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

Abstract

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.

Metabolomic profile of diet-induced obesity mice in response to humanin and small humanin-like peptide 2 treatment

“…These aforementioned metabolites were reduced in plasma from HNG and SHLP2 treated DIO mice, which is indicative of lower ROS levels in tissues and a decrease in demand for glutathione. This metabolic signature also agrees with previous studies demonstrating the anti-ROS benefits of humanin and SHLP2 (Paharkova et al 2015 ); (Cobb et al 2016 ; Klein et al 2013 ; Matsunaga et al 2016 ; Sreekumar et al 2016 ; Thummasorn et al 2017 ). Furthermore, suppression of the key intermediates in glutathione synthesis pathway may help explain why high SHLP2 levels are associated with lower risk of prostate cancer, as oxidative stress is involved in all aspects of tumor development (Xiao et al 2017 ).…”

Papel del péptido mitocondrial humanina como blanco terapéutico en cáncer y neurodegeneración

“…El efecto antiapoptótico de la HN aumenta la probabilidad de que este péptido posea un papel en la patogénesis del cáncer. Si bien la HN fue propuesta como un posible oncopéptido en el 2002 (53), continúa siendo escaso el número de trabajos que involucran a la HN en el desarrollo tumoral. Su expresión fue detectada en cáncer gástrico, por lo que fue propuesto que la sobreexpresión de este péptido podría constituir un evento molecular impor-tante de la tumorigénesis y la quimiorresistencia del tumor (54).…”

Protective Mechanisms of the Mitochondrial‐Derived Peptide Humanin in Oxidative and Endoplasmic Reticulum Stress in RPE Cells

“…Both OS and ER stress cause RPE apoptosis by increased generation of ROS, downregulation of mitochondrial GSH, and activation of caspase 3 [11, 47]. Increased ER stress from OS via the crosstalk by MAMs and ER-specific events of caspase 4 and CHOP activation are also shown (Figure 2()).…”