Induction of <i>VEGFA</i> mRNA translation by CoCl₂ mediated by HuR

<p>Vascular endothelial growth factor (VEGF) A is a master regulator of neovascularization and angiogenesis. VEGFA is potently induced by hypoxia and by pathological conditions including diabetic retinopathy and tumorigenesis. Fine-tuning of VEGFA expression by different stimuli is important for maintaining tissue vascularization and organ homeostasis. Here, we tested the effect of the hypoxia mimetic cobalt chloride (CoCl₂) on VEGFA expression in human cervical carcinoma HeLa cells. We found that CoCl₂ increased the levels of <i>VEGFA</i> mRNA and VEGFA protein without affecting <i>VEGFA</i> mRNA stability. Biotin pulldown analysis to capture the RNA-binding proteins (RBPs) bound to <i>VEGFA</i> mRNA followed by mass spectrometry analysis revealed that the RBP HuR [human antigen R, a member of the embryonic lethal abnormal vision (ELAV) family of proteins], interacts with <i>VEGFA</i> mRNA. <i>VEGFA</i> mRNA-tagging experiments showed that exposure to CoCl₂ increases the interaction of HuR with <i>VEGFA</i> mRNA and promoted the colocalization of HuR and the distal part of the <i>VEGFA</i> 3′-untranslated region (UTR) in the cytoplasm. We propose that under hypoxia-like conditions, HuR enhances <i>VEGFA</i> mRNA translation.</p

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

<div><p>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 <i>SQSTM1/p62</i> 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.</p></div

Sections of eyes with clinically diagnosed AMD immunostained for ubiquitin.

<p>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).</p

Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and autophagic vesicles quantification.

<p>(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.</p

Sections of foveomacular areas of age-matched control eyes for SQSTM1/p62, ubiquitin and ELAVL1/HuR.

<p>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).</p

Transmission electron micrographs of ARPE-19 cells exposed to AICAR or/and MG-132 and aggregate quantification.

<p>(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.</p

Cytoplasmic SQSTM1/p62, ubiquitin and ELAVL1/HuR proteins in foveomacular, perimacular and peripheral RPE cells.

<p>Cytoplasmic SQSTM1/p62, ubiquitin and ELAVL1/HuR proteins in foveomacular, perimacular and peripheral RPE cells.</p

Summary of the results.

<p>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 <i>SQSTM1/p62</i> 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.</p

The MG-132-mediated upregulation of SQSTM1/p62 protein expression requires the specific presence of ELAVL1/HuR protein.

<p>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.</p

<i>SQSTM1/p62</i> transcript as a new target of the RNA-binding ELAVL1/HuR protein.

<p>(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 <i>TNF</i>neg and <i>SQSTM1/p62</i>neg, that we designated as negative controls, against 1 nM of rELAVL1/HuR. Calculated dissociation constants (K_d) for <i>TNFalpha</i> (3.83±0.69 nM, R² = 0.97), and <i>SQSTM1/p62</i> (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 <i>SQSTM1/p62</i> gene expression. Determination of <i>SQSTM1/p62</i> mRNA by real-time qPCR in human ARPE-19 cells following treatments with solvent (control) or 5 µM MG-132 for 24 hrs. <i>SQSTM1/p62</i> 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 <i>SQSTM1/p62</i> transcript increases in the cytoplasm following MG-132 stimulus. Fold enrichment detected by quantitative real-time RT-PCR of <i>SQSTM1/p62</i> 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.</p