Effects Of NF-κB On Growth And Secretion Of Human Trabecular Meshwork Cells

Background and Objective:Trabecular meshwork(TM) cells,located in the TM tissues of the chamber angle of eye,contain multiple cytokines and could secret various substances,such as matrix metalloproteinases(MMPs) and extracellular matrix(ECM) components.It plays an important role in maintaining TM structure and function,and facilitating aqueous outflow. Dysfunctions of TM cells would increase aqueous outflow resistance and lead to elevation of intraocular pressure and development of glaucoma.Previously researchers have reported significant loss of TM cells and obviously increased extracellular matrix in TM tissues of patients with primary open-angle glaucoma,compared with normal controls.So,we suggest that if the molecular mechanisms of glaucoma pathogenesis,such as TM cells proliferation, apoptosis and secretion,could be clearly elucidated,glaucoma could be effectively treated.Nuclear factor-κB(NF-κB) regulates the transcription of a wide array of gene products that are involved in many pathological and physiological pathways,such as cell differentiation,proliferation and apoptosis,and immune/inflammatory responses. Activation of NF-κB may be a pivotal event in proinflammatory signal transduction.But, its excessive activation would lead to many diseases with severe chronic/acute inflammatory responses.Inflammatory responses commonly occur during the glaucoma onset,development and treatment,especially in that caused by trauma and uveitis.High levels of inflammatory mediators and cytokines,produced in inflammatory responses in anterior chamber,would inevitably initiate the activation of NF-κB,which also could be induced by stress reactions.It have been found that the expression of NF-κB of TM cells in patients with primary glaucoma is significantly higher than that in normal controls. Unfortunately,the effects of NF-κB activation on glaucoma still remain unclear.NF-κB,critical to cell survival,could mediate anti-apoptosis and cell proliferation by regulating the expressions of relative genes.Additionally,it also participates in controlling MMP gene activity.In clinic,chronic administration of glucocorticoid could result in secondary glaucoma.Because glucocorticoid belongs to one of antagonists of NF-κB,we suggest that glucocorticoid abuse could heavily inhibit the activity of NF-κB and consequently induce TM cells apoptosis.However,no experimental evidences show that the cascade processes described above are responsible for the occurrence of glucocorticoidinduced glaucoma.In our research,to evaluate the effects of NF-κB on TM cells' proliferation and apoptosis,and the transcription and expression of MMP and TIGR genes,we transfected RelA/P65 subunit gene into cultured TM cells and used oligodeoxynucleotides(ODNs), homologous to the sequence of cis-elements of NF-κB,to inhibit the activation of NF-κB in TM cells,according to the decoy strategy.At the same time,we tried to explain NF-κB role in TM cells dysfunction,and to evaluate the effect of different NF-κB activity on TM cells and its relationship with glaucoma occurrence.Materials and Methods:1.Primary culture and identification of TM cells:TM cells were cultured using tissue-explant technique with eye tissues of donors in corneal grafting.SP immunocytochemical method was used to identify fibronectin(FN),laminin(LN), CollagenⅣ,TIGRand FactorⅧ-related antigens.2.Cell grouping:The cells were divided into five groups,i.e.,normal control group (C),control plasmid group(P65-C),pRSV-RelA/P65 plasmid group(P65) and Random-decoy-ODNs control group(ODN-C) and NF-κB decoy-ODNs group(ODN)3.Transfection of P65 and NF-κB decoy-ODNs:After pRSV-RelA/P65 plasmid and NF-κB decoy-ODNs were transfected with liposome,the transfection efficiency was estimated by fluorescence microscope and Western Blot analysis.4.Detection of TNF-αlevel in pRSV-RelA/P65 plasmid transfected TM cells:At 24 hours after transfection,TNF-αlevel was measured by ELISA in the culture supematant of P65-transfected TM cells.5.Effect of NF-κB on TM cells' proliferation and apoptosis:MTT assay and ~3H-TdR incorporation were applied respectively for the detection of TM cells' proliferation,while Tunel method for their apoptosis. 6.Effect of NF-κB on the expressions of MMPs and their tissue inhinitors(TIMPs) in TM cells:RT-PCR was adopted to evaluate the expressions of mRNAs of MMPs(MMP-1, -2 and-3) and TIMPs(TIMP-1,-2 and-3).7.Effect of NF-κB on TIGR gene expression and regulation:The levels mRNA and protein expression of TIGR were detected respectively by RT-PCR and Western Blot.Results:1.We successfully cultured TM cells in DMEM.medium.Immunochemical analysis revealed that these cells were positive for LN,FN,CollagenⅣand TIGR gene expression, and negative for FactorⅧ-related antigens.2.At 24 hours after P65 gene transfection,the expression of P65 was dramatically leveled up,meanwhile,TNF-αlevel was also significantly increased in the culture supernatant of TM cells.3.Both relatively high expression of P65 and NF-κB decoy-ODNs could inhibit proliferation and DNA synthesis of TM cells,and consequently induce cell apoptosis.4.High P65 expression also had an evident expression-promoting effect on the mRNAs of MMP-1 and-3,while,NF-κB decoy-ODNs had an inhibitory one.At the same time,Both of them had no obvious effect on the expressions of MMP-2 and TIMP-1 and-2.5.The gene and protein expression of TIGR could be downregulated by High P65 expression,and upregulated by NF-κB decoy-ODNs.Conclusions:1.P65 obviously promotes TNF-αsecretion in TM cells,which indicates that excessive NF-κB activation may lead to the high-level inflammatory responses.2.Upregulated NF-κB expression would inhibit the proliferation and DNA synthesis of TM cells cultured in vitro and promote their apoptosis,which suggests that exuberant inflammatory responses in anterior chamber have a negative effect on TM cells survival and excessive activation of NF-κB may is one of the pathways involved in secondary glaucoma.NF-κB decoy-ODNs also suppress TM cells proliferation and DNA synthesis,and induce apoptosis,which suggests that NF-κB could maintain normal TM cell numbers and functions and chronic inhibition of NF-κB may cause glucocorticoid-induced glaucoma.NF-κB activation/inhibition could upregulate/downregulate the expressions of MMP-1 and-3 in TM cells,which indicates that NF-κB plays a critical role in ECM maintenance and chronic administration of NF-κB inhibitor may result in the decrease of secretion of MMP-1 and-3,and ECM accumulation in TM cells.NF-κB activation/inhibition also could cause changes of TIGR gene expression,which suggests that NF-κB plays a key role in regulating the expression of glaucoma-related TIGR gene and the inhibition of NF-κB function could lead to the increase of expression of TIGR protein in TM of glucocorticoid-induced glaucoma.