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Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

ABSTRACT: Increased production and assembly of extracellular matrix proteins during transdifferentiation of epithelial cells to a mesenchymal phenotype contributes to diseases such as renal and pulmonary fibrosis. TGF-β and hypoxia, two cues that initiate injury-induced fibrosis, caused human kidney cells to develop a mesenchymal phenotype, including increased fibronectin expression and secretion. However, upon hypoxia, assembled extracellular fibronectin fibrils were mostly absent, whereas treatment with TGF-β led to abundant fibrils. Fibrillogenesis required cell-generated force and tension. TGF-β, but not hypoxia, increased cell contractility, as determined by phosphorylation of myosin light chain and quantifying force and tension generated by cells plated on engineered elastomeric microposts. Additionally, TGF-β, but not hypoxia, increased the activation of integrins. However, experimentally activating integrins markedly increased the levels of phosphorylated myosin light chain and fibronectin fibril assembly upon hypoxia. Our findings show that deficient integrin activation and subsequent lack of cell contractility are mechanisms that mediate a lack of fibrillogenesis upon hypoxia and they challenge current views on oxygen deprivation being sufficient for fibrosis.

SUBMITTER: Rana MK

PROVIDER: S-EPMC4359919 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

Rana Manish K MK Srivastava Jyoti J Yang Michael M Chen Christopher S CS Barber Diane L DL

Journal of cell science 20150122 6

Increased production and assembly of extracellular matrix proteins during transdifferentiation of epithelial cells to a mesenchymal phenotype contributes to diseases such as renal and pulmonary fibrosis. TGF-β and hypoxia, two cues that initiate injury-induced fibrosis, caused human kidney cells to develop a mesenchymal phenotype, including increased fibronectin expression and secretion. However, upon hypoxia, assembled extracellular fibronectin fibrils were mostly absent, whereas treatment with ...[more]

PMID: 25616899

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Project description:Transforming growth factor-beta 1 (TGF-β1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-β1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-β1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-β1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-β1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Dataset Information

Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

Publications

Hypoxia increases the abundance but not the assembly of extracellular fibronectin during epithelial cell transdifferentiation.

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