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?II-spectrin and ?II-spectrin do not affect TGF?1-induced myofibroblast differentiation.

ABSTRACT: Mechanosensing of fibroblasts plays a key role in the development of fibrosis. So far, no effective treatments are available to treat this devastating disorder. Spectrins regulate cell morphology and are potential mechanosensors in a variety of non-erythroid cells, but little is known about the role of spectrins in fibroblasts. We investigate whether ?II- and ?II-spectrin are required for the phenotypic properties of adult human dermal (myo)fibroblasts. Knockdown of ?II- or ?II-spectrin in fibroblasts did not affect cell adhesion, cell size and YAP nuclear/cytosolic localization. We further investigated whether ?II- and ?II-spectrin play a role in the phenotypical switch from fibroblasts to myofibroblasts under the influence of the pro-fibrotic cytokine TGF?1. Knockdown of spectrins did not affect myofibroblast formation, nor did we observe changes in the organization of ?SMA stress fibers. Focal adhesion assembly was unaffected by spectrin deficiency, as was collagen type I mRNA expression and protein deposition. Wound closure was unaffected as well, showing that important functional properties of myofibroblasts are unchanged without ?II- or ?II-spectrin. In fact, fibroblasts stimulated with TGF?1 demonstrated significantly lower endogenous mRNA levels of ?II- and ?II-spectrin. Taken together, despite the diverse roles of spectrins in a variety of other cells, ?II- and ?II-spectrin do not regulate cell adhesion, cell size and YAP localization in human dermal fibroblasts and are not required for the dermal myofibroblast phenotypical switch.

SUBMITTER: Piersma B

PROVIDER: S-EPMC6132645 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation.

Piersma Bram B Wouters Olaf Y OY Bank Ruud A RA

Cell and tissue research 20180503 1

Mechanosensing of fibroblasts plays a key role in the development of fibrosis. So far, no effective treatments are available to treat this devastating disorder. Spectrins regulate cell morphology and are potential mechanosensors in a variety of non-erythroid cells, but little is known about the role of spectrins in fibroblasts. We investigate whether αII- and βII-spectrin are required for the phenotypic properties of adult human dermal (myo)fibroblasts. Knockdown of αII- or βII-spectrin in fibro ...[more]

PMID: 29725768

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Project description: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.

Dataset Information

?II-spectrin and ?II-spectrin do not affect TGF?1-induced myofibroblast differentiation.

Publications

αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation.

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