The Effects Of Bradykinin On The Proliferation Of Rabbit Corneal Endothelial Cells Trigerring By The ZO-1/ZONAB Pathway

BackgroundCorneal endothelial cells(CECs)are arranged as a monolayer on the posterior surface of the cornea.By exerting effective barrier and pump functions,CECs play a pivotal role in regulating corneal stromal hydration and hence transparency.CECs are notorious for having limited proliferative capacity in vivo because of the mitotic block at the G1 phase of the cell cycle in adults.Consequently,diseases,inflammation,aging,injuries or surgeries,frequently result in endothelial cell density decrease and subsequently lead to corneal blindness as a result of corneal irreversible edema and dysfunction.Therefore,to explore the underlying mechanisms of endothelial cell proliferation becomes a research hotspot at the field of cornea transplantation.Nowadays,there is an impending increase in demand for developing novel therapeutic methods and agents in clinical practice.Bradykinin(BK)is a member of Kallikrein-Kinin family that displaying a wide range of biological activities and mediating a number of pathophysiological responses,including inflammation,pain,angiogenesis,cell proliferation,tumorigenesis and regulation of the permeability of blood brain barrier.Bradykinin B1 receptors and bradykinin B2 receptors were found in cornea.Furthermore,BK has been shown to induce cell proliferation through BK B2 receptor in several cell types of cornea,including corneal epithelial cells,keratocytes,fibroblasts and CECs.ZONAB(zonula occludens 1(ZO-1)-associated nucleic-acid-binding protein)is a Y-box transcription factor that is recruited to tight junction by binding to the Src homology 3 domain of ZO-1.The interaction between ZO-1 and ZONAB is regulated by various types of cyclin-related proteins/genes/enzymes or transcription factors,which forms the ZO-1/ZONAB signaling.ZO-1 and ZONAB interact to modulate cell proliferation,cell differentiation,genetic expression and morphogenesis,which were studied in renal proximal tubule,retinal pigment epithelium,choroid,alveolar epithelium,neuroglia and tumor.Evidence showed that lentivirus-mediated interference with the ZO-1 and ZO-1 downregulation induced cell density increase and cell cycle progression in human CECs.In type II alveolar epithelial cells,the overexpression of ZONAB promoted cell proliferation and repressed transdifferentiation while the downregulation of ZONAB promoted cell transdifferentiation and repressed proliferation.The role of ZO-1/ZONAB pathway in the BK-induced cell proliferation of CECs still remains unclear.On the basic of previous in vitro cell cuture model of rabbit corneal endothelial cells(RCECs),we aim to explore the effect of BK on the proliferation of RCECs,and to determine the contribution of the ZO-1/ZONAB pathway involved in the RCECs proliferation of BK.Methods1.The effect of different concentration of BK on the proliferation of RCECs: By the methods of distripping Descemet's membrane combining with collagenase digestion,purified primary RCECs were collected and cultured,and then subcultured when eighty percent to ninty pecent of cells were adherent cultured.The cultures were randomly divided into different concentrations of BK treatment groups(0.01?0.1?1?10 ?M)and the normal control group of RCECs.The morphological changes of cells in each group were examined under invert microscope and MTT assays were performed to detect cell proliferation after 24 h,48 h,72 h,96 h culturing.Cells of all groups were collected at 72 h,the expression level of nucleus ZONAB and ZO-1 on the tight junction were detected using Western Blot analysis,meanwhile,the level of ZONAB was examined by immunofluorescence.2.Cell transfection of ZONAB-si RNA and effective gene targets screening: By the method of liposome transfection,transfection-specific ZONAB-si RNA(1195,1051 and 685 sequences)and transfection-nonspecific sequences(si RNA-negative control,si RNA-NC)were introduced into RCECs.Then,the m RNA relative expression of ZONAB was measured by real-time PCR and the protein expression of ZONAB was examined by Western Blot.Finally,the most effective gene sequence was identified by gene silencing efficacy.3.The effect of ZONAB-si RNA transfection combining with BK on the proliferation of RCECs: During logarithmic phase of RCECs,the cultures were randomly divided into different groups,including control group,1 ?M BK treatment group,NC-si RNA group,ZONAB-si RNA group,NC-si RNA+BK group and ZONAB-si RNA+BK group.Cell samples were collected at 72 h.MTT assays were performed to detect cell proliferation and the cell cycle of RCECs was detected using flow cytometry.The expression of ZO-1?ZONAB?PCNA and cyclin D1 were detected by Western Blot.Results1.The effect of different concentration of BK on the proliferation of RCECs: 1)With BK treatment at 0.01~1 ?M,the cultured cells of 72 h grew against the wall of flask,formed a monolayer after fusion and showed a mosaic arrangement;However,10 ?M BK inhibited cell growth and promoted cell apoptosis.2)MTT results showed that there was a proliferation trend during the period of 72 h when treated within 0.01~1 ?M,and 1 ?M of BK exhibited the best proliferation-promoting effect;cells growth with 10 ?M BK was significantly restricted at different times.3)Immunofluorescence results showed that 0.01~1 ?M of BK increased the expression or the positive cells ratio of ZONAB;while 10 ?M of BK decreased the positive cells ratio of ZONAB.4)Western Blot results showed that 0.01~10 ?M of BK upregulated the expression of ZO-1 at tight junction in a concentration-dependent manner;0.01~1 ?M of BK enhanced ZONAB nuclear translocation and 10 ?M of BK inhibited it.2.Cell transfection of ZONAB-si RNA and effective gene targets screening: After cell transfection of ZONAB-si RNA,the m RNA and the protein expression levels of ZONAB was significantly decreased,especially in 1051 sequence group.Thus,ZONAB-1051 sequence was the optimal gene target sequence.3.The effect of ZONAB-si RNA transfection combining with BK on the proliferation of RCECs: 1)BK promoted the proliferation and induced mitosis cell cycle progression in RCECs,coupled with the upregulation of ZO-1 and ZONAB expression,and increase of PCNA and cyclin D1 expression.2)With ZONAB-si RNA transfection,the expression of ZO-1?ZONAB? PCNA and cyclin D1 was down-regulated,exerting an inhibitory effect on cell proliferation and cell cycle.3)Before the BK administration,the pretreatment with ZONAB-si RNA transfection completely reversed these proliferation-promoting effect of BK.Conclusions1.Low and moderate concentration(0.01?M~1?M)of BK promoted cell proliferation while high concentration(10 ?M)of BK inhibited cell growth.1?M of BK exerted the preferable proliferation-promoting effect.It enlightened us that adding BK or BK analogues into the culture may be a new method to promote cell proliferation of CECs.2.BK upregulated the expression of ZO-1 at tight junction and enhance ZONAB nuclear translocation,and then promote the proliferation of RCECs.ZO-1/ZONAB signaling may be one of underlying mechanisms involved in the RCECs proliferation of BK.3.After ZONAB-si RNA transfection,the m RNA and the protein expression levels of ZONAB was significantly decreased.Hence,RNAi successfully led to the post-transcriptional gene silencing of ZONAB.4.After ZONAB-si RNA transfection,the expression of ZO-1 and ZONAB,and the expression of PCNA and cyclin D1(commonly used proliferation bio-markers)was significantly decreased,leading to the repression of cell proliferation.5.ZONAB-si RNA transfection reversed the proliferation-promoting effect of BK,which suggested that the ZO-1/ZONAB pathway may play a crucial role in the RCECs proliferation of BK.The promising results of this study may provide a new insight and a novel therapeutic target for the cornea regeneration and transplantation.