Involvement of Cx43 phosphorylation in 5'-N-ethylcarboxamide-induced migration and proliferation of mouse embryonic stem cells.
Despite a lot of gap junction research, the complex connection between gap junction and cell proliferation remains an exciting area of investigation. Thus, we examined the effect of connexin 43 (Cx43) on the migration and proliferation of embryonic stem (ES) cells and its related signaling pathways following stimulation with the adenosine analogue 5'-N-ethylcarboxamide (NECA). NECA increased phosphorylation of Cx43 which was blocked by caffeine, a non-selective adenosine receptor antagonist. In experiment to measure the gap junctional intercellular communication, NECA blocked transfer of Lucifer yellow to neighboring cells in a scrape loading/dye transfer (SL/DT) assay. In addition, NECA-induced phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and nuclear factor-kappa B (NF-kappaB) signal pathways. Inhibition of these signaling pathways reduced NECA-induced phosphorylation of Cx43. Moreover, NECA-treated cells demonstrated phosphorylation of Src, which was blocked by caffeine. In this experiment, a disruption of Cx43 using Cx43-specific small interfering RNA (siRNA) also enhanced Src phosphorylation. In a further study, phosphorylations of integrin beta1, focal adhesion kinase (FAK), and paxillin by NECA were restrained by caffeine as well as the Src blocker, PP2. Finally, we identified that NECA-stimulated cell migration and expressions of cell-cycle regulatory proteins [cyclin D1, cyclin-dependent kinase (CDK) 4, cyclin E, and CDK2]; these increases were inhibited by caffeine, or PP2. We conclude that NECA-stimulated Cx43 phosphorylation mediated by PI3K/Akt, PKC, MAPKs, and NF-kappaB, which subsequently stimulated cell migration and proliferation through Src, integrin beta1, FAK, and paxillin signal pathways.
Department of Veterinary Physiology, Biotherapy Human Resources Center (BK 21), College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.
Mi Ok
MO
Kim
Yu Jin
YJ
Lee
Ho Jae
HJ
Han
D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D019061:src-Family Kinases / Q000378:metabolism
D019830:Adenosine-5'-(N-ethylcarboxamide); D002110:Caffeine; D018031:Connexin 43; D019938:Cyclin D1; D019927:Cyclin E; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D051417:Focal Adhesion Kinase 1; D019012:Integrin beta1; D020928:Mitogen-Activated Protein Kinases; D016328:NF-kappa B; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D019061:src-Family Kinases; C530105:Ccnd1 protein, mouse; C543076:GJA1 protein, mouse; C496062:Pxn protein, mouse; C496051:Ptk2 protein, mouse; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse