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RSU-1 interaction with prohibitin-2 links cell-extracellular matrix detachment to downregulation of ERK signaling.

ABSTRACT: Cell-extracellular matrix (ECM) detachment is known to decrease extracellular signal-regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell-ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell-ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell-ECM adhesion-induced ERK activation. Furthermore, cell-ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150-206) effectively suppressed the cell-ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell-ECM detachment and links it to decreased ERK signaling.

SUBMITTER: Wang M

PROVIDER: S-EPMC7948471 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration.MethodsWe evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting.ResultsWe demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin.ConclusionsWe demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

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RSU-1 interaction with prohibitin-2 links cell-extracellular matrix detachment to downregulation of ERK signaling.

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