Dataset Information

Activation of AMP-activated protein kinase prevents TGF-?1-induced epithelial-mesenchymal transition and myofibroblast activation.

ABSTRACT: Transforming growth factor (TGF)-? contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-? exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-?1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and ?-smooth muscle actin expression and decreased in E-cadherin. TGF-?1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-?-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-?1 functions. Conversely, inhibition of AMPK with adenine 9-?-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-?1 and enhanced basal and TGF-?1-induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-?1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-? transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-? exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-?. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease.

SUBMITTER: Thakur S

PROVIDER: S-EPMC4530134 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Activation of AMP-activated protein kinase prevents TGF-β1-induced epithelial-mesenchymal transition and myofibroblast activation.

Thakur Sachin S Viswanadhapalli Suryavathi S Kopp Jeffrey B JB Shi Qian Q Barnes Jeffrey L JL Block Karen K Gorin Yves Y Abboud Hanna E HE

The American journal of pathology 20150610 8

Transforming growth factor (TGF)-β contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-β exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-β1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. T ...[more]

PMID: 26071397

Similar Datasets

Project description:Introduction:Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-? signalling pathway, which is involved in corneal epithelial wound healing. Methods:HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-? secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-?2 treatment. Results:KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-? canonical signalling pathway, TGF-?2 secretion by HCECs increased up to 5 folds, and several TGF-?-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-? signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-?2 treatment. Conclusions:These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-?2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-? signalling.

Project description:Transforming growth factor-? (TGF-?) acts as a key cytokine in epithelial-mesenchymal transition (EMT) and myofibroblast differentiation, which are important for normal tissue repair and fibrotic diseases. Ubiquitylation and proteasomal degradation of TGF-? signaling proteins acts as a regulatory mechanism for the precise control of TGF-? signaling. SMAD-specific ubiquitin E3 ligase (SMAD ubiquitination regulatory factor 2, SMURF2) controls TGF-? signaling proteins including the TGF-? receptor (TGFR) and SMAD2/3. Here, we report that tetratricopeptide repeat domain 3 (TTC3), a ubiquitin E3 ligase, positively regulates TGF-?1-induced EMT and myofibroblast differentiation, through inducing ubiquitylation and proteasomal degradation of SMURF2. In human bronchial epithelial cells (BEAS-2B) and normal human lung fibroblasts, TTC3 knockdown suppressed TGF-?1-induced EMT and myofibroblast differentiation, respectively. Similarly, when TTC3 expression was suppressed, the TGF-?1-stimulated elevation of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were inhibited. In contrast, overexpression of TTC3 caused both EMT and myofibroblast differentiation in the absence of TGF-?1 treatment. TGF-?1 reduced SMURF2 levels and TTC3 overexpression led to a further decrease in SMURF2 levels, while TTC3 knockdown inhibited TGF-?1-induced SMURF2 reduction. In cell and in vitro ubiquitylation assays demonstrated TTC3-mediated SMURF2 ubiquitylation, and coimmunoprecipitation assays established the binding between SMURF2 and TTC3. TGF-?1-induced TTC3 expression was inhibited by the knockdown of SMAD2 and SMAD3. Finally, Ttc3 mRNA levels were significantly increased and Smurf2 protein levels were significantly decreased in the lungs of mice treated with bleomycin as compared with the lungs of control mice. Collectively, these data suggest that TTC3 may contribute to TGF-?1-induced EMT and myofibroblast differentiation, potentially through SMURF2 ubiquitylation/proteasomal degradation and subsequent inhibition of SMURF2-mediated suppression of SMAD2 and SMAD3, which in turn induces TTC3 expression.

Dataset Information

Activation of AMP-activated protein kinase prevents TGF-?1-induced epithelial-mesenchymal transition and myofibroblast activation.

Publications

Activation of AMP-activated protein kinase prevents TGF-β1-induced epithelial-mesenchymal transition and myofibroblast activation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure