Chemical chaperone 4-phenylbutyrate prevents endoplasmic reticulum stress induced by T17M rhodopsin
© Jiang et al.; licensee BioMed Central Ltd. 2014
Received: 5 September 2014
Accepted: 25 November 2014
Published: 4 December 2014
Rhodopsin mutations are associated with the autosomal dominant form of retinitis pigmentosa. T17M mutation in rhodopsin predisposes cells to endoplasmic reticulum (ER) stress and induces cell death. This study aimed to examine whether chemical chaperone 4-phenylbutyrate prevents ER stress induced by rhodopsin T17M.
ARPE-19 cells were transfected with myc-tagged wild-type (WT) and T17M rhodopsin constructs. Turnover of WT and T17M rhodopsin was measured by cycloheximide chase analysis. The activity of ubiquitin-proteasome system was evaluated by GFPU reporter. We found that T17M rhodopsin was misfolded, ubiqutinated and eliminated by ER-associated degradation pathway (ERAD) in ARPE-19 cells. Accumulated T17M rhodopsin induced unfolded protein response, but had no effect on the activity of ubiquitin proteasome system. Moreover, chemical chaperone 4-phenylbutyrate facilitated the turnover of T17M rhodopsin and prevented apoptosis and ER stress induced by T17M rhodopsin.
Chemical chaperone could attenuate UPR signaling and ER stress induced by T17M rhodopsin and has potential therapeutic significance for retinitis pigmentosa.
KeywordsRetinitis pigmentosa Rhodopsin UPR Misfolded ERAD
Retinitis pigmentosa (RP) is considered the most commonly inherited retinal dystrophy with an estimated prevalence of approximately 1:4000 . RP is caused by the progressive loss of rod and cone photoreceptors with clinical hallmarks including the sensitivity to dim light, abnormal visual function and characteristic bone spicule deposits of pigment in the retina . Mutations in rhodopsin, a photon receptor that initiates phototransduction, have been linked to autosomal dominant retinitis pigmentosa (ADRP), accounting for about 10% of all reported cases of RP [3, 4]. Since the identification of the P23H mutation, more than 130 different mutations of rhodopsin have been shown to cause RP .
A mouse model of ADRP was created with a threonine-to-methionine mutation at the 17th residue of rhodopsin, which abolishes the glycosylation site at Asn15 and results in a class I RP phenotype [6, 7]. Transgenic mice carrying human T17M rhodopsin gene showed significant photoreceptor apoptosis as early as 24 h after illumination, while mice expressing a rhodopsin transgene with P23H mutation were only minimally affected . Further study showed that endoplasmic reticulum (ER) stress response is involved in retinal degeneration in T17M rhodopsin retinas in vivo, accompanied by the up-regulation of autophagy markers and the activation of mitochondrial apoptosis via the up-regulation of pro-apoptotic Bcl2 . Our previous study showed that T17M rhodopsin accumulated in ER, increased the cytotoxicity and predisposed the cells to ER stress induced cell death .
Misfolded proteins that do not pass ER quality control (ERQC) are selectively recognized and cleared during a process called ER-associated degradation (ERAD) that involves the export of misfolded proteins from ER followed by proteasomal degradation . Up to now, the role of ERAD in the clearance of T17M rhodopsin is unclear. We proposed that T17M rhodopsin may be misfolded and eliminated via ERAD, and chemical chaperone 4-phenylbutyrate may prevent ER stress induced by rhodopsin T17M.
In this study, we used a spontaneously arising retinal pigment epithelia (RPE) ARPE-19 cell line as the experimental model to investigate the role of ERAD in the clearance of T17M rhodopsin and the effects of 4-phenylbutyrate (4-PBA) on ER stress induced by rhodopsin T17M.
Cell culture and plasmid constructs
WT and T17M rhodopsin-myc constructs were described previously . p97/VCP QQ-HA construct was described previously . ARPE-19 cells were obtained from ATCC and cultured with DMEM supplemented with 10% FBS and penicillin-streptomycin (50 μg/ml) at 37°C in 5% CO2. 4-PBA (Sigma, St. Louis, MO, USA) was dissolved in filtered sterile water at 1 M stock concentration.
Cells were lysed with RIPA sample buffer, the supernatant was collected and protein concentration was determined using a Pierce protein assay kit (Thermo Scientific). 30 μg proteins were separated on SDS-PAGE gel and transferred to PVDF membrane (Millipore). The membrane was incubated for 1 h in a blocking solution (5% dry milk in 0.1% triton X-100/PBS buffer) followed by incubation with appropriate primary antibodies in a blocking solution. After being washed in 0.1% triton X-100/PBS buffer, the membrane was incubated in appropriate secondary antibodies for 1 h and visualized via an enhanced chemiluminescence kit (GE Health) according to the manufacturer’s instruction. Antibodies were as follows: actin, HA antibodies (Abcam), Myc, Erasin, GRP78, GRP94, CHOP, peIF-2α, eIF-2α, and active ATF-6α antibodies (Cell Signaling), GFP antibody (Invitrogen).
Analysis of protein turnover
ARPE-19 cells were transfected with erasin SMARTpool siRNAs (Dharmacon) using Dharmafect 1 reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. 48 h later, cells were transfected with Myc-tagged rhpdopsin using lipofection 2000 (Invitrogen). After 24 h, cells were treated with 50 μg/ml cycloheximide (Sigma) and collected at the indicated time points. For turnover experiments with p97/VCP QQ-HA, DNA construct was co-transfected with Myc-tagged rhpdopsin plasmid DNA followed by the inhibition of protein synthesis with cycloheximide. Equal amounts of protein in lysates from the different time points were separated by SDS–PAGE and immunoblotted.
RNA extraction, RT-PCR and XBP1 splicing assay
Total RNA was extracted from cells with TRIzol (Invitrogen) and cDNA was synthesized with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Primers encompassing the spliced sequences in XBP1 mRNA (5′-ACACGCTTGGGAATGGACAC-3′ and 5′-CCATGGGAAGATGTTCTGGG-3′) and loading control Actin (5’-GCGAGAAGATGACCCAGATC-3’, and 5’-CCAGTGGTACGGCCAGAGG-3’) were used for PCR, and products were separated by electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining.
ARPE-19 cells were transfected with myc-tagged wild-type or T17M mutant constructs. Twenty hours after transfection, apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit (Sigma). Briefly, cells were collected and washed twice with PBS. Then cells were resuspended in 500 μL Binding Buffer, and incubated with the addition of 5 μL Annex V-FITC and 10 μL propidium iodide at 37°C for 15 min in the dark. Next the stained samples were subjected to flow cytometry analysis to detect the apoptosis rate.
Determination of cellular 26S proteasome activity
A stable cell line overexpressing GFPU in ARPE-19 cells was established as a cellular model to test 26S proteasome activity after G418 selection. The reporter gene consisted of a short tag CL1 fused to the C-terminus of GFP. CL1, encoding a fragment of amino acids (ACKNWFSSLSHFVIHL), was shown to be a degradation substrate for ubiquitin-proteasome system . The expression level of GFPU after the transfection of rhodopsin T17M or WT was determined by immunoblotting.
All experiments were repeated at least three times. Results were presented as means ± standard deviation (SD). Statistical significance of differences was evaluated with one way ANOVA followed by the Tukey test and Dunnett’s test. p < 0.05 was considered as statistical significance. GraphPad Prism 5 software was used for the analysis.
Rhodopsin T17M mutant protein is ubiquitinated
Rhodopsin T17M mutant protein is degraded via ERAD pathway
We next examined p97/VCP, which is essential for the dislocation of proteins from ER during ERAD . We utilized a dominant-negative ATPase deficient p97/VCP-QQ mutant, which has been shown to slow the degradation of ERAD substrates. Accordingly, we cotransfected ARPE-19 cells with HA-tagged p97/VCP-QQ and myc-tagged WT or rhodopsin T17M, and assessed protein turnover by cycloheximide chase analysis. The results showed that the amounts of rhodopsin T17M was much higher in p97/VCP-QQ overexpressing cells than in control cells, indicating that p97/VCP-QQ inhibited the turnover of rhodopsin T17M (Figure 2c). Taken together, these results strongly indicate that rhodopsin T17M mutant protein is degraded via ERAD pathway.
Rhodopsin T17M mutant protein has no effect on ubiquitin-proteasome system
Rhodopsin T17M mutant protein induces unfolded protein response
4-phenylbutyrate prevents UPR induced by rhodopsin T17M
4-phenylbutyrate (4-PBA) is a small chemical chaperone that could reduce ER stress both in vivo and in vitro. Thus we used PBA to treat ARPE-19 cells overexpressing rhodopsin T17M. Western blot analysis showed that the levels of GRP78, GRP94, CHOP, peIF-2α, eIF-2α, and active ATF-6α were reduced after 4-PBA treatment (Figure 4a, b). We also detected the splicing of XBP-1 in ARPE-19 cells overexpressing rhodopsin T17M. The small size form of XBP-1(XBP-1 s) was observed in rhodopsin T17M overexpressing cells. As expected, 4-PBA reduces the splicing of XPB-1 induced by rhodopsin T17M (Figure 4c, d). Taken together, these data suggested that 4-PBA attenuates UPR signaling and ER stress induced by rhodopsin T17M in ARPE-19 cells.
Phenylbutyric acid facilitates rhodopsin T17M degradation and inhibits apoptosis induced by rhodopsin T17M
In this study, we found that rhodopsin T17M protein was ubiquitinated and the ubiquitination was increased following proteasome inhibitor treatment. Moreover, interference of ERAD either by overexpression of a dominant negative p97/VCP-QQ protein or by knockdown of erasin slowed the degradation of rhodopsin T17M mutant protein.
RPE is a monolayer of hexagonal cells separating the neural retina from the underlying choroidal vascular bed. RPE cells are essential for the development, survival, and physiological activity of photoreceptor cells . Mutations in genes that are expressed in the RPE can lead to photoreceptor degeneration. On the other hand, mutations in genes expressed in photoreceptor cells can lead to degenerations of the RPE. Thus RPE and photoreceptors cells are closely linked . It is found that rhodopsin is expressed in RPE cells . The human retinal pigment epithelial cell line (ARPE-19), a transformed human retinal pigment epithelial cell line, was employed to investigate the effect of rhodopsin mutant on RPE degeneration. In addition, some mammalian cell lines, such as 293 s , HeLa  and COS  were used to investigate biological functions of rhodopsin involved in RP mechanism.
T17M is a type II mutant rhodopsin that traffics abnormally and forms pigment inefficiently . Proper protein folding and processing is necessary to maintain cellular homeostasis. Protein misfolding could potentially not only affect function but also lead to protein aggregation and induce toxicity. Not surprisingly, cells have developed elaborate and complex systems to eliminate unwanted and potentially toxic proteins. One of the first quality control checkpoints is in the ER, where misfolded proteins are recognized and eliminated by ERAD. Although many mutations involved in human disease are thought to cause proteins to misfold, relatively few of them have been shown to be eliminated by ERAD. Here, we presented evidence that mutant rhodopsin protein linked to RP is degraded by ERAD.
Misfolded rhodopsin R32H is a substrate of the ERAD effector VCP, an ATP-dependent chaperone that extracts misfolded proteins from the ER and escorts them for proteasomal degradation . Inactivation of VCP/ter94 suppresses retinal pathology caused by misfolded rhodopsin in Drosophila . Co-expression of certain ERAD factors was sufficient to reduce Rh-1 protein levels and suppress ER stress reporter activation, indicating that ERAD acts as a protective mechanism against retinal degeneration in the Drosophila model for ADRP. These results suggest that manipulation of ERAD may serve as a powerful therapeutic strategy against a number of diseases associated with ER stress .
ER is responsible for the folding of secreted and membrane proteins in eukaryotic cells. Disruption of protein folding leads to ER stress. Chronic ER stress can cause cell death and is implicated in the pathogenesis of many human diseases. ER stress response is involved in retinal degeneration in T17M Rho mice and S334ter Rho rats . Ablation of CHOP, selective activation of ATF6 or PERK, disruption of CDK5 and MEKK1 pathway, or the stimulation of ERAD may serve as a powerful therapeutic strategy against rhodopsin mutants induced RP and reverse severe retinal degeneration [29–32]. Chemical chaperones reduce ER stress and are proposed as therapeutic target for various diseases . 4-PBA is a low molecular weight terminal aromatic substituted fatty acid approved for clinical use as an ammonia scavenger in children with urea cycle disorders . Recent reports showed that 4-PBA has protective effects against ER stress-induced neuronal cell death [35, 36]. In this study our results showed that 4-PBA reduced ER stress induced by T17M rhodopsin. Thus 4-PBA is a promising agent to treat RP because it could prevent RP induced by T17M rhodopsin mutant. Although 4-PBA facilitates the degradation of T17M mutant and attenuates apoptosis induced by ER stress, it does not affect the location of T17M mutant. Further studies are necessary to investigate whether intracellular trafficking of immature or misfolded rhodopsin is a potential target for RP therapy.
T17M rhodopsin is misfolded, ubiquitinated and eliminated by ER-associated degradation pathway. Chemical chaperone could attenuate UPR signaling and ER stress induced by T17M rhodopsin and has potential therapeutic significance for retinitis pigmentosa.
This study was supported by the grant from the National Natural Science Foundation of China (No. 81170844) and Hunan Provincial Science & Technology Department (No. 2014FJ3100).
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