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Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT.

ABSTRACT: Abscission is the final stage of cytokinesis during which the parent cell physically separates to yield two identical daughters. Failure of abscission results in multinucleation (MNC), a sign of genomic instability and a precursor to aneuploidy, enabling characteristics of neoplastic progression. Induction of epithelial-mesenchymal transition (EMT) causes MNC in mammary epithelial cells cultured on stiff microenvironments that have mechanical properties similar to those found in breast tumors, but not on soft microenvironments reminiscent of the normal mammary gland. Here we report that on stiff microenvironments, EMT signaling through Snail up-regulates the midbody-associated proteins septin-6, Mklp1, and anillin, leading to abscission failure and MNC. To uncover the mechanism by which stiff microenvironments promote MNC in cells undergoing EMT, we investigated the role of cell-matrix adhesion through β1-integrin and integrin-linked kinase (ILK). We found that ILK expression, but not kinase activity, is required for EMT-associated MNC in cells on stiff microenvironments. Conversely, increasing focal adhesions by expressing an autoclustering mutant of β1-integrin promotes MNC in cells on soft microenvironments. Our data suggest that signaling through focal adhesions causes failure of cytokinesis in cells actively undergoing EMT. These results highlight the importance of tissue mechanics and adhesion in regulating the cellular response to EMT inducers.

SUBMITTER: Rabie EM

PROVIDER: S-EPMC8684726 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71?kPa) and tissue culture plastic (TCP; > 1?GPa) in media containing 0 or 10?ng/ml TGF?1 for 72?h. Cells were treated with 0.4??M Lat-B or DMSO for 30?min every 24?h for 72?h. RNA was collected from cells and expression of alpha-smooth muscle actin (?-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess ?-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n?=?3) or 25% DMSO (vehicle control, n?=?3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less ?SMA mRNA (P???0.007) for RCF cells grown on 22 and 71?kPa substrates as well as TCP without or with TGF?1, and significantly decreased ?-SMA protein expression in RCFs cultured on the intermediate (22?kPa) stiffness in the absence (P?=?0.028) or presence (P?=?0.018) of TGF?1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P?>?0.05) in the absence or presence of TGF?1, but RCFs grown on stiff hydrogels (71?kPa) had significantly more keratocan mRNA expression versus the 22?kPa hydrogel or TCP (P?<?0.001) without TGF?1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of ?-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.

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Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT.

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