The Role Of ClC-3 In Trabecular Meshwork Cells

Primary open angle glaucoma (POAG) is one of the leading causes of irreversible blindness, and elevated intraocular pressure (IOP) is a critical risky factor for optic nerve injury in POAG. A large number of studies have shown that the IOP elevation is caused by the increased resistance of aqueous outflow, and trabecular meshwork is the major site of resistance. The alternation of number and phagocytosis function of trabecular meshwork cells create resistance for aqueous outflow, resulting in the occurrence and development of POAG. Thus, culturing and characterizing TMC in vitro is an important experimental way to study the pathogenesis of POAG. Recently, studies about ion channels especially chloride channels, which might be involved in POAG, have aroused widely attention. Chloride channels (ClC) have been shown extensively distributing in mammal's organs, tissues and cells. As one of the ClC subtype, ClC-3 plays an important role in many physiological and pathological functions including maintaining cells volume, proliferation,apoptosis,migration and so on. Although numerous researches have indicated that ClCs indeed expressed in TMC, the functions of ClC are still unclear now. In this study we were going to investigate the expression and functions of ClC-3 with an immortal trabecular meshwork cell line.Using RT-PCR and immunochemistry staining, we first confirmed that ClC-3 expressed in TMC at mRNA and protein level and located in cytoplasm of TMC. By using MTT, cell cycle synchronization, apoptosis inducer 5-FU, mitochondrial membrane potential(△Ψm), cell phagocytosis model, we further explored the role of NPPB, a chloride channel blocker, in cell proliferation, cell cycle, apoptosis, mitochondrial membrane potential and phagocytosis of TMC respectively. We found that NPPB inhibited proliferation of TMC in a concentration–dependent manner. Twenty-four hours after cell cycle synchronization, cell population increased in G0/G1 phase , while decreased in S phase induced by NPPB. Changes of Nucleoli indicated that cells were in a resting period. NPPB was able to increase the apoptosis induced by 5-FU and reduce the mitochondrial membrane potential in TMC. By establishing the model of phagocytose fluospheres beads in vitro, we found that NPPB reduced the phagocytosis of TMC at the same time point. In addition, to study the role of ClC-3 in TMC, we applied of antisense oligonucleotides(ASONS) transfected with Lipofectimine 2000, by which the expression of ClC-3 was inhibited specifically. Our results indicated that proliferation of TMC was reduced following the increase of ASONS transfecting concentration. Results about cell cycle showed that cell population increased in G0/G1 phase, but decreased in S phase in ASONS-transfected TMCs. Meanwhile, numerous ASONS-transfected TMCs were at a standstill in G0/G1 phase. Apoptotic rate of ASONS-transfected TMCs was higher than SONS-transfected TMCs and mitochondrial membrane potential reduced gradually. With the increase of ASONS transfecting concentration, phagocytosis of ASONS-transfected TMCs decreased either.In summary, our results indicated that ClC-3 positively correlated with proliferation of TMC and promoted TMC into S phase via G1/S check point. In addition, ClC-3 might be involved in anti-apoptosis mechanism through interfering internal mitochondrial pathway. ClC-3 was positively correlative with phagocytosis of TMC as well. Our studies pave a way for investigating the functions of ClC-3 in TMC and providing experimental evidences for exploring glaucoma pathogenesis.