The Expression And Significance Of GSK-3β In HKCs During The Tubular Epithelial To Mesenchymal Transition

Object:To investigate the expression and the significance of glycogen synthase kinase-3β(GSK-3β) in human renal tubular epithelial cells (HKCs) during undergoing tubular epithelial to mesenchymal transition (EMT) induceded by transforming growth factorβ₁ (TGFβ₁) and the result of intervention of phosphoinositide3-kinase (PI3K) inhibitor Wortmannin.Methods: The HKC cells were cultured in vitro with Dulbecco's modified Eagle's medium/F12 medium supplanted with 10% fetal bovine serum, and seeded on twenty-four-well culture plates or culture flasks to approximatedly 60%⁷0% confluence, and then shifted to serum-free medium for 24 hours. These cells were divided into five groups:â‘ Control (C) group: The HKC cells were treated with free serum medium;â‘¡TA group: The HKC cells were co-incubated different concentration TGFβ₁ (5 ng/ml,10 ng/ml,20 ng/ml) with free serum medium for 12 hours and were divided into TA1,TA2,TA3 for three subsections;â‘¢TB group: The HKC cells were co-incubated TGFβ₁(10 ng/ml) with free serum medium for 1,12,48 hours and were divided into TB1,TB2,TB3 for three subsections;â‘£T+W group: The HKC cells were co-incubated TGFβ₁(10 ng/ml) and Wortmannin (10nmol/L ) with free serum medium for 48 hours.⑤LiCl (L) group: The HKC cells were co-incubated LiCl (10mmol/L ) with free serum medium for 48 hours. The morphological changes in cells were observed through the invert phase-contrast microscope. At the same time, the protein and mRNA expression of E-cadherin,α-SMA, total GSK-3β, phosphorylated-GSK-3β(Ser 9) andβ-catenin was proved by immuno-histochemical staining, RT-PCR and Western blot.Result1. Cells shape observationâ‘ C group: The shape of HKC cells showed a classic cobblestone morphology and grew in a compact. mode.â‘¡T group: after induced 48 hours profound morphologic changes were observed in TA2(10ng/ml) and TA3(20ng/ml) groups, with cells becoming elongated in shape, and losing their cobblestone monolayer pattern; but incubated with 5ng/ml TGFβ₁, some cells become elongated, the others maintained cobblestone morphology.â‘¢T+W group: with the phosphoinositide3-kinase (PI3K) inhibitor Wortmannin (10nmol/L), TGFβ₁ (10ng/ml) could not induce HKC cells morphologic changes, cells showed a classic cobblestone morphology, similar to C group.2. To detect expression of E-cadherinâ‘ C group: the expression of E-cadherin protein was positive in cell membrane and endochylema ;â‘¡TA2 group: the expression of E-cadherin decreased markedly after cells were cultured with different concentration TGFβ₁ for 48 hours.â‘¢T+W group: the expression of E-cadherin protein in HKC cells was positive.3. To detect expression ofα-SMAâ‘ C group: the expression ofα-SMA protein in HKC cells was negative;â‘¡TA2 group: the expression ofα-SMA increased markedly after cells were cultured with differet concentration TGFβ₁ for 48 hours.â‘¢T+W group: the expression ofα-SMA protein in HKC cells was negative.4. To detect expression of GSK-3βin HKCs induced by TGFβ₁ and intervention of Wortmanninâ‘ C group: the expression of GSK-3βmRNA and protein in HKC cells was positive according to the result of detection of RT-PCR and Western blot respectively, but the protein expression of phosphorylated-GSK-3β(Ser 9) was microammount found by Western blot;â‘¡T group (including TA group and TB group): the expression of total GSK-3βand phosphorylated-GSK-3β(Ser 9) was posstive according to the results immuno-histochemical staining, There were no significant difference among groups at the levels of GSK-3βmRNA and protein detected by RT-PCR and Western blot (P>0.05); but T groups had been more phosphorylated-GSK-3β(Ser 9) than C group(P<0.01);â‘¢T+W group: the expression of phosphorylated-GSK-3β(Ser 9) was negtive found by immuno-histochemical staining. the expression of GSK-3βmRNA and protein in HKC cells was positive and had no significant difference according to the result of detection of RT-PCR and Western respectively(P>0.05); the expression level of phosphorylated -GSK-3β(Ser 9) in T+W group was low dramastically contrasted with T group(P<0.01).5. The effect of GSK-3βonβ-cateninâ‘ C group: the expression ofβ-catenin protein was positive in cell membrane and endochylema ;â‘¡TA2 group: the expression of β-catenin increased markedly after cells were cultured with 10 ng/ml TGFβ₁ for 48 hours in the cell nucleus, and the expression ofβ-catenin mRNA and protein increased strongly ;â‘¢T+W group: the expression ofβ-catenin protein in HKC cells was positive in cell membrane and endochylema and the expression ofβ-catenin mRNA and protein decreased strongly ;â‘£L group: the expression ofβ-catenin increased markedly after cells were cultured with 20mmol/L LiCl for 48 hours in the cell nucleus;Conclusion1. The expression of total GSK-3βmRNA and protein didn't change significantly and the level of phosphorylated GSK-3βincreased during HKC cells undergoing EMT induced by TGFβ₁;2. TGFβ₁ could significantly increase the level of phosphorylated GSK-3βso thatβ-catenin increased and enter the cell nucleus;3. PI3K inhibitor Wortmannin could significantly inhibit the level of phosphorylated GSK-3βand decrease the expression ofβ-catenin so that EMT was inhibited.