Idebenone Maintains Survival of Mutant Myocilin Cells by Inhibiting Apoptosis.

INTRODUCTION Glaucoma is a chronic progressive neurodegenerative disease and exhibits heterogeneity, polygenic inheritance, and incomplete penetrance.[1] Primary open-angle glaucoma (POAG) is the most common form of glaucoma. Although the underlying etiology of POAG is unknown, there is evidence that genetic mutations are closely associated with this disease.[2] Among myocilin (MYOC) mutations, Pro370Leu (P370L) is responsible for one of the most severe glaucoma phenotypes.[3] However, the function of MYOC protein is still not well understood. The MYOC mutation, P370L, is associated with features of mitochondrial dysfunction in trabecular meshwork cells and can cause impaired function and even cell death. These studies suggested that preventive mitochondrial protection might delay the onset of POAG in patients carrying the MYOC mutation, P370L.[4] CHOP (also called GADD153 or DDIT3) encodes a member of the CCAAT/enhancer-binding proteins, which is a family of transcription factors.[5] The CHOP protein is activated by endoplasmic reticulum (ER) stress and the mitochondrial unfolded protein response and can promote apoptosis. METHODS Small interfering RNA transfection According to our previous procedure, the P370L mutant MYOC gene was cloned into pDsRed2-N1 generating the pDsRed2-N1-mMYOC construct and transfected into COS7 cells.[3] CHOP/GADD153 protein levels were reduced using small interfering RNA (siRNA). The synthesized, scrambled siRNA oligonucleotides and siRNA targeting CHOP were purchased from QIAGEN (Shanghai, China). The siRNA target sequences for CHOP were as follows: GAA UCU AAU ACG UCG AUC AdTdT (dT is deoxythymidine). For transfection, Lipofectamine™ 2000Transfection Reagent (Invitrogen, Carlsbad, CA, USA) was used, and cells were seeded into 6-well plates for 24 h before the experiment. After 48 h transfection, the targeted gene expression levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and/or Western blotting analysis. Semiquantitative reverse transcription polymerase chain reaction analysis Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions and purified using an RNeasy Mini Kit (Qiagen, Germany). RT-PCR was performed using FastStart DNA Master SYBR Green I Reagent (Toyobo Co., Osaka, Japan) by means of a Roche LightCycler™ (Roche, Basel, Switzerland). A melting curve analysis was performed to verify the specificity of the products, and expression was quantified by the ΔΔCt (Ct [target gene] − Ct [β-actin]) method. Primers were purchased from Invitrogen. The primer sequences are listed in Table 1.Table 1: Primer sequences used for qPCR in this studyWestern blotting analysis For Western blotting analysis, rabbit polyclonal antibodies were used in all cases. Cellular proteins (20 μg) were separated on denaturing 10% polyacrylamide gels. The membrane was incubated with antibodies against MYOC (1:500; Abcam, polyclonal, UK), CHOP (1:500; Cell Signaling, L63F7, USA), BCL2 (1:500; Abcam, bcl2/100, UK), and caspase-3 (1:1000; Abcam, 3G2, UK) in blocking buffer overnight at 4°C. Secondary antibodies (peroxidase-conjugated streptavidin) were diluted 1:1000 in blocking buffer and incubated for 1 h at room temperature. As a loading control to ensure equal protein loading, the same blot was subsequently incubated with a GAPDH monoclonal antibody (1:1000; Abcam, 6C5, UK). Apoptosis assay Apoptosis was assessed in transfectants treated with CHOP-siRNA or scramble control. The cells were fixed for 30 min in 4% paraformaldehyde in PBS, followed by incubation with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen, USA) for 30 min, and analyzed by flow cytometry (Becton Dickinson FACSAria, San Diego, USA). Cell lines were gated by light scatter characteristics. Data were analyzed using FlowJo 7.6.1 software (FlowJo, Palo Alto, USA). Drug treatment assay The mutant MYOC (mMYOC) cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Idebenone was purchased from Sigma-Aldrich (I5659, Sigma, USA). The various concentrations of idebenone were used for the treatment of mMYOC cells, including 0, 1, 5, 10, 20, and 50 μmol/L (repeated 3 times). Cells were seeded into 6-well dishes and incubated at 37°C with 5% CO2; the medium was changed at the 3rd day combined with the same concentrations of idebenone, and the cells were collected for the further detection at the 5th day. Statistical analysis The gene expression levels were plotted using the error bar, and the difference between groups was compared using paired samples t-test. All analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). A P < 0.05 was considered statistically significant. RESULTS Apoptosis detection and expression of apoptosis- associated genes For investigating the pathogenesis of glaucoma induced by mutant MYOC gene, we established an in vitro model in which COS7 cells stably expressed either wild-type MYOC (wtMYOC) or mMYOC according to our previous procedure.[3] For evaluating the extent of apoptosis, DAPI was used for detecting dead cells. In wtMYOC cells, reduced apoptosis (<5%) was observed. However, the extent of apoptosis in mMYOC cells increased over time, and approximately, 30% of mMYOC cells were apoptotic at day 5, which was significantly greater than that of apoptotic wtMYOC cells (<3%). In addition, the expression levels of CHOP and caspase-3 were upregulated, and the expression of BCL2 was downregulated in mMYOC cells relative to wtMYOC cells. Treatment of mutant myocilin cells with CHOP-small interfering RNA or idebenone drug Because the expression of CHOP gene changed more significant in apoptosis situation, for inhibiting apoptosis, we tried to silence the expression of CHOP gene in mMYOC cells using CHOP-siRNA. Compared with the scrambled control [Figure 1a], the specific CHOP-siRNA decreased the RNA expression of CHOP (t = 12.35, P = 0.006). Accompanied with the downregulated CHOP, the RNA expression of caspase-3 was also decreased (t = 5.15, P = 0.026), but the RNA expression level of BCL2 was conversely without statistical significance (t = −2.87, P = 0.108). We also performed the protein detection of those genes using Western blotting; the similar results with the RNA expression were observed [Figure 1b]. Furthermore, apoptosis status of the mMYOC cells interfered by siRNA for CHOP was detected using flow cytometry; we found that the fraction of apoptotic mMYOC cells was reduced to 8.4% at the 50th day after CHOP silenced [Figure 1c and 1d].Figure 1: The mutant myocilin cells were transfected with scrambled control or CHOP-small interfering RNA, reverse transcription polymerase chain reaction detected the RNA expression level of apoptosis-associated genes (a), the protein expression level was detected using Western blotting (b), and flow cytometry analysis showed their apoptosis status (c and d). The mutant myocilin cells were treated with different concentrations of idebenone, the apoptosis status was detected using flow cytometry (e and f), and the expression level of protein and RNA was also shown in a and b, separately.The drug idebenone can release ER stress and has been reported to reduce apoptosis. Therefore, we tried to determine whether idebenone could reduce apoptosis induced by mutant MYOC gene. First, we assessed apoptosis in mMYOC cells treated with various concentrations of idebenone (1–50 μmol/L). We found that mMYOC cells treated with 5 μmol/L idebenone exhibited the least apoptosis (5.4%), and when the concentration was increased higher than 10 μmol/L, apoptotic cells number was increased gradually [Figure 1e and 1f]. After that, we detected the expression of RNA and protein of those genes, consistent with reduced apoptosis, we found that the mRNA expression level of CHOP (t = 11.72, P = 0.006) and caspase-3 (t = 9.25, P = 0.017) were downregulated, but the mRNA expression level of BCL2 was increased without statistical significance (t = −1.39, P = 0.130). The expression levels of CHOP and caspase-3 in mMYOC cells treated by idebenone were slightly higher than those in mMYOC cells treated with CHOP-siRNA, without statistical significance (t = −3.05, P = 0.065 and t = −0.62, P = 0.731, respectively) [Figure 1a and 1b]. DISCUSSION The product of the MYOC gene plays an important role in protein metabolism and secretion pathways. One of the risk factors for glaucoma is the elevation of intraocular pressure, which can cause damage to the optic nerve. In the current study, we tried to use idebenone and siRNA for CHOP gene to inhibit apoptosis induced by the MYOC protein. We observed upregulation of the CHOP gene and increased apoptosis in mMYOC cells, and the increased apoptosis situation was successfully decreased by inhibiting CHOP expression. The previous study has shown that the expression of CHOP correlated with ER stress and the mitochondrial unfolded protein response.[5] Our findings suggested that targeting the CHOP gene might provide an avenue for treating ER stress-derived apoptosis caused by mMYOC cells. However, the CHOP pathway may be not the only pathway for the apoptosis, JNK kinase might also be involved in the apoptosis procedure. Studies have revealed that idebenone has antioxidant and antiapoptotic properties.[6] In this study, idebenone was an effective inhibitor of apoptosis induced by MYOC mutation with a dose-dependent manner. As well, we observed reduced CHOP and caspase-3 expression at both protein and mRNA levels when idebenone played its role to inhibit apoptosis. It is difficult to confirm which one of them was the target gene of idebenone. However, CHOP has been regarded as a key mediator of cell death in response to ER stress,[7] and siRNA for CHOP was able to inhibit apoptosis of mMYOC cells in this study, which maybe imply that idebenone might rescue P370L mutant MYOC cells and might release ER stress by inhibiting apoptosis via the CHOP pathway. However, the further study should be designed to clarify that the CHOP gene is the target gene or only is the downstream gene changed following the target gene. Financial support and sponsorship The work was supported by a grant from Project of Science Foundation of Mudanjiang (No. G2012s0028). Conflicts of interest There are no conflicts of interest.