Butyrate Prevents TGF-β1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

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Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis

Cell Death and Disease ◽

10.1038/s41419-019-1873-x ◽

2019 ◽

Vol 10 (9) ◽

Cited By ~ 10

Author(s):

Peng Wei ◽

Yan Xie ◽

Peter W. Abel ◽

Yapei Huang ◽

Qin Ma ◽

...

Keyword(s):

Growth Factor ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Tissue Growth ◽

Negative Regulator ◽

Lung Fibroblasts ◽

Luciferase Reporter ◽

Myofibroblast Differentiation ◽

Dependent Manner ◽

Tgf Β1

Abstract Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

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Inhibition of lncRNA PFRL prevents pulmonary fibrosis by disrupting the miR-26a/smad2 loop

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00434.2017 ◽

2018 ◽

Vol 315 (4) ◽

pp. L563-L575 ◽

Cited By ~ 11

Author(s):

Hua Jiang ◽

Yingzhun Chen ◽

Tong Yu ◽

Xiaoguang Zhao ◽

Huitong Shan ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Fibrosis ◽

Feedback Loop ◽

Transforming Growth Factor ◽

Molecular Study ◽

Pulmonary Fibroblasts ◽

Proliferation And Differentiation ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with increasing mortality and poor prognosis. The current understanding of the role of long noncoding RNAs (lncRNAs) in IPF remains limited. In the present study, we identified a lncRNA NONMMUT022554, designated pulmonary fibrosis-regulatory lncRNA (PFRL), with unknown functions and found that its levels were increased in fibrotic lung tissues of mice and pulmonary fibroblasts exposed to transforming growth factor (TGF)-β1. Furthermore, we found that enforced expression of PFRL induced fibroblast activation and collagen deposition, which could be mitigated by the overexpression of microRNA (miR)-26a. By contrast, the inhibition of PFRL could markedly alleviate the TGF-β1-induced upregulation of fibrotic markers and attenuate fibroblast proliferation and differentiation by regulating miR-26a. Meanwhile, our study confirmed that PFRL inhibited the expression and activity of miR-26a, which has been identified as an antifibrotic miRNA in our previous study. Interestingly, our molecular study further confirmed that Smad2 transcriptionally inhibits the expression of miR-26a and that the miR-26a/Smad2 feedback loop mediates the profibrotic effects of PFRL in lung fibrosis. More importantly, knockdown of PFRL ablated bleomycin-induced pulmonary fibrosis in vivo. Taken together, our findings indicate that lncRNA PFRL contributes to the progression of lung fibrosis by modulating the reciprocal repression between miR-26a and Smad2 and that this lncRNA may be a therapeutic target for IPF.

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Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2015-0602 ◽

2016 ◽

Vol 94 (12) ◽

pp. 1315-1324 ◽

Cited By ~ 8

Author(s):

Xian-Wei Li ◽

Xiao-Hui Li ◽

Jie Du ◽

Dai Li ◽

Yuan-Jian Li ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Calcitonin Gene Related Peptide ◽

Collagen I ◽

Lung Fibroblasts ◽

Brdu Incorporation ◽

Pulmonary Fibroblasts ◽

Related Peptide ◽

Calcitonin Gene ◽

Collagen Iii ◽

Tgf Β1

We have found that eIF3a plays an important role in bleomycin-induced pulmonary fibrosis, and up-regulation of eIF3a induced by TGF-β1 is mediated via the ERK1/2 pathway. Whether ERK1/2 – eIF3a signal pathway is involved in calcitonin gene-related peptide (CGRP)-mediated pathogenesis of bleomycin-induced pulmonary fibrosis remains unknown. Pulmonary fibrosis was induced by intratracheal instillation of bleomycin (5 mg/kg) in rats. Primary pulmonary fibroblasts were cultured to investigate the proliferation by BrdU incorporation method and flow cytometry. Sensory CGRP depletion by capsaicin exacerbated bleomycin-induced pulmonary fibrosis in rats, as shown by a significant disturbed alveolar structure, marked thickening of the interalveolar septa and dense interstitial infiltration by inflammatory cells and fibroblasts, accompanied with increased expression of TGF-β1, eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts concomitantly with decreased expression of eIF3a, phosphorylated ERK1/2, α-SMA, collagen I, and collagen III. These effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the ERK1/2 – eIF3a signaling pathway.

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Anlotinib Inhibits PFKFB3-Driven Glycolysis in Myofibroblasts to Reverse Pulmonary Fibrosis

Frontiers in Pharmacology ◽

10.3389/fphar.2021.744826 ◽

2021 ◽

Vol 12 ◽

Author(s):

Weimou Chen ◽

Jinming Zhang ◽

Wenshan Zhong ◽

Yuanyuan Liu ◽

Ye Lu ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Lung Fibroblasts ◽

Current Evidence ◽

Extracellular Acidification ◽

Fatal Disease ◽

Growth Factor Beta ◽

Lactate Levels ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the normal alveolar network is gradually replaced by fibrotic scars. Current evidence suggests that metabolic alterations correlate with myofibroblast activation in IPF. Anlotinib has been proposed to have antifibrotic effects, but the efficacy and mechanisms of anlotinib against lung fibrosis have not been systematically evaluated. The antifibrotic effects of anlotinib were evaluated in bleomycin-induced mouse models and transforming growth factor-beta 1 (TGF-β1)-stimulated lung fibroblasts. We measured lactate levels, 2-NBDG glucose uptake and the extracellular acidification rate (ECAR) to assess glycolysis in fibroblasts. RNA-protein coimmunoprecipitation (RIP) and polysome analyses were performed to investigate novel mechanisms of glycolytic reprogramming in pulmonary fibrosis. We found that anlotinib diminished myofibroblast activation and inhibited the augmentation of glycolysis. Moreover, we show that PCBP3 posttranscriptionally increases PFKFB3 expression by promoting its translation during myofibroblast activation, thus promoting glycolysis in myofibroblasts. Regarding mechanism, anlotinib exerts potent antifibrotic effects by downregulating PCBP3, reducing PFKFB3 translation and inhibiting glycolysis in myofibroblasts. Furthermore, we observed that anlotinib had preventative and therapeutic antifibrotic effects on bleomycin-induced pulmonary fibrosis. Therefore, we identify PCBP3 as a protein involved in the regulation of glycolysis reprogramming and lung fibrogenesis and propose it as a therapeutic target for pulmonary fibrosis. Our data suggest that anlotinib has antifibrotic effects on the lungs, and we provide a novel mechanism for this effect. Anlotinib may constitute a novel and potent candidate for the treatment of pulmonary fibrosis.

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TGF-β-induced α-SMA expression is mediated by C/EBPβ acetylation in human alveolar epithelial cells

10.21203/rs.3.rs-76632/v3 ◽

2021 ◽

Author(s):

Hui Ding ◽

Jinjun Chen ◽

Jingping Qin ◽

Ruhua Chen ◽

Zili Yi

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

A549 Cells ◽

Alveolar Epithelial Cells ◽

Collagen I ◽

Sirtuin 1 ◽

Mesenchymal Transition ◽

Matrix Deposition ◽

Tgf Β1

Abstract Background: Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF. Object: Through in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein β (C/EBPβ) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF. Methods: The expression of EMT and C/EBPβ in A549 cells treated with transforming growth factor-beta (TGF-β) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPβ with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPβ was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored. Results: It was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. Conclusion: The EMT and fibrotic effect of TGF-β1 is dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. Thus, C/EBPβ acetylation may play a central role in pulmonary fibrosis.

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Activated mTORC1-SIRT6 Mediates Myofibroblast Differentiation and Idiopathic Pulmonary Fibrosis

10.21203/rs.3.rs-587874/v1 ◽

2021 ◽

Author(s):

Ji Zhang ◽

Yi Hu ◽

Huiping Huang ◽

Qun Liu ◽

Yang Du ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathway ◽

Mtor Signaling ◽

Signalling Pathway ◽

Lung Fibroblasts ◽

Myofibroblast Differentiation ◽

Dependent Manner ◽

Mtor Signaling Pathway ◽

Mtorc1 Pathway ◽

Tgf Β1

Abstract BackgroundIdiopathic pulmonary fibrosis (IPF) is characterised by accumulation of myofibroblasts and deposition of extracellular matrix proteins. Fibroblast-to-myofibroblast transdifferentiation and myofibroblast hyperproliferation plays a major role in pulmonary fibrosis. Moreover, mTOR signaling pathway and SIRT6 play a critical role in pulmonary fibrosis. However, the mechanisms whether SIRT6 affect the myofibroblasts differentiation during IPF remain unclear.MethodWe investigated myofibroblast differentiation using a bleomycin-induced mouse pulmonary fibrosis model and TGF-b1 induced human fetal lung fibroblasts (MRC5) in vitro. We used both SIRT6 siRNA and rapamycin to study the role of SIRT6 and mTOR signaling pathway in the normal human lung fibroblasts and the myofibroblasts from human IPF lungs.ResultsOur data show that high level of SIRT6 was detected in IPF samples, and SIRT6 was signiﬁcantly upregulated by TGF-β1 in a time and concentration-dependent manner. SIRT6 expression and activation of mTORC1 signalling pathway were upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. Furthermore, rapamycin treatment inhibited mTORC1 pathway activity and SIRT6 protein expression. SIRT6 SiRNA failed to mediate the activity of mTORC1 pathway and autophagy induction. However, SIRT6 knockdown could promote TGF-b1 induced pro-fibrotic cytokines.ConclusionActivated mTORC1 signalling pathway regulated SIRT6 overexpression. Deficiency of SIRT6 mediated myofibroblasts differentiation through induced pro-fibrotic cytokines production in the present of TGF-β1. The study indicated that manipulations of SIRT6 expression may provide a new therapeutic strategy to prevent and reverse the progression of pulmonary fibrosis.

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Inhibition of miR-182-5p attenuates pulmonary fibrosis via TGF-β/Smad pathway

Human & Experimental Toxicology ◽

10.1177/0960327119895549 ◽

2019 ◽

Vol 39 (5) ◽

pp. 683-695 ◽

Cited By ~ 2

Author(s):

Y Chen ◽

Q Zhang ◽

Y Zhou ◽

Z Yang ◽

M Tan

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Lung Fibroblasts ◽

In Vitro Assays ◽

Luciferase Reporter ◽

High Morbidity ◽

Human Embryonic Lung ◽

Promising Therapeutic Approach ◽

Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with high morbidity and mortality. miR-182-5p is overexpressed in several fibrosis-related diseases but its effect in pulmonary fibrosis has not been reported yet. To investigate the function of miR-182-5p in pulmonary fibrosis, we established bleomycin (BLM)-induced fibrotic mice model and transforming growth factor-β1 (TGF-β1)-treated human embryonic lung fibroblasts model. In this study, miR-182-5p was highly expressed in pulmonary tissues of BLM-induced fibrotic mice. The content of hydroxyproline and TGF-β1 was decreased by downregulating the expression of miR-182-5p, indicating that fibrosis was alleviated in mice treated with Lentivirus-anti-miR-182-5p.Quantification of fibrosis-related proteins demonstrated that downregulation of miR-182-5p inhibited the expression of profibrotic proteins (fibronectin, α-smooth muscle actin, p-Smad2/p-Smad3) as well as enhanced the level of Smad7. In vitro assays validated that miR-182-5p was induced by TGF-β1 with the function of promoting fibrosis. In dual-luciferase reporter assay, Smad7 was demonstrated to be negatively regulated by miR-182-5p. Moreover, the effect of knocking down miR-182-5p on inhibiting fibrosis was achieved by upregulating the expression of Smad7. Therefore, miR-182-5p can be regarded as a biomarker of IPF and its inhibition may be a promising therapeutic approach in treating IPF.

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NADPH oxidase DUOX1 sustains TGF-β1 signalling and promotes lung fibrosis

European Respiratory Journal ◽

10.1183/13993003.01949-2019 ◽

2020 ◽

pp. 1901949

Author(s):

Ruy Andrade Louzada ◽

Raphaël Corre ◽

Rabii Ameziane El Hassani ◽

Lydia Meziani ◽

Madeleine Jaillet ◽

...

Keyword(s):

Extracellular Matrix ◽

Pulmonary Fibrosis ◽

Nadph Oxidase ◽

Lung Fibrosis ◽

Transforming Growth Factor ◽

Lung Fibroblasts ◽

Mouse Lung ◽

Epithelial Surface ◽

Tgf Β1

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of pulmonary fibrosis, and is governed by transforming growth factor (TGF-β1)/Smad signalling. TGF-β1 and oxidative stress cooperate to drive fibrosis. Cells can produce reactive oxygen species (ROS) through activation and/or induction of NADPH oxidases, such as dual oxidase (DUOX1/2). Since DUOX enzymes, as extracellular H2O2-generating systems, are involved in extracellular matrix formation and in wound healing in different experimental models, we hypothesised that DUOX-based NADPH oxidase plays a role in the pathophysiology of pulmonary fibrosis.Our in vivo data (IPF patients and mouse models of lung fibrosis) showed that the NADPH oxidase DUOX1 is induced in response to lung injury. DUOX1-deficient mice (DUOX1+/- and DUOX1-/-) had an attenuated fibrotic phenotype. In addition to being highly expressed at the epithelial surface of airways, DUOX1 appears to be also well expressed in the fibroblastic foci of remodelled lungs. By using primary human and mouse lung fibroblasts, we showed that TGF-β1 upregulates DUOX1 and its maturation factor DUOXA1 and that DUOX1-derived H2O2 promoted the duration of TGF-β1-activated Smad3 phosphorylation by preventing phospho-Smad3 degradation. Analysis of the mechanism revealed that DUOX1 inhibited the interaction between phospho-Smad3 and the ubiquitin ligase NEDD4L, preventing NEDD4L-mediated ubiquitination of phospho-Smad3 and its targeting for degradation.These findings highlight a role for DUOX1-derived H2O2 in a positive feedback that amplifies the signalling output of the TGF-β1 pathway and identify DUOX1 as a new therapeutic target in pulmonary fibrosis.

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TGF-β-induced α-SMA expression is mediated by C/EBPβ acetylation in human alveolar epithelial cells

Molecular Medicine ◽

10.1186/s10020-021-00283-6 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Hui Ding ◽

Jinjun Chen ◽

Jingping Qin ◽

Ruhua Chen ◽

Zili Yi

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

A549 Cells ◽

Alveolar Epithelial Cells ◽

Collagen I ◽

Sirtuin 1 ◽

Mesenchymal Transition ◽

Matrix Deposition ◽

Tgf Β1

Abstract Background Although the morbidity and mortality rates associated with idiopathic pulmonary fibrosis (IPF) are high, there is still lack of powerful and precise therapeutic options for IPF. Object Through in vitro model, this study sought to determine whether binding of acetylated CCAAT/enhancer binding protein β (C/EBPβ) to alpha-smooth muscle actin (α-SMA) promoter could affect the activity of the latter as well as assess if it is essential for epithelial-to-mesenchymal transition (EMT) and extracellular matrix deposition in IPF. Methods The expression of EMT and C/EBPβ in A549 cells treated with transforming growth factor-beta (TGF-β) as pulmonary fibrotic model was detected by western blotting and qPCR. Collagen-I expression using ELISA was performed. The luciferase activity was used to examine the activity of C/EBPβ. Knockdown of C/EBPβ was performed by siRNA. We also investigated the effect of deacetylation of C/EBPβ on EMT using sirtuin 1 (SIRT1). The binding ability of C/EBPβ with α-SMA promoter was affirmed via chromatin immunoprecipitation (ChIP) and electrophoresis mobility shift assay (EMSA). The relationship between α-SMA and acetylated C/EBPβ was determined with co-immunoprecipitation (Co-IP). SiRNA-mediated knockdown of C/EBPβ in A549 cells attenuated TGF-β1-induced myofibroblast differentiation and ECM deposition. The extent of association between acetylated C/EBPβ and α-SMA promoter was dynamically monitored. Results It was confirmed that deacetylation of C/EBPβ in A549 cells successfully ameliorated TGF-β1-induced EMT, as shown by reduction in α-SMA expression and excessive collagen-I accumulation. Conclusion The EMT and fibrotic effect of TGF-β1 is dependent on acetylated C/EBPβ-mediated regulation of α-SMA gene activity. Thus, C/EBPβ acetylation may play a central role in pulmonary fibrosis.

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HuR drives lung fibroblast differentiation but not metabolic reprogramming in response to TGF-β and hypoxia

Respiratory Research ◽

10.1186/s12931-021-01916-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Joshua Trivlidis ◽

Noof Aloufi ◽

Fatmah Al-Habeeb ◽

Parameswaran Nair ◽

Ilan Azuelos ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Rna Binding ◽

Transforming Growth Factor ◽

Metabolic Reprogramming ◽

Proton Nuclear Magnetic Resonance ◽

Lung Fibroblasts ◽

Myofibroblast Differentiation ◽

Mrna Levels ◽

Metabolic Shift ◽

Tgf Β1

Abstract Background Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-β1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs). Methods Primary HLFs were cultured in the presence (or absence) of TGF-β1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down. Results Hypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFβ1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-β1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion. Conclusions Hypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-β. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.

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