Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea.
Stem Cells Dev. 2012 Jul 20;21(11):2058-70. doi: 10.1089/scd.2011.0505. Epub 2012 Jan 26.
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.
细胞外基质 (ECM) 界定的间隙连接确保了即将成为调节细胞行为的功能介质的细胞之间的同步活动。然而，ECM 在调节小鼠胚胎干细胞 (mESC) 间隙连接蛋白 (Cx) 功能中的作用尚未阐明。因此，我们研究了层粘连蛋白-111 在控制 mESC 中 Cx43 功能和相关信号通路中的作用。ECM 成分（层粘连蛋白-111、纤维连接蛋白和胶原 I）增加了 Cx43 的磷酸化，并减少了 Lucifer yellow（Ly）的扩散。此外，层粘连蛋白-111 通过减少缝隙连接细胞间通讯 (GJIC) 来增加增殖指数，这一点通过 18α-甘草次酸（18α-GA）得到了证实。层粘连蛋白-111 增加了粘着斑激酶 (FAK)/Src 和蛋白激酶 C (PKC) 的磷酸化，这分别被整合素 β1 抗体 (Ab) 和层粘连蛋白受体-1 (LR-1) Ab 抑制。此外，抑制 FAK/Src 和 PKC 均可阻断 Cx43 的磷酸化。层粘连蛋白-111 增加了 Ras 同源基因家族成员 A (RhoA) 的激活，该激活被 FAK/Src 和 PKC 抑制剂阻断，表明存在着在 RhoA 处合并的平行途径。RhoA 抑制剂的抑制作用逆转了层粘连蛋白-111 诱导的 Cx43 磷酸化增加和 GJIC 减少。层粘连蛋白-111 还刺激 Cx43/ZO-1 复合物的解离，随后破坏 Cx43/drebrin 和 Cx43/F-肌动蛋白复合物，这可被 C3（RhoA 抑制剂）逆转。ZO-1 小干扰 (si) RNA 显著减少了 Ly 的扩散。此外，层粘连蛋白-111 减少了细胞间连接处的 Cx43 标记，而用降解抑制剂（溶酶体蛋白酶抑制剂氯喹；蛋白酶体抑制剂乳清酸）预处理则增加了 Cx43 的表达，呈相反效果。总之，层粘连蛋白-111 通过 RhoA 介导的 Cx43 磷酸化和 Cx43/ZO-1/drebrin 复合物不稳定性介导的 Cx43 降解来减少 GJIC，从而刺激 mESC 的增殖。
