Role of the ClC-2 Chloride Channel in TGF-β1-induced Proliferation, Collagen Synthesis, and Collagen Gel Contraction Mediated by Human Conjunctival Fibroblasts

Background: Excessive scar tissue can reduce postoperative survival of filtering blebs in patients with glaucoma. Previous studies have highlighted the role of chloride channels in wound healing. whereas The role of chloride channels in the formation of follicular scar has not been studied. Objectives: To investigate the effects of the ClC-2 chloride channel on scar formation of filtering blebs after glaucoma filtering surgery. Methods: We Inhibited ClC-2 chloride channels of Human Conjunctival Fibroblasts (HConFs) by transfecting HConFs with ClC-2 siRNA, Then cell proliferation, cycle and collagen synthesis of HConFs were measured. ClC-2 siRNA-transfected HConFs were cultured in type I collagen gels in the presence of transforming growth factor (TGF)-β1. Collagen gel contraction was evaluated based on the gel area. The expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in HConFs were assessed by western blotting and q-PCR. Results: TGF-β1induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction in HConFs. TGF-β1 increased MMP-2 and MMP-9 levels but inhibited the expression of TIMPs. ClC-2 siRNA transfection inhibited TGF-β1-induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction, mediated by HConFs. TGF-β1-induced increases in MMP-2 and MMP-9 were also inhibited by NPPB and ClC-2 siRNA transfection, but TIMP expression was increased by ClC-2 siRNA transfection. Conclusions: These findings demonstrate that ClC-2 gene knockout inhibited TGF-β1-induced cell proliferation, collagen synthesis, and collagen gel contraction of HConFs by attenuating MMP-2 and MMP-9 production and by stimulating TIMP-1 production.


Background:
Excessive filtering bleb scar formation after glaucoma filtration surgery is the most frequent cause of surgical failure. Human conjunctival fibroblasts (HConFs) are key cells involved in the subconjunctival wound healing response, which involves the activation, proliferation, and migration of local fibroblasts as well as the synthesis of extracellular matrix components and its subsequent contraction [1,2]. Excessive deposition of matrix components and contraction of the subconjunctival tissue at the wound site, resulting in destruction of the normal tissue architecture, determine scar formation and surgery failure TGF-β is secreted during the early stages of scar formation by inflammatory cells and acts as a chemoattractant to promote fibroblast transformation to myofibroblasts, which synthesize extracellular matrix [3,4]. Accumulation of ECM structural proteins increases bleb capsule fibrosis. Matrix metalloproteinases (MMPs) constitute a large family of enzymes that remodel ECM by degrading ECM molecules during the wound-healing process. The action of MMPs is regulated by a group of endogenous tissue inhibitors of metalloproteinases (TIMPs) [5]. Imbalance between MMPs and TIMPs may lead to excessive degradation or increased accumulation of the ECM, resulting in excessive wound healing.
Chloride channels (ClCs) are a type of permeable channel protein for chloride ions or other anions on the cell membrane, and ClC proteins are encoded by genes of the ClC family [6].
ClC-2 is a member of the ClC voltage-gated chloride channel superfamily and is ubiquitously expressed in organs act as an regulator of cell volume and control the response to swelling and post-synaptic responses to GABA and glycine [7,8]. In the prevention of hyperplasia and remodeling of cerebrovascular smooth muscle cells ClC-2 plays an important role by inhibiting cell proliferation and migration [9]. ClC-2 also modulates wound epithelialization by regulating the migratory ability of rat keratinocytes [10]. In this study, the effects of the ClC-2 chloride channel on cell proliferation, collagen synthesis, collagen contraction, and MMP and TIMP production by HConFs were evaluated.

Cell Culture
HConFs were obtained from ScienCell Research Laboratories (San Diego, CA, USA), where they were isolated from human conjunctiva. HConFs were characterized by their spindle morphology and immunoreactivity with antibodies to fibronectin. Cells were grown at 37 °C in a humidified incubator with 5% CO 2 in fibroblast medium (ScienCell Research Laboratories) containing 2% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and fibroblast growth supplement (undisclosed formulation).

Transfection of HConFs with siRNA
To knockdown ClC-2 expression, we obtained commercially available stealth siRNA duplex oligoribonucleotides targeting the human ClC-2 gene. The sequence was 5′-UCCUCAUGAGGAAACGCCUGCUCUU-3′, and its corresponding complementary strand was 5′-AAGAGCAGGCGUUUCCUCAUGAGGA-3′. The negative control consisted of a non-silencing scrambled sequence that did not recognize any known sequence available in the GenBank.
To examine the uptake of ClC-2 siRNA by HConFs, siRNA was labeled with Alexa Fluor 488 HConFs were plated at a density of 5,000 cells/well in a 96-well plate. After treatment, 10 µL of CCK-8 solution (BestBio, Jiangsu, China) was added to each well, followed by incubation for another 3 h at 37 °C. Absorbance at 450 nm was measured using an automated microplate reader (Model 3001 − 1387; Thermo Fisher, Waltham, MA, USA).
Each group was provided with 6 duplicate holes, and the experiment was repeated 3 times.

Cell Cycle Analysis
Cell cycle status was assessed by flow cytometry using a Cell Cycle and Apoptosis Analysis

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted from HConFs at 60-80% confluency with RNAiso Plus (Takara Bio Inc., Shiga, Japan), and 500 ng RNAwas used to synthesize single-stranded cDNA with the RT for PCR Kit (Takara Bio Inc.). Quantitative real-time PCR was performed using methods similar to those previously described [11]. Oligonucleotide primers for the collagen III, MMP2, MMP9, TIMP1, and GAPDH genes were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/), and sequences are listed in Table 1.  Following two washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Beyotime) or anti-mouse IgG secondary antibody (Beyotime) for 1 h at room temperature. Final detection was accomplished with enhanced chemiluminescence western blotting reagents (Beyotime).

Assay of Collagen Gel Contraction
The collagen gel contraction assay was performed according to previously described methods [9], with some modifications. HConFs were harvested by treatment with trypsin-EDTA, washed twice with serum-free MEM, and resuspended in the same medium. Briefly, collagen I (from a rat tail) stock solution (5 mg/mL) (Solarbio, Beijing, China), 0.1 M NaOH, 10 × concentrated PBS, and the cell suspension were mixed on ice at a volume ratio of 200:12:23:765 (final concentration of type I collagen, 1 mg/mL; final cell density, 2 × 10 5 /mL). A portion (0.5 mL) of the mixture was added to each well of the 24-well culture plate and allowed to solidify by incubation at 37 °C under 5% CO 2 for 20 min. The collagen gels were freed from the sides of the wells using a 10-µL tip, and serum-free MEM (0.5 mL) containing TGF-β1 (10 ng/mL) was then added to the top of each gel. The gel area was examined at 48 h using ImageJ. For normalization, the area of the collagen gel containing untreated HConFs was set to 100%, and the fold changes in the area for each treatment group were determined. Four gels were assayed for each experimental condition, and all experiments were repeated four times.

Data Analyses
Results are expressed as means ± standard error (number of observations), and differences among groups were evaluated using analysis of variance (ANOVA). Statistical significance was defined as p < 0.05. All experiments were repeated four times.

Discussion
In the present study, we induced a series of conjunctival scarring responses with TGF-β1.
Subsequently, the effects of ClC-2 chloride channel on HConF scarring responses were measured. We found that ClC-2 siRNA transfection inhibited TGF-β1-induced HConF proliferation, collagen synthesis, and collagen gel contraction. Exaggerated wound contraction induced by elevated TGF-β levels has become a therapeutic target in fibrosis [12]. A study by Arslan et al. [13] showed that filtration surgeries increased TGF-β expression. Similar to our results, Liu et al. [14] found that TGF-β-induced collagen gel contraction was mediated by human Tenon fibroblasts.
Chloride channels are expressed in nearly all eukaryotic cells and play an important role in cell proliferation, apoptosis, and migration [15]. The regulatory effect of ClC chloride channels on cell proliferation has been shown in many studies [16]. A study by Warsi et al. [17] showed that downregulation of the chloride channel ClC-2 by Janus kinase 3 influenced cell proliferation and apoptosis. In osteosarcoma cells, ClC-3 silencing inhibited cell proliferation and migration via the AKT/GSK3β signaling pathway [18]. Li et al. [19] found that overexpression of the chloride intracellular channel 5 (CLIC5) inhibited proliferation of C2C12 cells and drove more C2C12 cells into the G0/G1 phase. We previously blocked the chloride channel of ARPE19 in human adult RPE cells using NPPB and tamoxifen (TAM) and found that this inhibited proliferation by arresting cells in the G0/G1 phase[20]. ClC-2, a ubiquitously expressed member of the superfamily of ClC transporters and channels, has been confirmed to be expressed in human conjunctival fibroblasts [11]. Our result showed that ClC-2 siRNA transfection inhibited TGF-β1-induced HConF cell proliferation by arresting cells in the G0/G1 phase.
Recent studies have highlighted the role of chloride channels in wound healing. Fuchigami et al. [21] showed that the chloride channel blocker NPPB reduced epidermal wound closure by blocking the CIC-3 chloride channel. Further, local enhancement of CIC-3 expression at the leading edge of the wounded epidermis was specific to closing wounds, suggesting that the intracellular translocation of CIC-3 was involved in wound closure.
Schiller et al. [22] found that CFTR chloride channels positively regulated wound healing in cell culture models of the airway surface epithelium. Thus, inhibition of conjunctival fibroblast proliferation is the main strategy for treatment of anti-glaucoma filtration scarring.
Cell-mediated tissue contraction is a complex process that involves various cellular activities, including cell migration and reorganization of the ECM, and MMPs have been implicated in wound contraction and matrix reorganization [23]. Collagen contraction involves the cell-directed reorganization of collagen fibrils and plays a significant role in wound healing. In the present study, a three-dimensional model of wound healing that closely mimics the in vivo situation was established. We found that TGF-β1 promoted HConF collagen synthesis and collagen gel contraction. ClC-2 gene knockout inhibited TGF-β1-induced HConF collagen synthesis and collagen gel contraction. Multiple studies have shown that chloride channels are involved in collagen synthesis [24][25][26]. Qi et al. [24] showed that the chloride channel blocker DIDS inhibited the collagen release of HFL1 fibroblasts. Downregulation of CLIC4 also reduced TGF-β1-induced expression of ECM components in primary fibroblasts [25]. In the development of atrial fibrillation, CLIC 1, 4, and 5 played an important role by interacting with collagen IV [26,27].
Inhibition of MMPs has been shown to limit subconjunctival scarring after experimental glaucoma filtration surgery in rabbits [27]. The balance between MMPs and TIMPs in the ECM is important for regulation of the ECM and wound healing [23]. The ratio of MMPs to TIMPs is an important factor in various biological activities, including cell migration, angiogenesis, and remodeling of the ECM [28]. Du and Yang [21] found that ClC-3 knockout attenuated osteosarcoma cell migration via downregulation of MMP-2 and MMP-9.
Wang et al. [30] silenced the ClC-3 gene and found that MMP-3 and MMP-9 levels were reduced. Our results showed that the ClC-2 knockout decreased TGF-β1-induced proliferation, collagen synthesis, and contractility of HConFs cultured in a three-dimensional collagen gel, in part by blocking MMP production. Further in vivo studies are needed to confirm the preventive effect of ClC-2 chloride channels on filtering bleb scarring after filtration surgery.

Conclusions:
These findings demonstrate that knockdown of ClC-2 expression inhibited TGF-β1-induced cell proliferation, collagen synthesis, and collagen gel contraction of HConFs by attenuating MMP-2 and MMP-9 production and by stimulating TIMP-1 production. ClC-2 chloride channels may therefore prove to be a new control target to inhibit scar formation of filtering blebs.

Consents to publish
Not applicable

Availability of data and material
The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.     Protein and mRNA expression of collagen III in Human conjunctival fibroblasts (HConF) after different treatments as determined by western blotting. Results are presented as means ± SD (n = 4); *p < 0.05, vs. control; #p < 0.05, vs. TGF-β1.