Abstract
Tubulointerstitial fibrosis (TIF) is the final pathway through which chronic kidney disease progresses toward renal failure. Tubular injury is a key feature of TIF but the exact role of the epithelium remains a matter of debate. In vivo only a small subset of myofibroblasts (MFs) appears to originate from the epithelium, through epithelial-MF transition (EMyT). The emerging view is that the injured epithelium is a critical driver of fibrosis via acquiring a profibrotic epithelial phenotype (PEP), characterized by cytoskeletal remodeling, ROS production, and fibrogenic cytokine secretion, priming interstitial cells for fibroblast-MF transition. The cytoskeleton- and TGFβ-regulated transcriptional coactivators, myocardin-related transcription factor (MRTF) and the Hippo pathway effector TAZ (and its paralog YAP) are central for EMyT, and are implicated in organ fibrosis; however, their roles in epithelial reprogramming are unclear. The aim of this thesis was to elucidate (i) the mechanism of TGFβ-induced TAZ expression in kidney fibrosis, (ii) the roles of MRTF and TAZ in PEP, (iii) how MRTF and TAZ regulate the oxidative state of the epithelium, and (iv) if the ensuing ROS production regulates TAZ.
In Paper I we investigated the regulation of NADPH oxidase Nox4 by MRTF and TAZ in a cellular model of EMyT using TGFβ-stimulation and cell-contact uncoupling. We found that ROS production increased during EMyT and that this correlated with increased Nox4 expression. Inhibition of MRTF and TAZ prevented this, linking the cytoskeleton to the oxidative state of the cell.
In Paper II TGFβ-induced increase in TAZ expression was investigated. Using pharmacological inhibition we show that non-canonical signaling via p38 and its downstream target MK2 mediates this upregulation. Furthermore, MRTF regulates TAZ expression in a translocation-independent manner. Pharmacological inhibition of Nox4, a known activator of p38, resulted in decreased TAZ, suggesting a feedback loop in which Nox4 regulates TAZ and MRTF, which in turn regulates Nox4.
In Paper III we investigated cytokine expression in the unilateral ureteral obstruction (UUO) model of obstructive nephropathy. UUO resulted in increased TGFβ, PDGF-B, CTGF, and IHH mRNA expression. Inhibition of MRTF by CCG-1423 decreased TGFβ and TAZ expression. Importantly, tubular expression of TGFβ, CTGF, and TAZ increased after UUO and was prevented by CCG-1423. In an in vitro model of mechanical stress we showed that epithelial cytokine expression increased upon stretch and this was prevented by inhibition/knockdown of MRTF and TAZ. Thus, MRTF and TAZ regulate cytokine production a key feature of PEP.
In Paper IV we investigated the mechanism by which ROS production regulates protein expression of TAZ and YAP. The singlet oxygen producers Verteporfin and Rose Bengal dose-dependently reduced monomeric protein levels of TAZ and YAP, as well as of several other proteins, likely via oligomer/aggregate formation. UV radiation and to a lesser extent H2O2 exposure, also decreased TAZ protein level, yet via at least partially different mechanisms. Inhibition of proteasomal or lysosomal degradation did not prevent the loss of TAZ expression. Thus, further studies are needed in order to clarify the mechanism by which ROS regulates TAZ.
Collectively, the work in this thesis shows that MRTF and TAZ are key regulators of the transcriptional reprogramming events necessary for the acquisition of PEP. Specifically, MRTF and TAZ regulate both the oxidative state of the epithelium and the expression of key fibrogenic cytokines in the epithelium. The work also revealed an intricate regulation of MRTF and TAZ by oxidative stress and the key fibrogenic cytokine TGFβ, pointing to MRTF and TAZ as valuable pharmacological targets in the treatment of fibrotic kidney disease.
In Paper I we investigated the regulation of NADPH oxidase Nox4 by MRTF and TAZ in a cellular model of EMyT using TGFβ-stimulation and cell-contact uncoupling. We found that ROS production increased during EMyT and that this correlated with increased Nox4 expression. Inhibition of MRTF and TAZ prevented this, linking the cytoskeleton to the oxidative state of the cell.
In Paper II TGFβ-induced increase in TAZ expression was investigated. Using pharmacological inhibition we show that non-canonical signaling via p38 and its downstream target MK2 mediates this upregulation. Furthermore, MRTF regulates TAZ expression in a translocation-independent manner. Pharmacological inhibition of Nox4, a known activator of p38, resulted in decreased TAZ, suggesting a feedback loop in which Nox4 regulates TAZ and MRTF, which in turn regulates Nox4.
In Paper III we investigated cytokine expression in the unilateral ureteral obstruction (UUO) model of obstructive nephropathy. UUO resulted in increased TGFβ, PDGF-B, CTGF, and IHH mRNA expression. Inhibition of MRTF by CCG-1423 decreased TGFβ and TAZ expression. Importantly, tubular expression of TGFβ, CTGF, and TAZ increased after UUO and was prevented by CCG-1423. In an in vitro model of mechanical stress we showed that epithelial cytokine expression increased upon stretch and this was prevented by inhibition/knockdown of MRTF and TAZ. Thus, MRTF and TAZ regulate cytokine production a key feature of PEP.
In Paper IV we investigated the mechanism by which ROS production regulates protein expression of TAZ and YAP. The singlet oxygen producers Verteporfin and Rose Bengal dose-dependently reduced monomeric protein levels of TAZ and YAP, as well as of several other proteins, likely via oligomer/aggregate formation. UV radiation and to a lesser extent H2O2 exposure, also decreased TAZ protein level, yet via at least partially different mechanisms. Inhibition of proteasomal or lysosomal degradation did not prevent the loss of TAZ expression. Thus, further studies are needed in order to clarify the mechanism by which ROS regulates TAZ.
Collectively, the work in this thesis shows that MRTF and TAZ are key regulators of the transcriptional reprogramming events necessary for the acquisition of PEP. Specifically, MRTF and TAZ regulate both the oxidative state of the epithelium and the expression of key fibrogenic cytokines in the epithelium. The work also revealed an intricate regulation of MRTF and TAZ by oxidative stress and the key fibrogenic cytokine TGFβ, pointing to MRTF and TAZ as valuable pharmacological targets in the treatment of fibrotic kidney disease.
| Original language | English |
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| Publisher | Department of Biology, Faculty of Science, University of Copenhagen |
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| Number of pages | 182 |
| Publication status | Published - 2017 |