Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione

<div><p>Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.</p></div

Activation of caspase 3 by TM-induced ER stress in hRPE cells and suppression of activated caspase 3 by HN.

<p>Confluent hRPE cells were pretreated for 12 hours with or without 10 μg/mL HN. Cells were then treated with 10 μg/mL HN and/or 10 μg/mL TM for 12 hours. (<b>A</b>) Western blot analysis of total cell lysates probed with active caspase 3 antibody showed increased amounts of active caspase 3 with TM, and attenuation of active caspase 3 with HN. (<b>B</b>) Protein expression quantified by densitometry as shown as a ratio normalized with GAPDH. Data are mean ± SEM (n = 3). Asterisks represent *p<0.05, ***p<0.001.</p

Increased expression of γ-GCLC by TM-induced ER stress in hRPE cells and the effect of HN treatment.

<p>Confluent hRPE cells were pretreated for 12 hours with or without 10 μg/mL HN. Cells were then treated with 10 μg/mL HN and/or 10 μg/mL TM for 12 hours. (<b>A</b>) RT-PCR analysis of γ-GCLC showed a significant increase in mRNA expression with TM compared to control (n = 4, ***p<0.001, **p<0.01). (B) Western blot analysis of total cell lysates probed with γ-GCLC antibody showed increased expression of γ-GCLC protein with TM compared to control. The γ-GCLC expression remained elevated with HN co-treatment. (<b>B</b>) Figure shows a representative western blot from protein expression experiment in the presence and absence of TM and HN. (C) Bar graph showing γ-GCLC protein expression quantified by densitometry as shown as a ratio normalized to GAPDH. Data are mean ± SEM (n = 3). Asterisks represent *p<0.05</p

HN restores mitochondrial GSH from depleted levels due to ER stress.

<p>Confluent hRPE cells were pretreated for 12 hours with or without 10 μg/mL HN. Cells were then treated with 10 μg/mL HN and/or 10 μg/mL TM for 12 hours. Cells were then fractionated into mitochondrial and cytosolic components using a mitochondrial/cytosol fractionation kit. (A) Western blots of COX IV (mitochondrial marker) and β-Tubulin (cytosolic marker) revealed there is negligible cross-contamination between the two fractions. (B) Mitochondrial GSH measurements showed decreased mitochondrial GSH with TM treatment, and repleted mitochondrial GSH with HN co-treatment. Data presented in the bar graph are mean +/- SD of 3 experiments. (*p<0.05, **p<0.01, ***p<0.001)</p

Increased expression of the ER stress markers GRP78 and CHOP by TM treatment of hRPE cells and the effect of HN.

<p>Confluent hRPE cells were pretreated for 12 hours with or without 10 μg/mL HN. Cells were then treated with 10 μg/mL HN and/or 10 μg/mL TM for 12 hours. (<b>A</b>) Expression of GRP78 by Western blot analysis was significantly higher in TM and HN plus TM groups compared to control. (n = 4, *p < 0.05). (<b>B</b>) Expression of CHOP by Western blot analysis was significantly different between TM and HN plus TM groups compared to controls. However, treatment with HN along with TM reduced the expression of CHOP as compared to TM alone. Bar graph represents protein expression quantified by densitometry normalized to GAPDH. Data are mean ± SEM (n = 3). Asterisks represent ***p < 0.001, **p < 0.01. C- Control.</p

TM-induced apoptosis in RPE cells and protection by HN.

<p>Confluent hRPE cells were treated with varying concentrations (1–20 μg/mL) of TM for 12 hours. (<b>A</b>) Percentage of TUNEL positive cells increased in a dose-dependent manner with TM treatment. (<b>B</b>) Representative images of TUNEL positive cells (red) and nuclei (blue) are shown for each treatment condition. (<b>C</b>) Pre-incubation of HN (1–20 μg/mL) for 12 hours protected RPE cells from apoptosis induced by TM (10 μg/mL). (<b>D</b>) Representative images are shown for each HN treatment group. Data are mean ± SEM (n = 3). Asterisks represent *p<0.05, **p<0.01, ***p<0.001). Scale bar: 20 μm in B and D.</p

Elevation of mitochondrial superoxide by TM-induced ER stress and decreased mitochondrial superoxide with HN co-treatment.

<p>Confluent hRPE cells were pretreated for 12 hours with or without 10 μg/mL HN. Cells were then treated with 10 μg/mL HN and/or 10 μg/mL TM for 12 hours. Increased superoxide in mitochondria is shown by Mitosox labeling (red) against an ER counter-stain (blue). Scale bar: 10 μm in ER Tracker, MitoSox and Merged panels, and 5 μm in enlarged panels.</p

Clinical Characteristics of Study Population.

<p>BMI indicates body mass index; CAD indicates coronary artery disease; LDL indicates low density lipoprotein; HDL indicates high density lipoprotein; ARB indicates angiotensin II receptor blocker; Values are expressed as mean±SEM for continuous variables;</p><p>*p<0.05 vs. asymptomatic.</p

HN is expressed in M2 phase macrophages.

<p>HN-secreting macrophages in the M2 polarization phase (indicated by arginase-1) were present, but less in number than HN-secreting M1 macrophages. No differences were seen between groups.</p

Histological immunoreactivity of HN is greater in symptomatic patients.

<p>HN expression is greater in carotid plaques from TIA (n = 12) and stroke (n = 10) symptomatic patients than in asymptomatic patients (n = 12; †p<0.001).</p