Investigation Of The Role Of AKT Kinases In Epithelial-Mesenchymal Transition Of Human Lens Epithelial Cells

Posterior Capsule Opacity(PCO)is the most common complication after intraocular lens implantation,result in impaired vision sight or even blindness.The epithelial-mesenchymal transition(EMT)of lens epithelial cells is a critical molecular event in the process of PCO.The elucidation of mechanisms underlying the EMT of lens epithelial cells will shed new light on therapeutic approaches of PCO treatments.AKT belongs to the threonine/serine protein kinase family,which includes three isomers: AKT1,AKT2 and AKT3.They are encoded by three independent genes but have high homology of amino acid sequence.In the case of AKT1,the amino terminus contains a PH domain,the middle is the catalytic domain and the carboxy terminus contains a FPQFSY hydrophobic motif.Phosphorylation of threonine 308(Thr308)and serine 473(Ser473)in the carboxy terminalare necessary for the activation of AKT kinase.Even though a few studies have shown that PI3K/AKT signaling pathway is associated with EMT in lens epithelial cells,which AKT kinase isoforms are involved and how they regulate EMT gene expression remain unclear.Previous studies from our lab revealed that AKT1 and AKT2 are predominantly expressed AKT kinases in mouse lens epithelium.To explore the molecular mechanism by which AKT1 and AKT2 regulate the EMT of lens epithelial cells,first of all,a model in which the EMT of human lens epithelial(HLE)cells induced TGFβ1 treatment was established.Then,PI3 K inhibitor LY294002 was used to inhibit the activation of endogenous AKT kinase in HLE cells,and LY294002 treatment can significantly attenuated the mRNA upregulaiton of TGFβ1-induced EMT marker gene Fibronectin and transcription factor SNAIL1.Consistently,in the stable HLE-AKT1 knockdown cell line and HLE-AKT2 knockdown cell line,TGFβ1-induced upregulation of Fibronectin and SNAIL1 mRNA expression level were also significantly attenuated.Finally,we constructed the wild-type human AKT1 and AKT2 expression plasmids,as well as the dominant negative mutation plasmids: AKT1-T308A/S473 A and AKT2-T309A/S474 A.The over expression of wild-type AKT1 or AKT2 plasmid in the HLE cells could significantly enhance TGFβ1-induced upregulation of Fibronectin and SNAIL1 mRNA level,while the dominant negative mutant plasmids failed to do so.These results demonstrate that AKT1 and AKT2 kinases can promote the TGFβ1-induced EMT of in HLE cells.Additionally,our results suggested that GSK3β is not the major downstream target in AKT kinases-mediated EMT in HLE cells.Currently,we were exploring the mechanisms of other potential substrates of AKT kinase in regulating EMT gene expression.Our study defined the function of AKT protein kinase in the EMT process of human lens epithelial cells,and we believe that the underlying mechanisms would be instructive for the therapeutic strategies of PCO.