MTOR In TGF-β₂Induced Emt In HLECs

Lens epithelial cells (LECs) are essential to the normal development and homeostaticto maintenances of the lens. Aberrant growth, proliferation, migration, transdifferentiation and extracellular matrix secretion of LECs contribute to various lens diseases such as posterior capsule opacification (PCO).TGF-β is one of the key factors which can induce cells undergone epithelial to mesenchymal transition (EMT) in various cells, recent studies showed among several subforms of TGF-β, TGF-β2is the major isoform within the eye, and can induce human lens epithelial cells (HLECs) undergo EMT As a result, TGF-P2has been commonly used to create PCO cell model.mTOR is a large serine/threonine protein kinase which performs like a central regulator of cell growth, proliferation and also regulates EMT. But, it is unkonw that whether mTOR exsit in HLECs and plays a role in the EMT process in HLECs or not.ObjectivesTo find out the phosphorylation state (S2448-P, S2481-P) and the role of mTOR in TGF-β2-induced EMT in HLECs.Methods1TGF-β2induces HLECs undergo EMTHLECs were stimulated with10ng/ml TGF-β2for0,1,6,12,24, and48h in serum-free medium when the cell cultures reach80%confluence, observed under inverted phase contrast microscope, and checked the expression levels of connexin43(Cx43), E-cadherin(E-cad), fibronectin (FN), and a-smooth muscle actin(a-SMA) with western blot to determine whether TGF-β2induces HLECs undergo EMT or not.2Checking the dynamic changes of the expression levels and the phosphorylation levels of mTOR during TGF-β2induced EMT in HLECs. 3The phosphorylation levels and the role of mTOR in HLECs during TGF-β2induced EMT process.3.1Choosing the most suitable concentration for rapamycin and KU-0063794to treat HLECsRapamycin and KU-0063794both are mTOR inhibitors.10,30,100,300,1000nM rapamycin or KU-0063794treated HLECs cultured in DMEM supplemented with10%FBS for24h before observing under inverted phase contrast microscope and checking the phosphorylation levels of mTOR to choose proper concentration for rapamycin and KU-0063794.3.2The phosphorylation levels of mTOR and the effect of mTOR inhibitors in HLECs during TGF-P2induced EMT process.3.2.1Experiment was divided into the following groupsControl group: when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, and continued to culture24h.TGF-β2group:normally cultured HLECs, when the cell cultures reach80%confluence, fetal bovine serum were abolished, and then cultured in DMEM containing10ng/ml TGF-P2for24h.Rapamycin group:when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, and then cultured in DMEM containing rapamycin, and the concentration of rapamycin depends on the results "experiment3" previously described.KU-0063794group:when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, and then cultured in DMEM containing KU-0063794, and the concentration of KU-0063794depends on the results "experiment3" previously described.Rapamycin+TGF-β2group:when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, then, rapamycin pretreated1h before added TGF-P2into the culture system, and in this group, the final concentration of TGF-P2is10ng/ml, and the final concentration of rapamycin depends on the results "experiment3" previously described. KU-0063794+TGF-β2group:when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, then, KU-0063794pretreated1h before added TGF-β2into the culture system, and in this group, the final concentration of TGF-β2is lOng/ml, and the final concentration of KU-0063794depends on the results of "experiment3" which were previously described.TGF-β2+Rapamycin group:when the normally cultured HLECs reached to80%confluence, fetal bovine serum were abolished, then, added TGF-β2into the culture system and continued cultivation for24h before added rapamycin to co-treat HLECs for another24h. in this group, the final concentration of TGF-P2is10ng/ml, and the final concentration of rapamycin depends on the results of "experiment3" which were previously described.TGF-β2+KU-0063794group:when the normally cultured HLECs reached to80%c onfluence, fetal bovine serum were abolished, then, added TGF-β2into the culture system and continued cultivation for24h before added KU-0063794to co-treat HLECs for another24h. in this group, the final concentration of TGF-P2is lOng/ml, and the final concentration of KU-0063794depends on the results of "experiment3" which were previously described.3.2.2Checking the phosphorylation levels of mTOR(S2448-P, S2481-P) in each group with western blot.3.2.3Checking the epithelial markers (E-cad, Cx43) and mesenchymal markers (FN, α-SMA) in each group with western blot.4SPSS13.0was used to statistically analyse the experiment data, all the experiment data value are expressed as x±s. Data which came from experiment described in chapter "1","2" and "3.1" in the department of "Methods" in this abstract were analysed by one way ANOVA, comparation between the control group and other experiment groups were analysed by Dunnett t. Data which came from experiment described in chapter "3.2.2" and "3.2.3" in the department of "Methods" in this abstract were analysed by one way ANOVA, comparation between the groups were analysed by LSD. P values less than0.05were considered to be significantly.Results 1During the lOng/ml TGF-β2-induced HLECs EMT process, HLECs displayed an altered morphology and became flattened and stretched, from oval-like to spindle-shaped cells with reduce adherent cells, increased floating cells and the cell gaps. Compared with nomal control HLECs (the expression of E-cad,Cx43, FN, a-SMA were1.245±0.001,0.941±0.169,0.516±0.049and0.196±0.071respectively), Stimulation HLECs with TGF-β2suppressed the expression of E-cad and Cx43protein at6h and12h respectively(E-cad:1.059±0.004, P=0.000; Cx43:0.477±0.076, P=0.032), while increased the expression of FN and a-SMA at24h (FN:0.872±0.134, P=0.011; α-SMA:0.516±0.049,P=0.000).2With the treatment of10ng/ml TGF-(32, phosphorylation level of mTOR in HLECs dramatically increased at6h (S2448-P:0.542±0.124, P=0.006, S2481-P0.519±0.122, P=0.006) and peaked at24h in a time-dependent manner, but the expression of total mTOR was not affected.3mTOR is involed in TGF-β2induced EMT in HLECs.3.1Observed under inverted phase contrast microscope, HLECs decreased and floated according to increased concentration of rapamycin or KU-0063794. when KU-0063794reach to300nM, only a few cells remained adherent and survival after24h treatment, when KU-0063794reach to1000nM, almost all the cells have died. Western blot showed lOnM rapamycin or KU-0063794can inhibit the phosphorylation of mTOR in a concentration-dependent manner. Take all these aspect into consideration,100nM rapamycin and KU-0063794were choose for the follow-up experiments.3.2mTOR inhibitors rapamycin and KU-0063794both can inhibit TGF-β2induced mTOR activation in HLECs.compared to the "control group", mTOR was activated in "TGF-β2group"(S2448-P0.884±0.015, P=0.000; S2481-P0.874±0.115, P=0.000). The activation of mTOR was suppressed in "rapamycin+TGF-β2group","KU-0063794+TGF-β2group","TGF-β2+rapamycin group", and "TGF-β2+KU-0063794group" compared to "TGF-β2group"("rapamycin+TGF-β2group" vs "TGF-β2group":mTOR S2448-P and S2481-P:P=0.000;"KU-0063794+TGF-β2group" vs "TGF-β2group":mTOR S2448-P and S2481-P:P=0.000;"TGF-β2+rapamycin group" vs "TGF-β2group" mTOR S2448-P and S2481-P:P=0.000;"TGF-β2+KU-0063794group" vs "TGF-β2group":mTOR S2448-P and S2481-P:P=0.000;). The phosphorylation levels of mTOR is higher in "TGF-(32+rapamycin group" than in "rapamycin+TGF-(32group", and is higher in "TGF-β2+KU-0063794group" than in "KU-0063794+TGF-β2group"("TGF-β2+rapamycin group" vs"rapamycin+TGF-β2group":mTOR S2448-P and S2481-P P=0.000;"TGF-β2+KU-0063794group" vs "KU-0063794+TGF-β2group" mTOR S2448-P and S2481-P:P=0.000).The inhibition effect of KU-0063794on TGF-β2induced mTOR activation in HLECs is higher than rapamycin ("KU-0063794+TGF-β2group" vs "rapamycin+TGF-β2group", and "TGF-β2+KU-0063794group" vs "TGF-β2+rapamycin group", mTOR S2448-P and S2481-P:P=0.000).3.3rapamycin and KU-0063794both can inhibit TGF-β2induced epithelial cell markers(E-cad, Cx43) decrease and mesenchymal markers elevation(FN、α-SMA).In "control group", HLECs contain abundant E-cad,Cx43and a few of FN and a-SMA, in "rapamycin group" and "KU-0063794group", the expression levels of E-cad, Cx43, FN and a-SMA is similar to this in "control group"("rapamycin group" vs"control group":E-cad P=0.863, Cx43P=0.807, FN P=0.859, a-SMA P=0.782;"KU-0063794group" vs "control group":E-cad P=0.773, Cx43P=0.834, FN P=0.395,α-SMA P=0.245). In"TGF-β2group", the expression levels of E-cad, Cx43significantly decreased and the expression levels of FN、β-SMA significantly increased compared to the "control group"("TGF-β2group" vs "control group", these four epithelial/mesenchymal markers P=0.000). The expression levels of E-cad, Cx43increased and the expression levels of FN, a-SMA decreased in"rapamycin+TGF-β2group","KU-0063794+TGF-β2group","TGF-β2+rapamycin group" and"TGF-β2+KU-0063794group" compared to the "TGF-β2group"(these four epithelial/mesenchymal markers P=0.000). The expression levels of E-cad, Cx43increased and the expression levels of FN, α-SMA decreased in "TGF-β2+rapamycin group" than in "control group"(these four epithelial/mesenchymal markers P=0.000). In "TGF-β2+KU-0063794group", the expression levels of FN is similar to the "control group"(FN P=0.071), but the expression levels of E-cad, Cx43was lower, and the expression levels of a-SMA was higher(E-cad P=0.017, Cx43P=0.001, a-SMA P=0.000). In"TGF-β2+rapamycin group", the expression levels of E-cad, Cx43decreased and the expression levels of FN、 α-SMA increased compared to the "rapamycin+TGF-β2group"(these four epithelial/mesenchymal markers P=0.000). the expression levels of E-cad in "TGF-β2+KU-0063794group" is similar to "KU-0063794+TGF-β2group"(P=0.063), but the expression levels of Cx43was few(P=0.003), and the expression levels of FN and a-SMA higher in "TGF-β2+KU-0063794group" than that in "KU-0063794+TGF-β2group"(FN and a-SMA, P=0.000). The inhibition effect of KU-0063794on TGF-β2induced epithelial markers decreasing and mesenchymal markers increasing in HLECs is higher than rapamycin ("KU-0063794+TGF-β2group" vs "rapamycin+TGF-β2group":E-cad P=0.006, Cx43P=0.012, FN and a-SMA P=0.000; and "TGF-β2+KU-0063794group" vs "TGF-β2+rapamycin group":E-cad, FN and α-SMA P=0.000, Cx43P=0.014).Conclusion110ng/ml TGF-β2can induces HLECs experience EMT.2mTOR is activated in HLECs during the TGF-β2induced EMT process, though the expression levels of total mTOR is not affected.3lOnM rapamycin or KU-0063794can effectively inhibits the mTOR (S2448-P, S2481-P) phosphorylation, but do not affect the expression levels of total mTOR.4100nM rapamycin or KU-0063794can effectively inhibits and reverse TGF-β2induced mTOR activation and EMT in HLECs, But the effect is better in100nM rapamycin or KU-0063794pretreated1h before TGF-β2stimulated cells than in cells have undergone TGF-β2induced EMT before100nM rapamycin or KU-0063794treatment. The inhibition effect of KU-0063794on TGF-β2induced mTOR activation and EMT in HLECs is higher than rapamycin.