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 18a-glycyrrhetinic acid (18a-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin b1 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.

Autotaxin-LPA Axis Regulates hMSC Migration by Adherent Junction Disruption and Cytoskeletal Rearrangement Via LPAR1/3-Dependent PKC/GSK3β/β-Catenin and PKC/Rho GTPase Pathways

“…Previous studies have shown that the activation of Ga q or Gbc i results in the stimulation of phospholipase C activity, leading to phosphatidylinositol-(4,5)bisphosphate hydrolysis and Ca 21 mobilization from internal stores, and the subsequent activation of protein kinase C (PKC) [53,54]. In addition, LPA stimulates Ca 21 signaling and PKC activation, which are involved in proliferation, migration, or gap junction regulation in both embryonic stem cells and MSCs [55][56][57][58][59]. In this study, LPA stimulates PKC a, d, and f, which were abolished by Ga i and Ga q siRNA transfections as well as by Ptx pretreatment.…”

Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells

“…Further, loss of Cx43 from neural crest cells results in a loss of β1-integrin immunoreactivity and a reduction in focal contacts (Xu et al, 2006). In contrast, gap junctional communication is reduced in embryonic stem cells (ESC) upon the activation of β1-integrins by laminin binding (Suh et al, 2012) while loss of β1-integrin from ESC derived cardiomyocytes results in the upregulation of connexin expression (Czyz, Guan, 2005). Here we find that loss of β1-integrin from the lens fibers results in an apparent redistribution of Cx50 from the broad faces of the fiber cell to all fiber cell membranes, perhaps explaining the increase in gap junctional coupling observed in cKO lenses.…”

The role of laminins in cartilaginous tissues: from development to regeneration

“…Stage-dependent expression of laminin in cartilage and cartilage-like tissue Increasing evidence indicates that chondrocytes are responsible for the production of various laminins ( Table 1a,b) that mainly locate in the PCM of cartilage SundarRaj et al, 1995). The expression of laminins varies during different developmental stages of cartilage and cartilage-like tissues ( Fig.…”

“…The interactions between laminins and chondrocytes can regulate many cell-biological functions, such as adhesion, migration and survival (Bulić, 1996;Francisco et al, 2014;SundarRaj et al, 1995). Laminin and cartilage regeneration chondrocytes and NP cells (Dürr et al, 1996;Gilchrist et al, 2011a;Gilchrist et al, 2011b).…”