J H Park1, M Y Lee, J S Heo, H J Han. 1. Department of Veterinary Physiology, Biotherapy Human Resources Center (BK21), College of Veterinary Medicine, Chonnam National University, Gwangju, South Korea.
Abstract
OBJECTIVES: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. MATERIALS AND METHODS: Expressions of Cx43, mitogen-activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [(3)H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. RESULTS: The results showed that EGF increased the level of Cx43 phosphorylation in a time- (> or =5 min) and dose- (> or =10 ng/mL) dependent manner. Indeed, EGF-induced increase in phospho-Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca(2+) influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre-treatment with BAPTA-AM (an intracellular Ca(2+) chelator), EGTA (an extracellular Ca(2+) chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF-induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18alpha-glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c-fos, c-jun and c-myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and p-Rb], [(3)H]thymidine incorporation and cell number, but decreased expression levels of the p21(WAF1/Cip1) and p27(Kip1), CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [(3)H]thymidine incorporation and cell number. EGF, 18alpha-GA or transfection of Cx43 siRNA increased 2-DG uptake and GLUT-1 protein expression. CONCLUSIONS: EGF-induced phosphorylation of Cx43, which was mediated by the Ca(2+)/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.
OBJECTIVES: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. MATERIALS AND METHODS: Expressions of Cx43, mitogen-activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [(3)H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. RESULTS: The results showed that EGF increased the level of Cx43 phosphorylation in a time- (> or =5 min) and dose- (> or =10 ng/mL) dependent manner. Indeed, EGF-induced increase in phospho-Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca(2+) influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre-treatment with BAPTA-AM (an intracellular Ca(2+) chelator), EGTA (an extracellular Ca(2+) chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF-induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18alpha-glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c-fos, c-jun and c-myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and p-Rb], [(3)H]thymidine incorporation and cell number, but decreased expression levels of the p21(WAF1/Cip1) and p27(Kip1), CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [(3)H]thymidine incorporation and cell number. EGF, 18alpha-GA or transfection of Cx43 siRNA increased 2-DG uptake and GLUT-1 protein expression. CONCLUSIONS:EGF-induced phosphorylation of Cx43, which was mediated by the Ca(2+)/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.
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