Studies On Mechanism Of Transforming Growth Factor Beta 2-induced Epithelial Mesenchymal Transition In Human Lens Epithelial Cells

Cataract is the leading cause of blindness worldwide.At present there are still no effective medicine therapies which can inhibit or delay the progress of cataract and surgical removal of cataracts is the main treatment for cataract.Early clinical studies revealed that about 50%of patients after cataract surgery would appear posterior capsular opacification(PCO).Although great improvements have acquired in surgical technique and intraocular lenses in the past decade,approximately 20-25%of patients who undergo modern cataract surgery experience PCO and YAG laser capsulotomy within 2 years.PCO becomes the main complication of cataract surgery and also the main cause for poor vision post-operation.Most of theses patients are youth,especially children,and the YAG laser capsulotomy has certain risks,such as retinal detachment and macular edema.Therefore,it is very important to clarify the mechanism of PCO and take safe and effective therapeutic measurements.Many factors have significant effect on PCO,including complex and traumatic surgeries,remnant natural lens,inflammatory mediators released by broken blood-aqueous barrier,the material,design and position of intraocular lenses implanted. Emerging evidence suggests that the main reasons of PCO are the proliferation and transition of lens epithelial cells(LECs),decomposition of extracellular matrix(ECM) and regeneration of lens fiber.Epithelial mesenchymal transition(EMT)is part of a wound healing response in LECs and is characterized by induced expression of numerous mesenchymal proteins,as well as loss of epithelial genes.EMT bears morphological and molecular resemblance to forms of human cataract,including anterior subcapsular cataract(ASO)and PCO.However,it still remains unknown about the signal factors which stimulate the abnormal proliferation and EMT in LECs. Numerous studies indicate that many growth factors are involved in process of EMT, including fibroblast growth factor(FGF),epidermal growth factor(EGF)and transforming growth factorβ(TGF-β).TGF-βis one of the key factors that can induce cell differentiation.As one member of TGF-βsuperfamily,TGF-βregulate expression of various target genes and play an important role in embryo development,cell differentiation, proliferation and apoptosis.Normally TGF-βis abundant in human humor aqueous, vitreous and lens albeit predominantly in the latent,inactive form and has critical effect in LECs' physiological and pathological differentiation.There are five isoforms of TGF-βfound at present and only TGF-β₁,TGF-β₂ and TGF-β₃ exist in mammal animals. The TGF-β₂,ten times as effective as TGF-β₁,is the most potent of the 3 forms of TGF-β.Many eye diseases can cause the change of TGF-β₂ concentration in aqueous humor and the active form of TGF-β₂ ranges from 11%to 61%.Jampel has reported that the concentration of TGF-βis 2.3-8.1ng/ml during cataract surgeries and 61%of TGF-βis active.In 1994,Liu et al.were the first to show the effect of TGF-βin LECs and found TGF-βinduced cells to adopt an abnormal spindle shape and produce aberrant ECM.Hales et al.also reported that TGF-βinducedα-smooth muscle actin expression in LECs,as well as localized capsule wrinkling.It was very similar to the formation of ASC and PCO.From then on,the phenomenon of EMT was widely studied in cataract research.The Smad signaling pathway has been widely considered to be central to the transduction of TGF-βsignals.TGF-βexerts its biological actions by binding to and activating specific transmembrane,serine-threonine kinases.The activated receptor complexes recruit and phosphorylate specific members of the Smad family of cytoplasmic proteins.Besides,increasing evidence suggests that TGF-βsignals may also be transmitted via alternate pathways,including Ras and RhoA pathways and then take part in cell proliferation,transdifferentiation and apoptosis.Phosphatidylinositol 3-kinase(PI3K)is an important member of growth factor receptors superfamily in signal transduction.The Akt kinase,acting as major downstream of PI3K,is activated by recruitment to the plasma membrane through direct contact of its pleckstrin homology domain with phosphatidylinositol triphosphate,and phosphorylation at Thr308 and Ser473.PI3K/Akt can be activated by growth factors and oncogenes and regulate cell survival,proliferation,growth and other biological activities in both normal development and tumor pathogenesis.Previous studies have reported its role in fibrosis,but the role in EMT in LECs hasn't been reported yet.Previous work has revealed that TGF-βplay an important role in ASC and PCO. Though it is extensively studied,the mechanism of TGF-βon lens is complex and not completely understood yet.So it is critical to know the mechanism of TGF-β,the potent growth factor in ocular media,in process of EMT in LECs.On the basis of previous studies,we analyzed the effect of TGF-β₂ on proliferation and EMT in LECs, established a research model for PCO in vitro,and also found the involvement of PI3K/Akt signal transduction pathway in TGF-β₂-mediated EMT.All of these findings will benefit the further studies of revealing the effect of TGF-βon lens in physiological and pathological conditions. Partâ… Proliferation and epithelial mesenchymal transition induced by transforming growth factor beta 2 in human lens epithelial cells【Objective】The aim of study in this part is to investigate the effect of TGF-β₂ on proliferation and epithelial mesenchymal transition(EMT)in human lens epithelial cells(HLECs) and establish a research model for posterior capsular opacification(PCO)in vitro.【Methods】HLE B-3 cells were treated with different concentrations of TGF-β₂.The cell proliferation was assayed by MTT.The cell cycle distribution was assayed by flow cytometry(FCM).The expression changes of connexin43,fibronectin,desmin and integrinβ1 were examined by RT-PCR,Western blot and immunofluorescence.【Results】Transforming growth factor-β₂ is an important growth regulator in HLECs.The proliferation of cells was suppressed significantly after treatment of TGF-β₂ in a doseand time- dependent manner.The effect of inhibition was most significant with TGF-β₂ at a concentration with 100 pg/ml.The percentage of cells in S phase was significantly decreased after treatment with TGF-β₂ while the percentage of cells in G1 phase was increased.The cells underwent morphological alteration after treatment with TGF-β₂.The expression of connexin43 was decreased after treatment of TGF-β₂,while the expression of fibronectin,desmin and integrinβ₁ was increased.【Conclusion】TGF-β₂ inhibited the proliferation and induces growth arrest in HLECs.TGF-β₂ induced the epithelial mesenchymal transition in HLECs. Partâ…¡Involvement of PI3K/Akt signal pathway in transforming growth factor beta 2-mediated epithelial mesenchymal transition in human lens epithelial cells【Objective】In the first part of study,our results demonstrated that TGF-β₂ inhibited proliferation and induced EMT in HLECs.Mitogen activated protein kinase(MAPK) and phosphatidylinositol 3-kinase(PI3K)/Akt signal pathways play a critical role in regulating cell proliferation,differentiation,metabolism,apoptosis and other biological activities.The aim of this study is to determine the involvement and possible mechanism of MAPK and PI3K/Akt pathways in TGF-β₂-mediated EMT in HLECs.【Methods】HLE B-3 cells were treated with 100 pg/ml TGF-β₂ at different concentration for indicated time.Activation of PI3K/Akt,mitogen activated protein kinase(MAPK)p38 and ERK pathways was also detected by Western blot.LY294002,the specific inhibitor of PI3K,was used to block the activation of PI3K/Akt pathway.The expression changes of connexin43,fibronectin,desmin and integrinβ1 were examined by Western blot and immunofluorescence.【Results】 TGF-β₂ significantly activated PI3K/Akt in a time-dependent manner in HLECs (P＜0.05),but not ERK and p38 MAPK.The coincubation with LY294002 and TGF-β₂ markedly abrogated the suppression of connexin43(P＜0.05)and the induction of fibronectin,desmin and integrinβ1 in HLECs.【Conclusion】The activation of PI3K/Akt was necessary for the TGF-β₂-stimulated downregulation of connexin43,which in turn was necessary for TGF-β₂-induced EMT in HLECs.