TY - JOUR
T1 - TGF-β1-induced miR-424 promotes pulmonary myofibroblast differentiation by targeting Slit2 protein expression
AU - Huang, Yapei
AU - Xie, Yan
AU - Abel, Peter W.
AU - Wei, Peng
AU - Plowman, Jocelyn
AU - Toews, Myron L.
AU - Strah, Heather
AU - Siddique, Aleem
AU - Bailey, Kristina L.
AU - Tu, Yaping
N1 - Funding Information:
We thank Dr. Reynold A. Panettieri (Rutgers University) for providing HLFs and Lyudmila Batalkina for her excellent technical assistance. This work was supported, in part, by the National Institutes of Health (NIH) grants R01HL116849 and 5R21ES029566 (Y.T. and P.W.A), and R01AG053553 (K.B.), and Nebraska State LB595 program (Y.T.). Immunofluorescence staining images of HLFs were obtained at the Integrated Biomedical Imaging Facility of Creighton University. This facility is supported by the Creighton University School of Medicine and the National Institute of General Medical Science (NIGMS) grants GM103427 and GM110768. The facility was constructed with support from the National Center for Research Resources (RR016469) and the NIGMS (GM103427). This investigation is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH.
Funding Information:
We thank Dr. Reynold A. Panettieri (Rutgers University) for providing HLFs and Lyudmila Batalkina for her excellent technical assistance. This work was supported, in part, by the National Institutes of Health (NIH) grants R01HL116849 and 5R21ES029566 (Y.T. and P.W.A), and R01AG053553 (K.B.), and Nebraska State LB595 program (Y.T.). Immunofluorescence staining images of HLFs were obtained at the Integrated Biomedical Imaging Facility of Creighton University . This facility is supported by the Creighton University School of Medicine and the National Institute of General Medical Science ( NIGMS ) grants GM103427 and GM110768 . The facility was constructed with support from the National Center for Research Resources ( RR016469 ) and the NIGMS ( GM103427 ). This investigation is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH.
Publisher Copyright:
© 2020 Elsevier Inc.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/10
Y1 - 2020/10
N2 - Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. Myofibroblasts in the lung are key cellular mediators of IPF progression. Transforming growth factor (TGF)-β1, a major profibrogenic cytokine, induces pulmonary myofibroblast differentiation, and emerging evidence has established the importance of microRNAs (miRs) in the development of IPF. The objective of this study was to define the pro-fibrotic roles and mechanisms of miRs in TGF-β1-induced pulmonary myofibroblast differentiation. Using RNA sequencing, we identified miR-424 as an important TGF-β1-induced miR in human lung fibroblasts (HLFs). Quantitative RT-PCR confirmed that miR-424 expression was increased by 2.6-fold in HLFs in response to TGF-β1 and was 1.7-fold higher in human fibrotic lung tissues as compared to non-fibrotic lung tissues. TGF-β1-induced upregulation of miR-424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of this canonical TGF-β1 signaling pathway. Transfection of a miR-424 hairpin inhibitor into HLFs reduced TGF-β1-induced expression of classic myofibroblast differentiation markers including ɑ-smooth muscle actin (ɑ-SMA) and connective tissue growth factor (CTGF), whereas a miR-424 mimic significantly enhanced TGF-β1-induced myofibroblast differentiation. In addition, TGF-β1 induced Smad3 phosphorylation in HLFs, and this response was reduced by the miR-424 inhibitor. In silico analysis identified Slit2, a protein that inhibits TGF-β1 profibrogenic signaling, as a putative target of regulation by miR-424. Slit2 is less highly expressed in human fibrotic lung tissues than in non-fibrotic lung tissues, and knockdown of Slit2 by its siRNA enhanced TGF-β1-induced HLF differentiation. Overexpression of a miR-424 mimic down-regulated expression of Slit2 but not the Slit2 major receptor ROBO1 in HLFs. Luciferase reporter assays showed that the miR-424 mimic represses Slit2 3′ untranslated region (3′-UTR) reporter activity, and mutations at the seeding regions in the 3′-UTR of Slit2 abolish this inhibition. Together, these data demonstrate a pro-fibrotic role of miR-424 in TGF-β1-induced HLF differentiation. It functions as a positive feed-back regulator of the TGF-β1 signaling pathway by reducing expression of the negative regulator Slit2. Thus, targeting miR-424 may provide a new therapeutic strategy to prevent myofibroblast differentiation and IPF progression.
AB - Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. Myofibroblasts in the lung are key cellular mediators of IPF progression. Transforming growth factor (TGF)-β1, a major profibrogenic cytokine, induces pulmonary myofibroblast differentiation, and emerging evidence has established the importance of microRNAs (miRs) in the development of IPF. The objective of this study was to define the pro-fibrotic roles and mechanisms of miRs in TGF-β1-induced pulmonary myofibroblast differentiation. Using RNA sequencing, we identified miR-424 as an important TGF-β1-induced miR in human lung fibroblasts (HLFs). Quantitative RT-PCR confirmed that miR-424 expression was increased by 2.6-fold in HLFs in response to TGF-β1 and was 1.7-fold higher in human fibrotic lung tissues as compared to non-fibrotic lung tissues. TGF-β1-induced upregulation of miR-424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of this canonical TGF-β1 signaling pathway. Transfection of a miR-424 hairpin inhibitor into HLFs reduced TGF-β1-induced expression of classic myofibroblast differentiation markers including ɑ-smooth muscle actin (ɑ-SMA) and connective tissue growth factor (CTGF), whereas a miR-424 mimic significantly enhanced TGF-β1-induced myofibroblast differentiation. In addition, TGF-β1 induced Smad3 phosphorylation in HLFs, and this response was reduced by the miR-424 inhibitor. In silico analysis identified Slit2, a protein that inhibits TGF-β1 profibrogenic signaling, as a putative target of regulation by miR-424. Slit2 is less highly expressed in human fibrotic lung tissues than in non-fibrotic lung tissues, and knockdown of Slit2 by its siRNA enhanced TGF-β1-induced HLF differentiation. Overexpression of a miR-424 mimic down-regulated expression of Slit2 but not the Slit2 major receptor ROBO1 in HLFs. Luciferase reporter assays showed that the miR-424 mimic represses Slit2 3′ untranslated region (3′-UTR) reporter activity, and mutations at the seeding regions in the 3′-UTR of Slit2 abolish this inhibition. Together, these data demonstrate a pro-fibrotic role of miR-424 in TGF-β1-induced HLF differentiation. It functions as a positive feed-back regulator of the TGF-β1 signaling pathway by reducing expression of the negative regulator Slit2. Thus, targeting miR-424 may provide a new therapeutic strategy to prevent myofibroblast differentiation and IPF progression.
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U2 - 10.1016/j.bcp.2020.114172
DO - 10.1016/j.bcp.2020.114172
M3 - Article
C2 - 32712053
AN - SCOPUS:85088991469
VL - 180
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
SN - 0006-2952
M1 - 114172
ER -