF-ß responses. Here, we investigated the role of YAP/TAZ in the matrix stiffness dependence of fibroblast responses to TGF-ß In contrast to growth on a stiff surface, fibroblast growth on a soft matrix led to YAP/TAZ sequestration in the cytosol and impaired TGF-ß-induced Smad2/3 nuclear accumulation and transcriptional activity. YAP knockdown or treatment with verteporfin, a drug that was recently identified as a potent YAP inhibitor, elicited similar changes. Furthermore, verteporfin reduced YAP/TAZ levels and decreased the total cellular levels of Smad2/3 after TGF-ß stimulation. Verteporfin treatment of mice subjected to unilateral ureteral obstruction similarly reduced YAP/TAZ levels and nuclear Smad accumulation in the kidney, and attenuated renal fibrosis. Our data suggest that organ stiffening cooperates with TGF-ß to induce fibrosis in a YAP/TAZ- and Smad2/3-dependent manner. Interference with this YAP/TAZ and TGF-ß/Smad crosstalk likely underlies the antifibrotic activity of verteporfin. Finally, through repurposing of a clinically used drug, we illustrate the therapeutic potential of a novel mechanointerference strategy that blocks TGF-ß signaling and renal fibrogenesis.

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE.

TGFß-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFß is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application of contact uncoupling and TGFß. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses TGFß/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFß to facilitate MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFß-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFß-activated Smad3 and TAZ/YAP, two contact- and cytoskeleton-regulated Smad3-interacting coactivators. Down-regulation/inhibition of TAZ/YAP mitigates injury-induced epithelial Nox4 expression in vitro and in vivo. These findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition.