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. 2013 Jul 29;8(7):e69563.
doi: 10.1371/journal.pone.0069563. Print 2013.

Autophagy activation clears ELAVL1/HuR-mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human retinal pigment epithelial cells

Johanna Viiri  1 , Marialaura Amadio, Nicoletta Marchesi, Juha M T Hyttinen, Niko Kivinen, Reijo Sironen, Kirsi Rilla, Saeed Akhtar, Alessandro Provenzani, Vito Giuseppe D'Agostino, Stefano Govoni, Alessia Pascale, Hansjurgen Agostini, Goran Petrovski, Antero Salminen, Kai Kaarniranta
Affiliations

Autophagy activation clears ELAVL1/HuR-mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human retinal pigment epithelial cells

Johanna Viiri et al. PLoS One. .

Abstract

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called "drusen". Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MG-132-induced increase of SQSTM1/p62 but not ELAVL1/HuR protein levels is counteracted by AICAR treatment.
Representative western blotting (upper) and densitometric analysis (lower) of SQSTM1/p62 (A), ELAVL1/HuR (B) and MAP1LC3A/LC3-II (C) proteins in the total homogenates of ARPE-19 cells after starvation or/and exposure to AICAR (2 mM) or/and MG-132 (5 µM) for 0,5 h, 3 h, 12 h and 24 h. α-tubulin was used as a loading control. Control cells were exposed only to solvent (DMSO). Results are expressed as means ± S.E.M. The data were analyzed by ANOVA, followed by Dunnett’s Multiple Comparison Test; *p<0.05, **p<0.01, control vs. treated, n = 7 (SQSTM1/p62 and ELAVL1/HuR), n = 3 (MAP1LC3A/LC3-II).
Figure 2
Figure 2. SQSTM1/p62 transcript as a new target of the RNA-binding ELAVL1/HuR protein.
(A): RNA-binding activity of ELAVL1/HuR evaluated by AlphaScreen technology. Saturation binding experiments investigated by titrating a series of biotinylated single-stranded (BITEG-) RNAs, including TNFneg and SQSTM1/p62neg, that we designated as negative controls, against 1 nM of rELAVL1/HuR. Calculated dissociation constants (Kd) for TNFalpha (3.83±0.69 nM, R2 = 0.97), and SQSTM1/p62 (30.85±13.22 nM R2 = 0.91) are indicated. The plots represent mean ± SD of two independent experiments. (B): Saturation binding experiments as function of rELAVL1/HuR concentrations against four different type of RNA-substrates at 50 nM concentration. 1 nM of rELAVL1/HuR was enough to reach saturation of the binding. The plots represent Mean ± SD of two independent experiments. (C): Effect of MG-132 exposure on SQSTM1/p62 gene expression. Determination of SQSTM1/p62 mRNA by real-time qPCR in human ARPE-19 cells following treatments with solvent (control) or 5 µM MG-132 for 24 hrs. SQSTM1/p62 mRNA expression in control cells was taken as 100%. The values obtained from total cellular mRNA have been normalized to the level of RPL6 mRNA and expressed as mean ± S.E.M. ***p<0.001; Student’s t test; n = 3. (D): The binding of ELAVL1/HuR protein to SQSTM1/p62 transcript increases in the cytoplasm following MG-132 stimulus. Fold enrichment detected by quantitative real-time RT-PCR of SQSTM1/p62 mRNA in control and 24 h MG-132 RPE cells following immunoprecipitation with anti-ELAV antibody (IP) in the cytoplasm. ***p<0.001, Student’s t-test, n = 3. The data of SQSTM1/p62 were normalized with respect to the data obtained from immunoprecipitation with an irrelevant antibody as a negative control.
Figure 3
Figure 3. The MG-132-mediated upregulation of SQSTM1/p62 protein expression requires the specific presence of ELAVL1/HuR protein.
Representative western blotting (upper) and densitometric analysis (lower) of ELAVL1/HuR (A) and SQSTM1/p62 (B) proteins in the total homogenates of ELAVL1/HuR silenced ARPE-19 cells and negative control (NEG-siRNA) cells after exposure to 5 µM MG-132 for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.E.M. *p<0.05; **p<0.01; ***p<0.001, Tukey’s multiple comparison test; n = 5.
Figure 4
Figure 4. Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and aggregate quantification.
(A) Representative transmission electron micrographs of ARPE-19 untreated control cells, cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. Aggregates are indicated by arrows. (B) Quantification of aggregates in ARPE-19 cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. Eight parallel samples were measured in all treatments. **p<0.01, Mann–Whitney.
Figure 5
Figure 5. Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and autophagic vesicles quantification.
(A) Representative transmission electron micrographs of ARPE-19 cells exposed to MG-132 5 µM solely and with AICAR 2 mM for 3 h, 12 h and 24 h. Autophagic vesicles are indicated by arrows. (B) Quantification of autophagic vesicles in ARPE-19 exposed to MG-132 5 µM solely and with AICAR 2 mM for 3 h, 12 h and 24 h. Six parallel samples were measured in all treatments. *p<0.05, **p<0.01, Mann–Whitney.
Figure 6
Figure 6. Colocalization of SQSTM1/p62 and MAP1LC3A/LC3 and position changes of SQSTM1/p62, revealed by confocal microscopy analysis.
A light merge orange/yellow signal of colocalizing MAP1LC3A/LC3 (pDendra2-hLC3, green) and SQSTM1/p62 (pDsRed2-hp62, red) is detectable, usually near to the cell nuclei. Nuclei are stained with blue dye. The scale bar equals to 5 µm. (A): Confocal microscopy images of untreated control ARPE-19 cells and cells exposed to AICAR 2 mM or/and MG-132 5 µM for 24 h. A = Aggregates (B): Position of SQSTM1/p62 (pDendra2-hp62) in ARPE-19 cells, revealed by confocal microscopy analysis. SQSTM1/p62 in two different areas was photoconverted within approximately 7 sec (circled). Within 1 minute after photoconversion, the photoconverted SQSTM1/p62 is stationary. Cells have been exposed to 5 µM MG-132 for 24 h. N = Cell nucleus.
Figure 7
Figure 7. SQSTM1/p62 protein, but not ELAVL1/HuR protein, is degraded by autophagy in ARPE-19 cells.
Representative western blotting and densitometric analysis of SQSTM1/p62 (A) and ELAVL1/HuR (B) proteins in the total homogenates of ARPE-19 cells after exposure to bafilomycin (50 nM) or/and MG-132 (5 µM) for 24 h. α-tubulin was used as a loading control. Results are expressed as means ± S.D. The data were analyzed by ANOVA, followed by Mann-Whitney; **p<0.01, untreated control cells vs. treated cells, n = 7.
Figure 8
Figure 8. Sections of eyes with clinically diagnosed AMD immunostained for SQSTM1/p62.
The extent of cytoplasmic immunopositivity in the retinal pigment epithelial cells (RPE, shown by arrows) and in the drusen was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The SQSTM1/p62 staining in the foveomacular areas was more extensive as compared to the perimacular and peripheral areas (B and C, respectively). The drusen (shown by asterisks) were mostly SQSTM1/p62 negative. The nuclei of RPE cells were SQSTM1/p62 negative. (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).
Figure 9
Figure 9. Sections of eyes with clinically diagnosed AMD immunostained for ubiquitin.
The extent of Bruch’s membrane immunopositivity of the retinal pigment epithelial cells (RPE, shown by arrows) and in the drusen (asterisks) was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The uniform staining of ubiquitin in RPE cells Bruch’s membrane was observed in all these regions (arrows). There were no differences in the extent of staining between these regions in each group. The nuclei of RPE cells were mostly ubiquitin negative. Most of the drusen were strongly ubiquitin-positive (asterisks). (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).
Figure 10
Figure 10. Sections of eyes with clinically diagnosed AMD immunostained for ELAVL1/HuR.
The immunopositivity of the nuclei of RPE cells (shown by arrows) and the extent of immunopositivity in the drusen (asterisks) was evaluated microscopically (no staining or positive staining) by selecting 5 mm long areas of foveomacular (A), perimacular (B) and peripheral (C) regions. The foveomacular nuclei showed less extensive immunopositivity for ELAVL1/HuR than the perimacular and peripheral regions. However this difference was not statistically significant in any of the groups studied (p>0.1). The cytoplasms of RPE cells were ELAVL1/HuR negative. Most of the drusen were ELAVL1/HuR-negative. (Original magnifications of x 200 and in insets x 400; Bruch's membrane shown by arrow heads).
Figure 11
Figure 11. Sections of foveomacular areas of age-matched control eyes for SQSTM1/p62, ubiquitin and ELAVL1/HuR.
The extent of cytoplasmic immunopositivity for SQSTM1/p62, Bruch’s membrane immunopositivity for ubiquitin and the immunopositivity of nuclei of RPE cells for ELAVL1/HuR (foveomacular areas shown; A, B and C respectively) in the RPE cells (shown by arrows) was evaluated microscopically (no staining or positive staining). For SQSTM1/p62 there were only a few immunopositive cytoplasm’s randomly distributed through-out the RPE cell layer and the nuclei were negative. Bruch’s membrane (shown by arrowheads) was immunopositive for ubiquitin occasionally through-out the RPE cell layer in very small amounts while all of the nuclei were negative. Half of the nuclei showed minor immunopositivity for ELAVL1/HuR while the cytoplasm’s of RPE cells were negative. The foveomacular areas showed no difference in immunohistochemical stainings when compared to areas of perimacular and peripheral retina in all of the proteins studied. (Original magnifications of x 200 and in insets x 400).
Figure 12
Figure 12. Summary of the results.
The proteasome inhibitor MG-132 down-regulates (−) the ubiquitin-proteasome pathway (UPP). AMPK-activator AICAR and proteasome inhibitor MG-132 co-treatment activates (+) the autophagy. UPP inhibition significantly increases (+) SQSTM1/p62 protein levels, while autophagy induction during UPP inhibition robustly decreases (−) SQSTM1/p62 protein levels. Since SQSTM1/p62 localizes to aggregates, the decreasing of SQSTM1/p62 reveals its autophagy clearance. In addition, proteasome inhibition significantly increases ELAVL1/HuR protein levels by itself and during the autophagy induction (+). Proteasome inhibition induces a positive regulation of SQSTM1/p62 expression that occurs also at post-transcriptional level via ELAVL1/HuR protein (+). Activated autophagy is able to completely abolish the MG-132-induced protein aggregation (−), which, in turn improves the cell vitality. Ubiquitin is also found in intracellular aggregates in ARPE-19 cells as well as from drusens. In contrast, SQSTM1/p62 is found only in the intracellular aggregates in ARPE-19 cells, but not in drusens. However, SQSTM1/p62 levels were high in macular area of RPE cells revealing impaired autophagy. What is the relation between drusen and intracellular aggregates remains still unknown. Red lines with minus symbol represent inhibiting and decreasing events in ARPE-19 cells. Black lines and plus symbol represent activating and increasing events in the ARPE-19 cell. Zero (0) and dark blue line indicate neutral effects in the ARPE-19 cell. UPP: ubiquitin-proteasome pathway.
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