TGF-β₂Induces EMT Of Human Lens Epithelial Cells Through The Activation Of PI3K/Akt Signaling Pathway

BackgroundPosterior capsule opacification (PCO) is one of the most common complication after cataract surgery. It is still the main reason of visual acuity decreasing after cataract surgery, even though the PCO morbidity has been reduced as the development of operation technology and the progression of intraocular lens material as well as its design. At present, the after cataract pathogenesis is considered to be associated with aberrant growth, fibrosis, migration of lens epithelial cells as well as the secretion of extra cellular matrix. The phenomenon of epithelial cells getting characteristics of mesenchymal cells, which refers to epithelial mesenchymal transition (EMT), is the cytological basis of after-cataract. The connection of lens epithelial cells was disappeared, and the cell migration ability was enhanced through EMT.Transforming growth factor-P (TGF-P), which is a multifunctional protein belonging to the transforming growth factor-β superfamily, has the ability to regulate growth, differentiation, apoptosis,immune of variety of cells. It is also an important regulating factor interfere with normal lens structure, induced aberrant proliferation, differentiation, apoptosis of lens epithelial cells in lens in a variety of physiological and pathological conditions, and has a close relationship with the formation of subcapsular cataract and after cataract[1-3]. Transforming growth factor-β2contains three kinds of TGF TGF-β2homologues isomers:transforming growth factor TGF-β2, transforming growth factor-β2and transforming growth factor TGF-β2. Among them, transforming growth factor-β2, which has the highest concentration and activity in ocular aqueous and vitreous body, is one of the strongest cytokine in regulation of lens epithelial cells.TGF-β2can induce lens epithelial cells undergo EMT including loss of polarity and obtaining mesenchymal characteristics such as migration ability, strong strong invasion ability and anti-apoptotic ability. Thus cells contain the myofibroblast-like features. Currently, the lens epithelial cells EMT model, which is induced by TGF-β2,is widely studied as the cytological model of cataract forming (including subcapsular cataract and PCO).mTOR is a kind of serine/threonine protein kinase belonging to the phosphatidylinositol kinase associated with the protein kinase family. It exists extensively in the cytoplasm of mammal cells. mTOR signaling pathway is considered to be a signal convergence point regulating cell cycle progression and cell growth,cell proliferation as it is closely related with cell growth, proliferation, differentiation, migration, self-renewal and cell cycle in variety of cells. mTOR can accept many signals such as growth factors signal, nutrients (amino acid) signal, energy signal, and so on. It plays the roles through multiple pathways including the PI3K/Akt signal pathway.ObjectivesTo study the role and the mechanism of PI3K/Akt signaling pathway in TGF-β2inducing EMT of lens epithelial cells.To provide definite theoretical evidences for PCO pathogenesis.To provide new target points for drug intervention of PCO.Methods1TGF-β2induces HLECs to undergo EMTHLECs-B3cells were routine cultured, when the cell cultures reach80%confluence, removed the culture fluid.The cells were washed3times with PBS, cultured with DMEM medium without FBS. Adding in TGF-β2and making its final concentration of10ng/ml, cells were placed in the cell culture tank and cultured for24hours. Confocal immunofluorescence and Western blotting were used to detect the expression of connexin43(Cx43) and fibronectin (FN), which are the marker proteins of epithelium and mesenchymal cells, respectively.2To detect the expression changes of molecules in PI3K/Akt signal pathway in HLECs undergoning EMT induced by TGF-β2.Western blotting assay were used for the detection of the expression levels and the phosphorylation levels of PI3K, Akt and mTOR, which are the major signal molecules of PI3K/Akt signal pathway.3CCK-8experiments were used to determine the effect of LY294002, which is the inhibitor of PI3K, on the proliferation of HLEC-B3cells.HLEC-B3cells were cultured in96hole plate, LY294002was added and made it the final concentration of0μM,10μM,20μM,30μM,40μM,50μM,60μM,70μM and80μM, respectively. Incubating the cells for24hours in incubator, and adding10μL CCK-8solutions into each hole and continuing to culture, using MicroplateReader to detect absorbance (A) value of each hole and calculate the inhibitory rate of cell proliferation.4To observe the effect of PI3K inhibitor LY294002on the process of HLEC-B3epithelial mesenchymal transition which was induced by TGF-β2-4.1The cells were grouped by the following way:Control group:HLECs-B3cells were routine cultured, when the cell cultures reach80%confluence, replaced the medium by serum-free DMEM medium and then continued to culture for24h.TGF-β2group:HLECs-B3cells were routine cultured, when the cell cultures reach80%confluence, replaced the medium by serum-free DMEM medium containing10ng/ml TGF-β2for24h.LY294002+TGF-β2group:HLECs-B3cells were routine cultured, when the cell cultures reach80%confluence, replaced the medium by serum-free DMEM medium containing20μM LY294002for1h, and then continue to cultured in10ng/ml TGF-β2for24h. 4.2Checking the influence of PI3K inhibitor LY294002on the expression changes of main signal molecules in PI3K/Akt signal pathway in TGF-β2induced EMT in HLECs.Expression and phosphorylation levels of PI3K, Akt, mTOR were detected by Western blotting method.4.3Checking the effect of PI3K inhibitor LY294002on the EMT process in HLECs induced by TGF-β2.Cell immunofluorescence and western blotting were used to detect the expression level of Cx43and FN, which are the marker protein of epithelium and mesenchymal cell respectively.5SPSS13.0was used to statistically analyze the experiment data, all the experiment data value are expressed asx±s. The repeated measures analysis of variance is used to the compartments of A values of different concentrations of LY294002on the HLE-B3by detected by CCK-8. One way ANOVO was used to the compartments of the protein expressions among three groups by detected by Western blot. P＜0.05is considered significant difference.Results1The culture system construction of HLECs EMT induced by TGF-β2.1.1Cell Immunofluorescence method was used to detect the influence of TGF-β2on the expression of Cx43and FN, which are the marker proteins of epithelium and mesenchymal cells, respectively.To determine weather TGF-β2induce HLECs to undergo EMT, we used Cell Immunofluorescence method to detect the expressions of Cx43and FN.And we found that HLECs-B3cells without treated with TGF-β2(control group) expressed abundant of Cx43but a little of FN. And the result was exactly the opposite in the TGF-β2group, the HLECs express abundant of FN but a little of Cx43while treated by TGF-β2for24h. The quantitative detection of which is detailed in Western blot detection. 1.2Western blotting method was used to detect the influence of TGF-β2on the expression of Cx43and FN, which are the marker protein of epithelium and mesenchymal cells respectively.For further verification of weather TGF-β2can induce HLECs to undergo EMT, we used Western blotting of quantitative detection method to detect the expression changes of Cx43and FN, and the "target protein value/GAPDH gray value" was used to represent the protein expressing quantity. The results found that:HLECs-B3cells which were not treated with TGF-P2(control group) express abundant of Cx43(2.53±0.14) while the TGF-β2group were not,the Cx43(1.40±0.10) were decreased compare with control group. The control group express a little of FN (0.29±0.02) while the FN (0.97±0.09) expression level was much more higher in TGF-β2group compared with control group. The differences among two groups were all significant statistically (P＜0.05)2. The expression changes of main signal molecules in PI3K/Akt signal pathway in TGF-β2induced EMT in HLECs.2.1Cell immunofluorescence detection was used to detect the influence of TGF-β2on the expression of p-Akt, which is one of the main molecules in PI3K/Akt signaling pathway.To verify whether EMT in HLECs induced by TGF-β2was through the activation of PI3K/Akt signaling pathway, cell immunofluorescence detection was used to detect the expression changes of p-Akt, which is the main protein of PI3K/Akt signaling pathway, in HLECs EMT induced by TGF-β2. Results found that: HLECs-B3cells without TGF-β2treatment (control group) expressed a little p-Akt; while HLECs-B3cells with TGF-β2treatment for24h expressed much more higher p-Akt which get gather in the cell membrane compared with control group, and the quantitative detection of which was detailed by Western blot detection.2.2Western blot was used to detect the influence of TGF-β2on the expression of main molecules in PI3K/Akt signaling pathway.For the verification whether HLECs EMT induced by TGF-β2was through the activation of PI3K/Akt signaling pathway, we used Western blot of quantitative detection method to detect the expression changes of signaling molecules in PI3K/Akt signaling pathway during the HLECs EMT process induced by TGF-β2, and the "target protein value/GAPDH gray value" was used to represent the protein expressing quantity. The results found that:HLECs-B3cells which were not treated with TGF-β2(control group) express a little of p-PI3K (0.69±0.01) while the the expression level of p-PI3K (0.92±0.11) were much more higher in TGF-β2group compared with control group. The control group expressed a little of p-Akt (0.91±0.08) while the expression level of p-Akt (1.48±0.13) were much more higher in TGF-β2group treated with TGF-β2for24h compared with control group. The control group expressed a little of p-mTOR (0.64±0.02) while the expression level of p-mTOR (1.22±0.12) were much more higher in TGF-β2group compared with control group. The differences among two groups were all significant statistically (P＜0.05).3CCK-8experiments were used to determine the effect of LY294002, which is the inhibitor of PI3K, on the proliferation of HLEC-B3cells.CCK-8test was used to the determine effects of LY294002of different concentrations (0μM,10μM,20μM,30μM,40μM,50μM,60μM,70μM and80μM)on the proliferation of HLE-B3. Absorbance (A) value on each microplate reading hole was detected in1hour point,2hour point and3hour point. The statistical analysis results:With the increasing of LY294002concentration, A value decreased and proliferation inhibiting rates of LY294002to HLE-B3cells gradually increased. A values had significant differences in different concentration groups (F=9.72, P=0.000). There were significant differences among different time points measured A values (F=1737.54, P=0.000). There were significant interactions between concentration groups and measurement time points (F=5.60, P=0.000)4. Checking the influence of PI3K inhibitor LY294002on the expression of main signal molecules in PI3K/Akt signal pathway in HLECs EMT induced by TGF-p2. 4.1Cell immunofluorescence method was used to detect the influence of PI3K inhibitor LY294002on the expression of main signal molecules p-Akt in PI3K/Akt signal pathway in TGF-P2induced EMT in HLECs.To verify whether PI3K inhibitor LY294002can stop the process of EMT in HLECs induced by TGF-β2, cell immunofluorescence method was used to detect the expression changes of p-Akt in PI3K/Akt signal pathway in EMT induced by TGF-β2in HLECs. Results found that:HLECs-B3cells which were not treated with TGF-β2(control group) express a little of p-Akt; HLECs-B3cells treated with TGF-β2had an increased expression of p-Akt,which were get together to cell membrane; when given pretreatment with LY294002for1h before treated with TGF-β2for24h, the expression level of p-Akt decreased compared with the TGF-β2group.4.2.Western blot method was used to detect the influence of PI3K inhibitor LY294002on the expression of main signal molecules in PI3K/Akt signal pathway in TGF-β2induced EMT in HLECs.To verify PI3K inhibitor LY294002did block the activation of PI3K/Akt signal pathway induced by TGF-β2. WB was used to detect the expression changes of PI3K,Akt,mTOR in PI3K/Akt signal pathway in HLECs EMT induced by TGF-β2, and the "target protein value/GAPDH gray value" was used to represent the protein expressing quantity.Results found that:HLECs-B3cells which were not treated with TGF-β2(control group) express a little of p-PI3K (0.69±0.01) while the the expression level of p-PI3K (0.92±0.11) was much more higher in TGF-β2group compared with control group.The expression level of p-PI3K (0.48±0.03) is much lower when given pretreatment with LY294002for1h before treated with TGF-β2for24h compared with TGF-β2group.The differences among three groups were significant statistically (P＜0.05). The difference between control group and TGF-β2group was significant statistically (P＜0.05). The difference between TGF-β2group and LY294002+TGF-β2group was significant statistically (P＜0.05) Results found that:The control group express a little of p-Akt (0.91±0.08) while the expression level of p-Akt (1.48±0.13) was much more higher in TGF-P2group treated with TGF-β2compared with control group. The expression level of p-Akt (0.95±0.19) is much lower when given pretreatment with LY294002for1h before treated with TGF-β2for24h compared with TGF-β2group. The differences among three groups were significant statistically (P＜0.05). The difference between control group and TGF-β2group was significant statistically (P＜0.05). The difference between TGF-β2group and LY294002+TGF-β2group was significant statistically (P＜0.05)Results found that:The control group express a little of p-mTOR (0.64±0.02) while the expression level of p-mTOR(1.22±0.12) was much more higher in TGF-β2group treated with TGF-P2compared with control group. The expression level of p-mTOR(0.76±0.07) is much lower when given pretreatment with L Y294002for1h before treated with TGF-β2for24h compared with TGF-P2group.The differences among three groups were significant statistically (P<0.05). The difference between control group and TGF-β2group was significant statistically (P＜0.05). The difference between TGF-β2group and LY294002+TGF-β2group was significant statistically (P＜0.05)5. PI3K inhibitor LY294002can stop the process of HLEC-B3EMT which was induced by TGF-β2.5.1Cell immunofluorescence method was used to detect the Influence of PI3K inhibitor L Y294002on the expression of Cx43and FN, which are the marker protein of epithelium and mesenchymal cells respectively.To verify whether PI3K inhibitor LY294002can stop the process of EMT in HLECs induced by TGF-β2, Cell immunofluorescence method was used to detect the expression changes of Cx43and FN, which are the marker protein of epithelium and mesenchymal cells respectively. And the results found that:HLECs-B3cells which were not treated with TGF-β2(control group) express abundant of Cx43and a little of FN; while FN expression level of the TGF-β2group was increased and the Cx43 expression level was decreased compared with control group; when given pretreatment with LY294002for1h before treated with TGF-β2for24h, the expression level of FN decreased and the expression level of Cx43increased compared with TGF-β2group.5.2Western blotting method was used to detect the influence of PI3K inhibitor LY294002on the expression of Cx43and FN, which are the marker protein of epithelium and mesenchymal cells respectively.For further verification of whether PI3K inhibitor LY294002can stop the process of TGF-β2induced EMT in HLECs, we used Western blot method to quantitativly detect the expression changes of epithelium marker Cx43and mesenchymal cells marker FN, the "target protein value/GAPDH gray value" was used to represent the protein expressing quantity.Results found that:HLECs-B3cells which were not treated with TGF-β2(control group) express abundant of Cx43(2.53±0.14); HLECs-B3cells treated with TGF-β2for24h had a decreased level of Cx43(1.40±0.10) expression compared with control group; the expression level of Cx43(2.35±0.16) is much higher when given pretreatment with LY294002for lh before treated with TGF-β2for24h compared with TGF-β2group. The differences were statistically significant among the three groups (F=60.02, P=0.000); The difference between control group and TGF-β2group was statistically significant (P＜0.05). The difference between TGF-β2and LY294002+TGF-β2group was statistically significant (P<0.05).Results found that:HLECs-B3cells which were not treated with TGF-P2(control group) express a little of FN (0.29±0.02); HLECs-B3cells treated with TGF-β2for24h had a increased level of FN(0.97±0.09)expression compared with control group; the expression level of FN (0.38±0.04) is much lower when given pretreatment with LY294002for1h before treated with TGF-β2for24h compared with TGF-β2group. The differences were statistically significant among the three groups (F=117.01, P=0.000); The difference between control group and TGF-β2group was statistically significant (P＜0.05). The difference between TGF-β2and LY294002+TGF-β2 group was statistically significant (P＜0.05).Conclusion1.10ng/ml TGF-β2induces effectively the HLECs EMT experience.2. PI3K, Akt and mTOR are activated by phosphorylation in the HLECs EMT process induced by10ng/ml TGF-β2.3. With the increasing of LY294002concentrations, its proliferation inhibiting rates of the HLECs increased gradually.4. Pretreated with20μM L Y294002can effectively inhibit activation of PI3K, Akt and mTOR by phosphorylation.5. Pretreated with20μM LY294002can effectively inhibit the HLECs EMT process induced by10ng/ml TGF-β2.The experimental results demonstrated the scientific hypothesis:TGF-β2induced the epithelial mesenchymal transition of human lens epithelial cells through the PI3K/Akt signal pathway.