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Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

ABSTRACT: Extracellular matrix (ECM) stiffness influences gene expression, leading to modulation of various cellular functions. While ROCK2 regulates actomyosin activity as well as cell migration and proliferation, expression of ROCK2 is increased in response to stiffening ECM. However, the mechanism underlying rigidity-dependent ROCK2 expression remains elusive. Here, we show that YAP, a mechanically regulated transcription coactivator, upregulates ROCK2 expression in an ECM rigidity-dependent manner. YAP interacted with the ROCK2 promoter region in an actomyosin activity-dependent manner. Knockdown of YAP decreased ROCK2 expression while activity of the ROCK2 promoter was upregulated by expressing constitutively active YAP. Furthermore, we found that ROCK2 expression promotes transcriptional activation by YAP. Our results reveal a novel positive feedback loop between YAP and ROCK2, which is modulated by ECM stiffness.

SUBMITTER: Sugimoto W

PROVIDER: S-EPMC5927696 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

Sugimoto Wataru W Itoh Katsuhiko K Mitsui Yasumasa Y Ebata Takahiro T Fujita Hideaki H Hirata Hiroaki H Kawauchi Keiko K

Cell adhesion & migration 20180129 2

Extracellular matrix (ECM) stiffness influences gene expression, leading to modulation of various cellular functions. While ROCK2 regulates actomyosin activity as well as cell migration and proliferation, expression of ROCK2 is increased in response to stiffening ECM. However, the mechanism underlying rigidity-dependent ROCK2 expression remains elusive. Here, we show that YAP, a mechanically regulated transcription coactivator, upregulates ROCK2 expression in an ECM rigidity-dependent manner. YA ...[more]

PMID: 28686514

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Project description:BackgroundThe transforming growth factor-β (TGF-β) signaling pathway plays an essential role in maintaining homeostasis in joints affected by osteoarthritis (OA). However, the specific mechanism of non-SMAD and classical SMAD signaling interactions is still unclear, which needs to be further explored.MethodsIn ATDC5 cells, USP15 overexpression and knockout were performed using the transfected lentivirus USP15 and Crispr/Cas9. Western blotting and immunofluorescence staining were used to test p-SMAD2 and cartilage phenotype-related molecular markers. In rat OA models, immunohistochemistry, hematoxylin and eosin (HE)/Safranin-O fast green staining, and histology were used to examine the regulatory activity of USP15 in TGF-β/SMAD2 signaling and the cartilage phenotype. Then, ERK2 overexpression and knockout were performed. The expressions of USP15, p-SMAD2, and the cartilage phenotype were evaluated in vitro and in vivo. To address whether USP15 is required for ERK2 and TGF-β/SMAD2 signaling, we performed rescue experiments in vitro and in vivo. Immunoprecipitation and deubiquitination assays were used to examine whether USP15 could bind to ERK2 and affect the deubiquitination of ERK2. Finally, whether USP15 regulates the level of p-ERK1/2 was evaluated by western blotting, immunofluorescence staining, and immunohistochemistry in vitro and in vivo.ResultsOur results indicated that USP15 stimulated TGF-β/SMAD2 signaling and the cartilage phenotype. Moreover, ERK2 required USP15 to influence TGF-β/SMAD2 signaling for regulating the cartilage phenotype in vivo and in vitro. And USP15 can form a complex with ERK2 to regulate ubiquitination of ERK2. Interestingly, USP15 did not regulate the stability of ERK2 but increased the level of p-ERK1/2 to further enhance the TGF-β/SMAD2 signaling pathway.ConclusionsTaken together, our study revealed positive feedback regulation between USP15 and ERK2, which played a critical role in TGF-β/SMAD2 signaling to inhibit OA progression. Therefore, this specific mechanism can guide the clinical treatment of OA.

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Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

Publications

Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

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