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?V-integrins are required for mechanotransduction in MDCK epithelial cells.

ABSTRACT: The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding ?V-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that ?V-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. ?V-integrin-deficient MDCK cells bound collagen I (Col I) substrate via ?2?1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature ?2?1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in ?V-integrin deficient cells seeded onto Col I substrate.

SUBMITTER: Teravainen TP

PROVIDER: S-EPMC3747215 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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αV-integrins are required for mechanotransduction in MDCK epithelial cells.

Teräväinen Terhi P TP Myllymäki Satu M SM Friedrichs Jens J Strohmeyer Nico N Moyano Jose V JV Wu Chuanyue C Matlin Karl S KS Muller Daniel J DJ Manninen Aki A

PloS one 20130819 8

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epit ...[more]

PMID: 23977051

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Project description:Epithelial-mesenchymal transition (EMT) is an important process in embryonic development, especially during gastrulation and organ formation. Furthermore EMT is widely observed in pathological conditions, e.g., fibrosis, tumor progression and metastasis. Madin-Darby Canine Kidney (MDCK) cells are widely used for studies of EMT and epithelial plasticity. MDCK cells show an epithelial phenotype, while oncogenic Ras-transformed MDCK (MDCK-Ras) cells undergo EMT and show a mesenchymal phenotype.RNA-Seq and miRNA-Seq analyses were performed on MDCK and MDCK-Ras cells. Data were validated by qRT-PCR. Gene signature analyses were carried out to identify pathways and gene ontology terms. For selected miRNAs target prediction was performed.With RNA-Seq, mRNAs of approximately half of the genes known for dog were detected. These were screened for differential regulation during Ras-induced EMT. We went further and performed gene signature analyses and found Gene Ontology (GO) terms and pathways important for epithelial polarity and implicated in EMT. Among the identified pathways, TGF?1 emerged as a central signaling factor in many EMT related pathways and biological processes. With miRNA-Seq, approximately half of the known canine miRNAs were found expressed in MDCK and MDCK-Ras cells. Furthermore, among differentially expressed miRNAs, miRNAs that are known to be important regulators of EMT were detected and new candidates were predicted. New dog miRNAs were discovered after aligning our reads to that of other species in miRBase. Importantly, we could identify 25 completely novel miRNAs with a stable hairpin structure. Two of these novel miRNAs were differentially expressed. We validated the two novel miRNAs with the highest read counts by RT-qPCR. Target prediction of a particular novel miRNA highly expressed in mesenchymal MDCK-Ras cells revealed that it targets components of epithelial cell junctional complexes. Combining target prediction for the most upregulated miRNAs and validation of the targets in MDCK-Ras cells with pathway analysis allowed us to identify two novel pathways, e.g., JAK/STAT signaling and pancreatic cancer pathways. These pathways could not be detected solely by gene set enrichment analyses of RNA-Seq data.With deep sequencing data of mRNAs and miRNAs of MDCK cells and of Ras-induced EMT in MDCK cells, differentially regulated mRNAs and miRNAs are identified. Many of the identified genes are within pathways known to be involved in EMT. Novel differentially upregulated genes in MDCK cells are interferon stimulated genes and genes involved in Slit and Netrin signaling. New pathways not yet linked to these processes were identified. A central pathway in Ras induced EMT is TGF? signaling, which leads to differential regulation of many target genes, including miRNAs. With miRNA-Seq we identified miRNAs involved in either epithelial cell biology or EMT. Finally, we describe completely novel miRNAs and their target genes.

Dataset Information

?V-integrins are required for mechanotransduction in MDCK epithelial cells.

Publications

αV-integrins are required for mechanotransduction in MDCK epithelial cells.

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