Sodium 4-Phenylbutyrate Acts as a Chemical Chaperone on Misfolded Myocilin to Rescue Cells from Endoplasmic Reticulum Stress and Apoptosis

Abstract

purpose. To evaluate the effect of chemical chaperones on the trafficking of secretion-incompetent primary open-angle glaucoma–associated mutant myocilin and the possibility to rescue cells coexpressing mutant and wild-type myocilin from endoplasmic reticulum (ER) stress and apoptosis.

methods. CHO-K1, HEK293 and human trabecular meshwork cells were transfected to express wild-type or mutant (C245Y, G364V, P370L, Y437H) myocilin-green fluorescent protein fusion protein and were treated or not with various chemical chaperones (glycerol, dimethylsulfoxide, or sodium 4-phenylbutyrate) for different time periods. The secretion, Triton X-100 solubility, and intracellular distribution of wild-type and mutant myocilin were analyzed by immunoprecipitation, Western blotting, and confocal double immunofluorescence. The effect of sodium 4-phenylbutyrate on ER stress proteins and apoptosis was examined in cells coexpressing mutant and wild-type myocilin.

results. Treatment with sodium 4-phenylbutyrate, but not with glycerol or dimethylsulfoxide, reduced the amount of detergent-insoluble myocilin aggregates, diminished myocilin interaction with calreticulin, and restored the secretion of mutant myocilin. Heteromeric complexes formed by mutant and wild-type myocilin induced the ER stress–associated phosphorylated form of ER-localized eukaryotic initiation factor (eIF)-2α kinase and the active form of caspase 3, which resulted in an increased rate of apoptosis. Sodium 4-phenylbutyrate treatment of cells coexpressing mutant and wild-type myocilin relieved ER stress and significantly reduced the rate of apoptosis.

conclusions. These findings indicate that sodium 4-phenylbutyrate protects cells from the deleterious effects of ER-retained aggregated mutant myocilin. These data point to the possibility of a chemical chaperone treatment for myocilin-caused primary open-angle glaucoma.

Glaucoma is a heterogeneous group of optic neuropathies characterized by progressive degeneration of the optic nerve that eventually results in blindness. ¹ Glaucoma is, apart from retinal degenerations, the second-most frequent cause of severe vision loss or blindness. Primary open-angle glaucoma (POAG) is the most common form of this disease. ² Mutations of myocilin (MYOC) gene account for up to 35% of juvenile-onset and approximately 4% of adult-onset POAG. ³ To date, more than 70 MYOC mutations have been identified among patients with POAG (see Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/gene.php?gene=MYOC). Most of them were found in the third exon encoding the olfactomedin-like domain at the C terminus of MYOC. ^{4 5} Myocilin, a secretory glycoprotein, is synthesized in large amounts by trabecular meshwork (TM) cells and is a constituent of aqueous humor. ^{6 7} Secreted MYOC codistributes with extracellular matrix proteins of the TM, such as fibronectin, decorin, and flotillin-1. ^{8 9} The function of MYOC is enigmatic, though it was recently reported to modify signaling events mediated by the heparin II domain of fibronectin. ¹⁰  

The molecular pathogenesis of MYOC-caused POAG is still poorly defined. ¹¹ Glaucoma-associated MYOC mutants are secretion incompetent. ^{12 13 14 15 16} It has been proposed that the impairment of MYOC secretion results in an alteration of the extracellular matrix along the TM outflow pathway and impedes aqueous humor outflow. Furthermore, various glaucoma-associated MYOC mutants are retained in the ER, are detergent-resistant, and are inefficiently degraded inside cells. ^{14 15 17} Increasing evidence suggests that glaucoma-associated MYOC mutations operate through a pathologic gain-of-function mechanism. ^{18 19 20} The most direct evidence in favor of this mechanism is that mutant MYOC interacts with wild-type (WT) MYOC to form aggregation-prone complexes that are retained intracellularly. ^{21 22} Recent evidence suggests that various glaucoma-associated MYOC mutants may be misfolded; hence, MYOC-associated glaucoma is thought to be a protein folding disease, ^{15 23} such as cystic fibrosis, α1-antitrypsin deficiency, Parkinson disease, and Alzheimer disease. 

Attempts have been made to correct disease-causing protein misfolding and the associated defective protein trafficking. Lowering the temperature facilitates protein folding. ^{24 25} Indeed, defective trafficking of misfolded ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis could be corrected in vitro by shifting the temperature from 37°C to 30°C. ²⁶ Similarly, culturing of TM cells expressing different glaucoma-associated mutant MYOC at 30°C resulted in partial release of the secretion block and improved cell viability. ^{15 23} This approach may not be generally applicable, however, because ER-retained renal diabetes insipidus–causing aquaporin 2 mutants could not be rescued by low temperature. ²⁷ Another tactic is the application of chemical chaperones which are low molecular mass, protein-stabilizing agents that can help to correct protein folding abnormalities. ^{24 27 28} Chemical chaperones are diverse agents and include broadly acting compounds such as glycerol, polyols, dimethylsulfoxide (DMSO), deuterated water, and sodium 4-phenylbutyrate (PBA) as well as specific receptor antagonists and enzyme inhibitors. For instance, glycerol releases the secretion block of misfolded ΔF508 CFTR ²⁸ and of mutant aquaporin 2 ²⁷ and restores channel gating and water permeability, respectively. Furthermore, 1-deoxygalactonojirimycin, a competitive inhibitor of α-galactosidase A, was found to stabilize Fabry disease-causing enzyme mutants ²⁹ and to correct its defective lysosomal trafficking, which resulted in the clearance of the lysosomal storage in fibroblasts of Fabry patients harboring different α-galactosidase A mutants. ^{30 31}  

In the present study, we tested the potential of chemical chaperones to improve the solubility and secretion of glaucoma-associated MYOC mutants with the aim of overcoming the associated cytotoxic effects. We show that PBA is useful as a chemical chaperone because it causes the reduction of aggregates formed by mutant and WT MYOC and induces MYOC secretion. As a consequence, PBA reduces the ER stress and the rate of apoptosis and thereby protects the cells from the deleterious effects of mutant MYOC. 

Materials and Methods

Cells and Reagents

Immortalized human trabecular meshwork (HTM) cells ³² were kindly provided by Thai D. Nguyen and calreticulin^+/+ and calreticulin^−/− fibroblasts by Timothy J. Elliott. Other materials used were as follows: CHO-K1 and HEK293 cells (American Tissue Culture Collection, Manassas, VA); culture media, fetal bovine serum (FBS), normal goat serum, competent Escherichia coli DH5α cells, reagent (Lipofectamine 2000; Invitrogen, Basel, Switzerland), and antibiotic (Geneticin 418; Invitrogen); transfection reagent (FuGene 6; Roche Diagnostics, Rotkreuz, Switzerland), protease inhibitor cocktail, and cell death detection kit (In Situ Cell Death Detection kit TMR red; Roche Diagnostics); epitope-tagged expression vectors pcDNA3 (Invitrogen); pEGFP-N₃ (Clontech, Basel, Switzerland); p3xFLAG-myc-CMV-25 (Sigma, Buchs, Switzerland); synthesized oligonucleotides (MicroSynth, Balgach, Switzerland); mutagenesis kit (QuikChange II Site-Directed Mutagenesis kit; Stratagene, La Jolla, CA); PCR and other kits (QuantiTect SYBR Green PCR kit; RNeasy Mini kit; on-column RNase-free DNase kit; Qiagen, Basel, Switzerland); [³⁵S]cysteine and [³⁵S]methionine (Anawa, Wangen, Switzerland); magnetic separation material (protein A Dynabeads; Dynal, Hamburg, Germany); enhancer (En3hance; PerkinElmer, Boston, MA); ECL detection kit (Enhanced Chemiluminescence Western Blotting Detection kit; Amersham Biosciences, Buckinghamshire, UK); glycerol and DMSO (Merck, Basel, Switzerland); chemicals of analytical grade (sodium 4-phenylbutyrate, Triton X-100 [Tx] and all others; Sigma, St Louis, MO); mouse monoclonal antibodies anti-calreticulin and anti-ERp57 (Stressgen, La Jolla, CA), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion, Austin, TX), and anti–β-actin (Sigma); rabbit polyclonal antibodies anti–green fluorescent protein (GFP; Molecular Probes, Eugene, OR), anti-FLAG (Sigma), anti-BiP, and anti-calreticulin (BD Biosciences, San Jose, CA), anti-phosphorylated ER–localized eukaryotic initiation factor (eIF)-2α kinase (pERK; Santa Cruz Biotech Inc., Santa Cruz, CA), and anti–caspase 3 (Upstate, Charlottesville, VA); rabbit polyclonal anti–peptide antibody against the amino acids 188 to 204 of human MYOC raised in rabbits (Imgenex, San Diego, CA) and affinity-purified using the synthetic peptide; goat anti–rabbit immunoglobulin antibody (Alexa 488–conjugated; Molecular Probes), red X-conjugated Fab fragments of goat anti–mouse immunoglobulin or anti–rabbit immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove, PA), and horseradish peroxidase–conjugated donkey anti–rabbit immunoglobulin or sheep anti–mouse immunoglobulin antibodies (Amersham Biosciences). 

Plasmids and Mutagenesis

Human full-length MYOC cDNA was obtained from human skeletal muscle and cloned into the expression vectors pcDNA3, pEGFP-N₃, and p3xFLAG-myc-CMV-25 to generate the constructs p3-MYOC^WT, pEGFP-MYOC^WT, and pFLAG-MYOC^WT, respectively. Mutations in MYOC cDNA were introduced by a PCR-based method (QuikChange II Site-Directed Mutagenesis kit; Stratagene) and specific primers. Sense primer sequences with altered bases highlighted were 5′-GAG TGG AGA GGG AGA CAC CGG ATA TGG AGA ACT AGT TTG GGT AGG for C245Y MYOC; GAA GGA AAT CCC TGG AGC TGT CTA CCA CGG ACA GTT CCC G for G364V MYOC; 5′-CTG GCT ACC ACG GAC AGT TCC TGT ATT CTT GGG GTG GCT ACA CG for P370L MYOC; and 5′-CAT CAT CTG TGG CAC CTT GCA CAC CGT CAG CAG CTA CAC C for Y437H MYOC. All constructs were verified by direct sequencing. 

Cell Culture and Transfection

HEK293 and CHO-K1 cells were cultured in MEM supplemented with MEM nonessential amino acids (Gibco, Grand Island, NY), 10% FBS, 110 μg/mL sodium pyruvate, and antibiotics. HTM cells were grown in DMEM (1 mg/mL d-glucose) supplemented with FBS, sodium pyruvate, and antibiotics. Cells were transfected with plasmids containing WT or different mutant MYOC cDNAs by transfection reagent (FuGene 6; Roche Diagnostics) or reagent (Lipofectamine 2000; Invitrogen) according to manufacturer’s protocol with a ratio of 3 μL transfection reagent/1 μg DNA. For stable MYOC expression, the cells were selected with 100 μg/mL antibiotic (Geneticin 418; Invitrogen) in culture medium for 10 days. After examination by immunofluorescence, cells with suitable WT or different mutant MYOC-GFP expression levels (see Supplementary Fig. S1) were clonally expanded. For transient MYOC expression, cells were used within 5 days of transfection. 

Chemical Chaperone Treatment

Glycerol (0.5% and 5%, vol/vol), DMSO (0.5% and 5%, vol/vol), or PBA (0.2, 0.5, 1, 2, 5, and 10 mM) was added to the culture medium of cells stably or transiently expressing MYOC. The culture medium containing chemical chaperone was replenished daily. 

Monitoring Triton X-100 Solubility of MYOC

Cells expressing WT or mutant MYOC-GFP with or without chemical chaperone treatment were washed twice with ice-cold PBS and lysed in buffer (5 × 10⁶ cells/mL buffer) containing 100 mM Tris-HCl (pH 7.4), 3 mM EGTA, 5 mM MgCl₂, 0.5% Tx, protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 2 minutes on ice. ¹⁷ After centrifugation, the supernatant containing Tx-soluble proteins was denatured in SDS sample buffer containing 50 mM dithiothreitol (DTT). The pellet containing Tx-insoluble proteins was washed twice with ice-cold PBS, sonicated, and denatured in SDS sample buffer containing 9 M urea. Tx-soluble and Tx-insoluble proteins from samples equivalent to 2 × 10⁵ cells were analyzed by 10% SDS-PAGE and Western blotting using antibodies against MYOC (1:800 dilution), GFP (1:2000 dilution), GAPDH (1:3000 dilution), or β-actin (1:3000 dilution), respectively, and appropriate HRP-conjugated secondary antibodies. Signals were detected by ECL, and bands were quantified (Quantity One Image Analysis software; BioRad, Basel, Switzerland). MYOC expression was normalized with GAPDH (for Tx-soluble protein) or β-actin (for Tx-insoluble protein). 

Monitoring Myocilin Secretion

Cells expressing either WT or mutant MYOC-GFP treated or not with 1 mM PBA were incubated with media containing 25 μCi/mL [³⁵S]cysteine and [³⁵S]methionine for 24 hours. Culture media were collected and centrifuged to remove cell debris. The supernatant was immunoprecipitated with rabbit antibodies against GFP or MYOC bound to magnetic separation material (protein A Dynabeads; Dynal). Immunoprecipitated proteins were denatured in SDS sample buffer containing 20% β-mercaptoethanol and resolved by 10% SDS-PAGE. The gel was treated with enhancer (En3hance; PerkinElmer) and radioactivity was detected by bio-imaging (BAS-1800II; Fujifilm, Dielsdorf, Switzerland). Alternatively, GFP-immunoprecipitated proteins were analyzed by Western blotting using antibody to MYOC, and signals were detected by ECL. 

Combined Immunoprecipitation/Western Blot Analysis

CHO-K1 cells stably expressing WT or mutant MYOC-GFP treated or not with 1 mM PBA were washed twice with ice-cold PBS and homogenized in buffer containing 0.25 M sucrose, 10 mM PIPES (pH 6.8), 100 mM KCl, 3 mM MgCl₂, 10 mM CaCl₂, EDTA-free protease inhibitor cocktail, and 1 mM PMSF on ice for 2 minutes. ³⁰ After centrifugation, the supernatant was immunoprecipitated with antibodies to calreticulin or GFP. Immunoprecipitated proteins were denatured and analyzed by Western blotting with antibodies against MYOC or calreticulin. Signals were detected by ECL. 

Western Blot Analysis

CHO-K1 cells coexpressing WT or mutant MYOC-GFP, together with FLAG-WT MYOC treated or not with 1 mM PBA, were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitor cocktail, and 1 mM PMSF. To study pERK expression, phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium diphosphate) were added to the RIPA buffer. After centrifugation, the supernatant was denatured in SDS sample buffer containing 50 mM DTT, and samples (20 μg protein) were resolved by 8% or 10% SDS-PAGE followed by Western blotting with antibodies to pERK (1:300 dilution), caspase 3 (1:200 dilution), or GAPDH (1:3000 dilution). Signals were detected by ECL. 

Real-Time PCR Analysis

Stably MYOC-GFP–expressing CHO-K1 cells treated or not with 1 mM PBA for 2 or 24 hours were collected. Total RNA was obtained (RNeasy Mini kit; Qiagen) and purified (on-column RNase-free DNase kit; Qiagen). RNA (1 μg) was reverse transcribed with 10 ng/mL oligo(dT)₂₅ (Invitrogen) and reverse transcriptase (SuperScript III; Invitrogen). cDNAs were amplified with specific primers for MYOC (5′-CAT CTG GCT ATC TCA GGA GTG G; 5′-CCT TCA CTG TCT CGG TAT TCA G) or β-actin (5′-CAT CCA GGC TGT GCT GTC CCT GTA TG; 5′-GAT CTT CAT GGT GCT AGG AGC CAG AGC) and PCR kit (QuantiTect SYBR Green; Qiagen) according to manufacturer’s protocol on a sequencing detector (ABI 7700; Applied Biosystems, Foster City, CA). MYOC expression relative to β-actin was analyzed by the 2^−ΔΔCT method. ³³  

Confocal Double Immunofluorescence

Stably or transiently MYOC-expressing HEK293 cells treated or not with 1 mM PBA were grown on glass coverslips. They were fixed with freshly prepared 2% formaldehyde, permeabilized with 0.05% saponin, and processed for double immunofluorescence as described previously ³⁰ with the use of antibodies to FLAG (1:2000 dilution), calreticulin (1:300 dilution), or ERp57 (1:500 dilution) and appropriate Alexa 488– or red X–conjugated secondary antibodies (1:2000 dilution). Nuclei were stained with Hoechst 33258 (1:2000 dilution). Samples were examined under a confocal laser scanning microscope (CLSM SP2; Leica, Nidau, Switzerland) using a 63× (1.4) blue-corrected objective. 

Quantification of Apoptosis Rate

HEK293 cells expressing WT/WT or mutant/WT MYOC treated or not with 1 mM PBA were analyzed. Apoptosis rate was determined as the percentage of cells with fragmented nuclei in Hoechst 33258 stained cell cultures or cells that were TUNEL positive. For each experiment (n = 3), a minimum of 10 randomly taken images (40× objective) were analyzed. 

Results

Effect of Chemical Chaperones on the Solubility and Secretion of Mutant MYOC

Glaucoma-associated mutant MYOC can be distinguished from WT MYOC by its higher insolubility in Tx. ¹⁷ When expressed in CHO-K1 cells, we confirmed this for the Y437H and P370L mutant MYOC-GFP and established it for the novel C245Y mutant ¹⁶ (Figs. 1Aand Supplementary Fig. S2). At steady state, nearly 40% of C245Y mutant and 65% of Y437H mutant were resistant to extraction by 0.5% Tx compared with approximately 20% of WT MYOC. In the Tx-insoluble fraction of mutant MYOC, 12% of C245Y and 46% of Y437H could not be resolved by regular 10% SDS-PAGE and remained in the stacking gel, indicating aggregate formation independent of disulfide bonds. 

Treatment with diverse chemical chaperones has been shown to have a beneficial effect on the folding and trafficking of various disease-causing misfolded glycoproteins. ^{27 29 30 31 34 35} When we treated CHO-K1 cells stably expressing C245Y, Y437H, or P370L MYOC-GFP with glycerol or PBA for 2 days, a dose-dependent decrease of the Tx-insoluble MYOC-GFP fraction was observed (Figs. 1Aand Supplementary Fig. S2). PBA had a more pronounced effect than glycerol. Among the concentrations tested (0.2–10 mM), we estimated 1 mM PBA to be the minimal effective dose. After treatment with 1 mM PBA for 2 days, the Tx-insoluble fraction of C245Y and Y437H mutant MYOC-GFP was reduced to almost one third (P < 0.05, paired Student t test). Of the Tx-insoluble fraction, the amount of aggregated protein that could not be dissolved in SDS was reduced by approximately 3-fold for both the C245Y and the Y437H mutant (Supplementary Fig. S2). Moreover, the amount of Tx-soluble mutant MYOC-GFP was increased. As shown in Figures 1B and 1C , treatment with 1 mM PBA for 5 days increased the Tx-soluble fraction of the C245Y MYOC by approximately 1.5-fold, P370L MYOC by more than 4-fold, and Y437H MYOC by approximately 0.6-fold. This effect was maintained over 30 days, the longest time period tested (data not shown). Treatment with 5% glycerol for 5 days was not effective on SDS solubility of mutant MYOC-GFP and was toxic to cells, as indicated by reduced GAPDH expression (Fig. 1B)and diminished cell count (Supplementary Fig. S3). A similar toxic effect on CHO cells expressing mutant MYOC-GFP was observed with DMSO when used at a concentration of 0.5% or 5%. Thus, glycerol and DMSO were not tested in further experiments. 

The effect of PBA on the solubility of mutant MYOC-GFP in Tx could be attributed to its stabilizing action on protein folding or a higher steady state level of MYOC mRNA. By real-time PCR, we obtained no evidence of the latter because 1 mM PBA treatment on HEK cells stably expressing WT or C245Y mutant MYOC-GFP for 24 hours insignificantly increased WT MYOC mRNA (1.3- ± 0.2-fold) and C245Y MYOC mRNA (1.5- ± 0.4-fold). Similarly, 2-hour PBA treatment had no effect on the MYOC mRNA level. 

We extended the data obtained with CHO-K1 cells to HTM cells transiently expressing WT or C245Y MYOC-GFP. We verified the absence of endogenous WT MYOC in the used HTM cells by RT-PCR and Western blot analysis (data not shown). Treatment with 1 mM PBA for 30 minutes reduced the steady state level of Tx-insoluble C245Y MYOC-GFP to 50% and after 4 hours to 25% (Fig. 1D) . Similarly, a time-dependent reduction of C245Y and Y437H mutant MYOC-GFP resistant to extraction by 0.5% Tx, which remained in the stacking gel, was observed (Supplementary Fig. S4). Concomitantly, the intracellular level of the Tx-soluble fraction of C245Y MYOC-GFP increased with time (Fig. 1E)and reached a level close to that of WT MYOC-GFP after 4 hours of PBA treatment (Fig. 1F) . 

Secretion blockade of MYOC is assumed to be a major determinant of MYOC-induced glaucoma. ³⁶ Therefore, we next evaluated whether PBA increased not only Tx solubility of mutant MYOC but also its secretion. Note that MYOC tagged with GFP has been shown to be secreted with an efficiency similar to that of MYOC. ^{21 37} In contrast to WT MYOC-GFP, the different mutant (C245Y, P370L, Y437H) MYOC-GFP stably expressed in CHO cells had a much reduced secretion (Fig. 2A) . Treatment with 1 mM PBA improved secretion of mutant MYOC-GFP (Fig. 2A) . After 2 days, the amount of MYOC in the culture medium was increased by 1.4-fold for the C245Y MYOC, 1.7-fold for P370L MYOC, and 1.6-fold for Y437H MYOC (Fig. 2A) . In human TM cells transiently expressing the C245Y MYOC, treatment with 1 mM PBA induced its secretion, which increased with time (Fig. 2B) . Secreted WT and C245Y MYOC-GFP moved as doublets representing the glycosylated and unglycosylated forms of MYOC monomers (Fig. 2B) . ^{12 22}  

Effect of PBA on the Interaction of Mutant MYOC and Calreticulin

Misfolded glycoproteins are recognized by protein quality control and retained in the ER by chaperones. ^{38 39 40} This also appears to be the case for mutant MYOC. ^{14 15} We studied the distribution of MYOC and that of the ER chaperones calreticulin and ERp57 by confocal double immunofluorescence in HEK293 cells transiently expressing WT or C245Y MYOC (Figs. 3A 3B 3C 3D 3E 3F) . Unlike WT MYOC, which as expected showed little colocalization with calreticulin (Figs. 3A 3B) , C245Y MYOC exhibited prominent colocalization with calreticulin (Figs. 3C 3D)and ERp57 (data not shown). The same observations were made for P370L and Y437H MYOC (data not shown). When the cells were treated with 1 mM PBA for 2 days, codistribution between mutant MYOC and calreticulin was reduced to the extent observed for WT MYOC (Figs. 3E 3F) . Molecular interaction between the mutant MYOC and calreticulin was directly demonstrated by combined immunoprecipitation–Western blotting (Fig. 3G) . In general, more complexes between mutant MYOC and calreticulin were observed than between WT MYOC and calreticulin. The amount of MYOC–calreticulin complexes was reduced after PBA treatment. Further evidence for molecular interaction between mutant MYOC and calreticulin was obtained with mouse fibroblasts lacking calreticulin. As expected, WT MYOC was secreted by calreticulin^+/+ and calreticulin^−/− fibroblasts, whereas C245Y MYOC was secreted only by calreticulin^−/− fibroblasts (Supplementary Fig. S5). Together, the interaction with calreticulin indicated a folding problem of the studied mutant MYOC, which hindered their trafficking out of the ER and resulted in a secretion defect. This could be positively influenced by PBA treatment. 

PBA Reduces ER Stress and Apoptosis of Cells Expressing Mutant MYOC

Glaucoma-associated MYOC mutations appear to act through a pathologic gain-of-function mechanism. ^{18 19 20} Intracellular accumulation of dimers and oligomers formed by WT and mutant MYOC has been suggested to play an important role in the pathogenesis of MYOC-induced glaucoma. ^{21 22} To test the effect of PBA on cells coexpressing WT and mutant MYOC, we applied a double epitope-tagging protocol. As a control, HEK293 cells stably expressing WT MYOC-GFP were transfected with increasing amounts of pFLAG-MYOC. By confocal double fluorescence microscopy, the GFP- and the FLAG-tagged WT MYOC exhibited cytoplasmic colocalization (Fig. 4A) , and approximately 20% of the cells showed MYOC aggregates (Fig. 4C) . When HEK293 cells stably expressing C245Y MYOC-GFP were transfected with increasing amounts of pFLAG-MYOC, approximately 60% of the cells exhibited numerous MYOC aggregates containing WT and mutant MYOC in addition to fine reticular fluorescence (Fig. 4B) . The same observations were made for cells double expressing P370L or Y437H MYOC-GFP together with FLAG-WT MYOC (Fig. 4C) . Treatment with 1 mM PBA for up to 5 days reduced the number of cells containing MYOC aggregates by approximately 50% (Fig. 4C) . 

It is known that intracellular accumulation of aggregated proteins leads to ER stress, which may be followed by apoptotic cell death. ⁴¹ We analyzed by Western blotting pERK and caspase 3 expression levels as indicators of ER stress and apoptosis in double-transfected HEK293 cells. In control WT/WT cells, immunoreactivity for pERK was undetectable, and only the inactive form of caspase 3 was detectable (Fig. 5A) . In contrast, C245Y/WT, P370L/WT, and Y437H/WT cells exhibited a pERK immunoreactive band of intensity similar to that of mock-transfected HEK293 cells treated with DTT to induce ER stress (Fig. 5A) . Similarly, the active form of caspase 3 was detectable in the mutant/WT MYOC–expressing cells (Fig. 5A) . When cells were treated with 1 mM PBA for 2 days, immunoreactivity for PERK and for active caspase 3 became undetectable (Fig. 5A) . Next we quantified apoptosis by determining the percentage of cells showing fragmented nuclei by Hoechst 33258 staining or by the TUNEL method. In WT/WT HEK293 cells, serving as a control, the apoptosis rate was approximately 8% (Fig. 5B) , whereas it was significantly increased in mutant/WT MYOC–expressing cells: 27.7% ± 2.4% for C245Y/WT, 37.3% ± 9.6% for G364V/WT, 21.2% ± 2.1% for P370L/WT, and 27% ± 4% for Y437H/WT cells (Fig. 5B) . After treatment with 1 mM PBA for up to 5 days, the apoptosis rate was reduced to 17.7% ± 4.5% for C245Y/WT, 18.7% ± 14.6% for G364V/WT, 14.5% ± 4.3% for P370L/WT, and 15% ± 0.8% for Y437H/WT cells (Fig. 5B) . Similar results were obtained by TUNEL analysis. Treatment with 1 mM PBA for 5 days resulted in a decrease of TUNEL-positive cells from 25.5% to 10.6% for C245Y/WT and from 28.1% to 17.8% for P370L/WT. These apoptosis rates were close to the rate of WT/WT cells (14.3%). 

Discussion

The primary objective of the present study was to test chemical chaperones for their capability to reduce the adverse effects of glaucoma-associated mutant MYOC caused by the formation of MYOC aggregates and the MYOC secretion block. We made the following major observations. The formation of protein aggregates composed of glaucoma-associated heteromeric WT/mutant MYOC complexes results in apoptosis because of ER stress. Treatment with PBA, a butyrate analogue approved for clinical use in subjects with urea cycle disorders and thalassemia, ^{42 43 44} reduces the number of cells with MYOC aggregates and rescues the cells from apoptosis. 

Misfolded glycoproteins become retained inside cells by the protein quality control recognizing non-native conformers. ^{38 39} In the case of mutant MYOC, detergent insolubility and formation of intracellular aggregates, together with a secretion block, have been taken as positive evidence for protein misfolding. ^{13 14 17} Hence, it has been proposed MYOC-caused POAG belongs to the family of protein folding diseases. ¹⁵ Consistent with this, a molecular interaction between severe juvenile-onset POAG-causing mutant MYOC and the ER chaperone calreticulin was directly demonstrated by combined immunoprecipitation–Western blot analysis (Ref. ¹⁵ and present study). Furthermore, mutant MYOC singly expressed in TM cells appears to be cytotoxic, as indicated by a change in cell shape and reduced cell proliferation. ^{14 21} By mimicking the pathologic gain-of-function situation, we demonstrate that accumulation of heteromeric WT/mutant MYOC complexes results in a high rate of cell death by apoptosis. 

Human disease-causing protein misfolding can be positively influenced by low temperature or by chemical chaperones. ^{24 27 28} With regard to MYOC-caused POAG, incubation of cells expressing mutant MYOC at 30°C is beneficial because it results in reversal of the MYOC secretion block and lowers the cytotoxic MYOC effect. ^{15 23} Treatment with chemical chaperones such as PBA reversed the trafficking blockade of misfolded ΔF508 CFTR ³⁴ and Z variant α1-antitrypsin. ³⁵ Pilot clinical trails for the treatment of cystic fibrosis with PBA have been conducted with success, and no drug-induced toxicity was encountered by patients. ⁴⁵ In the present study, we demonstrated a beneficial effect of PBA on four mutant MYOCs that cause severe juvenile-onset POAG. PBA treatment improved the solubility of the mutant MYOC, reduced their interaction with the ER chaperone calreticulin, and eventually resulted in MYOC secretion. In parallel, the number of cells containing MYOC aggregates was reduced. The most striking consequence of PBA treatment, however, was on ER stress and apoptosis rate. pERK and active caspase 3 proteins became basically undetectable by Western blotting. The apoptosis rate was reduced close to levels observed in control cells expressing WT MYOC. 

What could be the mechanism through which PBA acts on mutant MYOC protein? Mutant MYOC is thermolabile and growth of cells at 30°C leads to its thermal stabilization, which allows secretion. ^{15 23} It is tempting to speculate that PBA may act by stabilizing the thermolabile mutant MYOC. The increase in thermodynamic stability of mutant MYOC could salvage it from denaturation and allow it to pass through ER quality control and to become secreted. PBA is a histone deacetylase inhibitor and has been shown to be a transcriptional regulator. ⁴² Through gene expression profiling, various heat-shock proteins were found to be transcriptionally regulated by PBA, whereas no significant change was observed for the ER unfolded protein response. ⁴⁶ On the other hand, a decrease in the expression of Hsc70 protein caused by decreased stability of mRNA was observed, indicating a mode of action different from that of inhibiting deacetylase activity. ⁴⁷ In our experiments, no evidence of transcriptional upregulation of MYOC by PBA treatment was obtained. Furthermore, expression levels of the ER chaperones calreticulin and BiP did not change under PBA treatment. Secretion of mutant MYOC could be observed as early as 30 minutes after the start of PBA treatment. This observation points to a direct effect of PBA on the folding state of mutant MYOC. There is good evidence that another, unrelated, chemical chaperone may directly influence the folding state of a misfolded glycoprotein. DGJ, a competitive inhibitor of α-galactosidase A, releases misfolded, Fabry disease-causing enzyme from BiP and restores its trafficking into lysosomes. ^{30 31}  

In summary, the present data indicate that PBA exerts a chaperone-like effect on misfolded MYOC. Given that PBA is a tissue- and cell-permeable molecule, it holds the potential for topical administration in the treatment of MYOC-caused POAG. 

²  Present affiliation: Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.

 Supported by the Velux Stiftung (Zurich), the Kamillo Eisner Stiftung (Hergiswil), and the Canton of Zurich.

 Submitted for publication August 9, 2006; revised October 19, 2006; accepted January 29, 2007.

 Disclosure: G.H.-F. Yam, None; K. Gaplovska-Kysela, None; C. Zuber, None; J. Roth, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Chemical chaperones improve Triton X-100 solubility of glaucoma-associated mutants of MYOC. (A) CHO-K1 cells expressing WT or mutant (C245Y, Y437H) MYOC-GFP were treated with glycerol (0.5% and 5%) or PBA (0.2 mM and 1 mM) for 2 days. After cell lysis in 0.5% Tx, detergent-insoluble fractions were analyzed by Western blotting for GFP and β-actin. A prominent reduction of the detergent-insoluble and SDS non-resolvable fractions of mutant MYOC-GFP was found after 1 mM PBA treatment. Only a slight reduction was observed after treatment with 5% glycerol. (B) The same cells as in (A) were treated for 5 days with 5% glycerol or 1 mM PBA. Tx-soluble fractions were immunoblotted for GFP and GAPDH. Glycerol treatment exerted an adverse effect on cell survival, as indicated by the low GAPDH content. PBA strongly increased the amount of soluble C245Y and P370L MYOC-GFP and only moderately increased the amount of WT and Y437H MYOC-GFP. (C) Quantification by band densitometry of the PBA effect shown in (B). (D, E) HTM cells transiently expressing C245Y MYOC-GFP were treated with 1 mM PBA for the time periods indicated. Cells collected at different time points were lysed with 0.5% Tx. Detergent-insoluble (D) and -soluble (E) fractions were assayed for GFP and either β-actin or GAPDH. Untreated WT MYOC-GFP expressing cells served as a control. PBA treatment induced a time-dependent reduction of Tx-insoluble mutant MYOC-GFP with a concomitant increase of the detergent-soluble mutant MYOC-GFP. (F) Quantification by band densitometry of the results presented (D, E) showed return to WT MYOC levels of the detergent-insoluble and soluble mutant MYOC.

Figure 2.

 

PBA treatment improves secretion of glaucoma-associated mutants of MYOC. (A) CHO-K1 cells stably expressing WT or mutant MYOC-GFP were treated or not with 1 mM PBA for 2 days in the presence of 25 μCi/mL [³⁵S]cysteine and [³⁵S]methionine. After culture media were immunoprecipitated with anti–GFP antibodies, immunoprecipitated samples were resolved by 10% SDS-PAGE and analyzed by phosphoimaging. (B) HTM cells transiently expressing WT or C245Y MYOC-GFP were treated with 1 mM PBA. Media collected at the indicated time points were immunoprecipitated with anti–GFP antibodies, and the immunoprecipitated samples were analyzed by immunoblotting with anti–MYOC antibodies. Asterisk: position of the MYOC band.

Figure 3.

 

PBA treatment reduces the interaction between mutant MYOC and calreticulin. (A, B) Confocal double immunofluorescence for MYOC and calreticulin in HEK293 cell stably expressing nontagged WT MYOC reveals little overlap, as indicated by the low level of yellow, which can be best appreciated at higher magnification in (B). (C, D) In contrast, in C245Y MYOC–expressing cells, considerable codistribution of MYOC and calreticulin (CRT) is indicated by the high level of yellow, which can be best appreciated at higher magnification in (D). (E, F) In C245Y MYOC–expressing cells treated with 1 mM PBA for 2 days, codistribution of MYOC with calreticulin was greatly reduced and resembled that of WT MYOC–expressing cells. Nuclei appear in blue due to Hoechst 33258 DNA staining. (G) Combined immunoprecipitation–Western blot analysis of CHO-K1 cells expressing WT or mutant MYOC-GFP demonstrated substantial complex formation between mutant MYOC-GFP and calreticulin and its reduction after treatment with 1 mM PBA for 2 days. Bottom, arrow: position of the heavy chain of secondary anti–mouse immunoglobulin antibody. Regions indicated by rectangles (A, C, E) are shown at higher magnification (B, D, F). Scale bars: (A, C, E) 2 μm; (B, D, F) 0.1 μm.

Figure 4.

 

PBA treatment of HEK293 cells coexpressing mutant and WT MYOC reduces MYOC aggregates. Cells stably expressing WT or C245Y MYOC-GFP were transiently transfected with pFLAG-MYOC^WT. (A) By confocal double-immunofluorescence, codistribution of GFP- and FLAG-tagged WT MYOC is obvious. (B) C245Y MYOC-GFP– and FLAG-WT MYOC–expressing cells exhibit distinct cytoplasmic aggregates containing mutant and WT MYOC. Blue color: Hoechst 33258 DNA staining. (C) Percentage of cells containing MYOC aggregates is reduced after treatment with 1 mM PBA for 5 days. Scale bars: 5 μm.

Figure 5.

 

Effect of PBA treatment on the expression of ER stress proteins and apoptosis. (A) HEK293 cells coexpressing WT and WT or mutant and WT MYOC were treated with 1 mM PBA for 2 days. This resulted in a reduced intensity of the pERK and active caspase 3 bands in mutant and WT MYOC–expressing cells. Mock-transfected HEK293 cells treated with 10 μM DTT for 2 hours to induce ER stress proteins served as control. (B) Mutant/WT MYOC–coexpressing cells had a significantly higher apoptosis rate than did WT/WT MYOC–coexpressing cells. PBA (1 mM) treatment for 5 days drastically reduced the apoptosis rate in mutant/WT MYOC cells.

Supplementary Materials

Supplementary Figure S1 - (PDF) 

Supplementary Figure S2 - (PDF) 

Supplementary Figure S3 - (PDF) 

Supplementary Figure S4 - (PDF) 

Supplementary Figure S5 - (PDF) 

The authors thank Thai D. Nguyen for providing HTM cells and T. J. Elliott for providing K41 calreticulin^+/+ and K42 calreticulin^−/− fibroblasts. They also thank Chi-Pui Pang for communicating the C245Y MYOC mutation before publication and Peter M. Lackie, Douglas J. Taatjes, and Charlotte Remé for critical evaluation of the manuscript.