Expression and regulation of aquaporin 3 in human amnion epithelial cells

Abstract

Objective To investigate the role of mitogen-activated protein kinases (MAPK)/extracellular signal regulated kinase1/2 (ERK1/2) signal transduction pathway in regulating the expression of aquaporin 3 (AQP3) in human amnion epithelial cells.Methods Primary cell culture of human amnion epithelial cells deriving from amnion of term pregnancy with normal amniotic fluid volume (AFV) or isolated oligohydramnios was conducted in the Second Affiliated Hospital of Wenzhou Medical College from January to November 2011.Either group included 10 elective cesarean cases.The primarily cultured cells were treated with different concentrations (0,5,10,20 and 40 mol/L) of ERK inhibitors (U0126) for 12 h,and then the optimal concentration of U0126 which resulted in the lowest expression of phospho~ERK1/2 (p-ERK1/2) was added for different durations(0,2,6,12 and 24 h).Immunocytochemistry was used to detect the localization of AQP3 and Western blot analysis was used to examine the expression of total ERK1/2,p-ERK1/2 and AQP3 in human amnion epithelial cells.Statistical analysis was performed by t-test and one-way ANOVA.Results (1) Compared with those in normal AFV group,p-ERK1/2 and AQP3 expression in human amnion epithelium cells decreased in oligohydramnios group,respectively (p-ERK1/2:3.46 ± 0.33 and 2.46±0.25;AQP3:2.34 ± 0.18 and 1.56±0.10,t=9.243 and 13.292,P＜0.01).(2) In oligohydramnios groups,after treated with different concentrations of U0126,the expressions of total ERK1/2 did not change (F=0.365,P＞0.05).The expression of p-ERK1/2 and AQP3 in 5,10,20,40 μmol/L U0126 (p-ERK1/2:0.96±0.16,1.12±0.13,0.98±0.17 and 1.02±0.26; AQP3:1.10±0.09,1.12±0.08,1.13±0.06 and 1.11±0.06) were all significantly lower than those in 0 μmol/LU0126 group (p-ERK1/2:2.46±0.25; AQP3:1.56±0.10,P＜0.05).However,the expression of p-ERK1/2 and AQP3 showed no significant difference among 5,10,20,and 40μmol/L U0126 groups (P＞0.05).The optimal concentration of U0126 was 5 μmol/L.After treated with 5 μmol/L U0126 for 2 h,the expressions of p-ERK1/2 and AQP3 (1.27±0.29 and 1.44±0.12)were lower than those after treated for 0 h (2.55±0.12 and 2.15±0.09,P＜0.05).However,there was no significant difference among groups treated for 2,6,12 and 24 h.Therefore,the optimal treatment time was 2 h.(3) The expression of AQP3 was distributed in both cell membrane and cytoplasm in amnion epithelial cells with normal amniotic fluid volume or isolated oligohydramnios,but mainly in cytoplasm.U0126 did not change the localization of AQP3 expression.Conclusions U0126 inhibits the phosphorylation of ERK1/2 and expression of AQP3 of women with oligohydramnios,indicating that the MAPK/ERK1/2 signal transduction pathway might regulate the expression of AQP3 in human amnion epithelial cells,and therefore affect the balance of amniotic fluid volume. Key words: Aquaporin 3 ; Extracellular signal-regulated MAP kinases ; Amnion; Epithelial cells ; Oligohydramnios