title: Laminin-111 stimulates proliferation of mouse embryonic stem cells through a reduction of gap junctional intercellular communication via RhoA-mediated Cx43 phosphorylation and dissociation of Cx43/ZO-1/drebrin complex.
abstract: Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation.
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea.
authors.forename: Han Na
authors.initials: HN
authors.lastname: Suh
authors.forename: Mi Ok
authors.initials: MO
authors.lastname: Kim
authors.forename: Ho Jae
authors.initials: HJ
authors.lastname: Han
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D049109:Cell Proliferation; D002738:Chloroquine / Q000494:pharmacology; D018031:Connexin 43 / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism; D005353:Fibronectins / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007546:Isoquinolines / Q000378:metabolism; D007797:Laminin / Q000494:pharmacology*; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017474:Receptors, Laminin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
chemical_list: D000199:Actins; D018031:Connexin 43; D005353:Fibronectins; D007546:Isoquinolines; D007797:Laminin; D046912:Multiprotein Complexes; D009479:Neuropeptides; D011480:Protease Inhibitors; D034741:RNA, Small Interfering; D017474:Receptors, Laminin; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C053386:drebrins; C067713:lactacystin; C119129:18alpha-glycyrrhetinic acid; D002738:Chloroquine; C017475:lucifer yellow; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D006034:Glycyrrhetinic Acid; D000111:Acetylcysteine