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Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

ABSTRACT: We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker ?-smooth muscle actin (?-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-?1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (?NLS kindlin-2) results in increased ?-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced ?-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the ?-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ?NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of ?-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

SUBMITTER: Godbout E

PROVIDER: S-EPMC7766948 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

Godbout Elena E Son Dong Ok DO Hume Stephanie S Boo Stellar S Sarrazy Vincent V Clément Sophie S Kapus Andras A Wehrle-Haller Bernhard B Bruckner-Tuderman Leena L Has Cristina C Hinz Boris B

Cells 20201217 12

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-β1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonist ...[more]

PMID: 33348602

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Project description:BACKGROUND & AIMS:Hepatic stellate cells (HSCs) contribute to desmoplasia and stiffness of liver metastases by differentiating into matrix-producing myofibroblasts. We investigated whether stiffness due to the presence of tumors increases activation of HSCs into myofibroblasts and their tumor-promoting effects, as well as the role of E1A binding protein p300, a histone acetyltransferase that regulates transcription, in these processes. METHODS:HSCs were isolated from liver tissues of patients, mice in which the p300 gene was flanked by 2 loxP sites (p300F/F mice), and p300+/+ mice (controls). The HSCs were placed on polyacrylamide gels with precisely defined stiffness, and their activation (differentiation into myofibroblasts) was assessed by immunofluorescence and immunoblot analyses for alpha-smooth muscle actin. In HSCs from mice, the p300 gene was disrupted by cre recombinase. In human HSCs, levels of p300 were knocked down with small hairpin RNAs or a mutant form of p300 that is not phosphorylated by AKT (p300S1834A) was overexpressed. Human HSCs were also cultured with inhibitors of p300 (C646), PI3K signaling to AKT (LY294002), or RHOA (C3 transferase) and effects on stiffness-induced activation were measured. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to identify HSC genes that changed expression levels in response to stiffness. We measured effects of HSC-conditioned media on proliferation of HT29 colon cancer cells and growth of tumors following subcutaneous injection of these cells into mice. MC38 colon cancer cells were injected into portal veins of p300F/Fcre and control mice, and liver metastases were measured. p300F/Fcre and control mice were given intraperitoneal injections of CCl4 to induce liver fibrosis. Liver tissues were collected and analyzed by immunofluorescence, immunoblot, and histology. RESULTS:Substrate stiffness was sufficient to activate HSCs, leading to nuclear accumulation of p300. Disrupting p300 level or activity blocked stiffness-induced activation of HSCs. In HSCs, substrate stiffness activated AKT signaling via RHOA to induce phosphorylation of p300 at serine 1834; this caused p300 to translocate to the nucleus, where it up-regulated transcription of genes that increase activation of HSCs and metastasis, including CXCL12. MC38 cells, injected into portal veins, formed fewer metastases in livers of p300F/Fcre mice than control mice. Expression of p300 was increased in livers of mice following injection of CCl4; HSC activation and collagen deposition were reduced in livers of p300F/Fcre mice compared with control mice. CONCLUSIONS:In studies of mice, we found liver stiffness to activate HSC differentiation into myofibroblasts, which required nuclear accumulation of p300. p300 increases HSC expression of genes that promote metastasis.

Dataset Information

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

Publications

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts.

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