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. 2016 Jul 26;7(30):46899-46912.
doi: 10.18632/onco_target.10380.

The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus

Su-Jeong Kim  1 , Noel Guerrero  1 , Gabriella Wassef  1 , Jialin Xiao  1 , Hemal H Mehta  1 , Pinchas Cohen  1 , Kelvin Yen  1
Affiliations

The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus

Su-Jeong Kim et al. Onco_target. .

Abstract

Humanin is a small secreted peptide that is encoded in the mitochondrial genome. Humanin and its analogues have a protective role in multiple age-related diseases including type 2 diabetes and Alzheimer's disease, through cytoprotective and neuroprotective effects both in vitro and in vivo. However, the humanin-mediated signaling pathways are not well understood. In this paper, we demonstrate that humanin acts through the GP130/IL6ST receptor complex to activate AKT, ERK1/2, and STAT3 signaling pathways. Humanin treatment increases phosphorylation in AKT, ERK 1/2, and STAT3 where PI3K, MEK, and JAK are involved in the activation of those three signaling pathways, respectively. Furthermore, old mice, but not young mice, injected with humanin showed an increase in phosphorylation in AKT and ERK1/2 in the hippocampus. These findings uncover a key signaling pathway of humanin that is important for humanin's function and also demonstrates an age-specific in vivo effect in a region of the brain that is critical for memory formation in an age-dependent manner.

Keywords: AKT; ERK1/2; Gero_target; STAT3; humanin; signaling pathway.

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Conflict of interest statement

Pinchas Cohen is a consultant and stockholder of CohBar Inc.

Figures

Figure 1
Figure 1. Deciphering the acute humanin signaling pathway
_targets were identified if there was a significant minimum of 20% increase or decrease in phosphorylation. A pie chart showing humanin _targets' (A) biological functions and (B) subcellular localization. The cytoplasm includes mitochondria, lysosome, and other subcellular compartment in the cytoplasm (C) Top molecular and cellular functional classes to which the humanin-_targeted proteins are associated.
Figure 2
Figure 2. HNG rapidly increases the phosphorylation of MAPK p44/42 (ERK 1/2), AKT, and STAT3 in SH-SY5Y cells
Quantification and representative western blots of (A) ERK activation, (B) AKT activation, and (C) STAT3 activation. Total cell lysates from SH-SY5Y cells following 100μM HNG treatment in DMEM or DMEM supplemented with 10% FBS for the indicated time periods were immunoblotted using anti-phospho- and total-ERK 1/2 (Thr202/Tyr204), AKT (Ser473), and STAT-3 (Tyr705). A densitometric analysis of ERK, AKT, and STAT-3 levels were performed using image J. Data are reported as mean ± SEM of three to six independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05. Abbreviations: CM, Complete medium; SF, Serum-free medium.
Figure 3
Figure 3. HNG rapidly increase the phosphorylation of MAPK p44/42 (ERK1/2) in HEK293 cells
Quantification and representative western blots of (A) ERK activation, (B) AKT activation, and (C) STAT3 activation. Total cell lysates from HEK293 cells following 100μM HNG treatment for the indicated time periods were immunoblotted using anti-phospho- and total-ERK 1/2 (Thr202/Tyr204), AKT (Ser473), and STAT3 (Tyr705). A densitometric analysis of ERK, AKT, and STAT3 levels were performed using Image J. Data are reported as mean ± SEM of four independent experiments. *p < 0.05. Abbreviations: CM, Complete medium; SF, Serum-free medium.
Figure 4
Figure 4. Inhibitors block the HNG-mediated activation of ERK, AKT, and STAT3. (
A) Representative western blots showing ERK, AKT, and STAT3 activation in conjunction with specific inhibitors under serum free conditions (n = 3). Quantification of (C) AKT and (D) ERK activation. Data are reported as mean ± SEM of 4 independent experiments. ***p < 0.001, *p < 0.05. Abbreviations: PD, PD98059; LY, LY294002; JAKI, JAK inhibitor I; GP, Anti-GP130 antibody.
Figure 5
Figure 5. Phosphorylated ERK 1/2 is localized to cytosol, nucleus, and other subcellular compartments upon HNG treatment
Representative western blots showing ERK1/2 subcellular localization (n = 3). (A) HEK293 cells were treated with 1μM HNG. Both cytosolic (15μg) and nuclear (15μg and 50μg) lysates were immunoblotted using phospho- and total-ERK antibody. (B) SH-SY5Y cells were treated with 100μM HNG. Mitochondria fractions were obtained 30min after HNG treatment. Cytosolic, nuclear, and mitochondrial (15μg) lysates were immunoblotted using phospho- and total-ERK antibody. Anti-GAPDH, Anti-Lamin B, anti-Tom20 antibody were used as the cytosolic, nuclear, mitochondrial fraction markers, respectively. The ratio is the fold change in phospho-MAPK normalized to total-MAPK compared to the cytosolic, time 0 phospho to total ratio.
Figure 6
Figure 6. HNG increases AKT and ERK phosphorylation in the hippocampus of old mice
(A) Western blot analysis for phospho- and total-AKT, ERK, and STAT3 in hippocampus of 12-week-old mice and 18-month-old mice. Quantitation of (B) normalized ERK activation and (C) normalized AKT activation in response to HNG. Total protein was used to normalize protein loading. Data are reported as mean ± SEM (n = 8 for 12-week-old mice, n = 5 for 18-month-old mice). *p < 0.05, ns, not significant.
Figure 7
Figure 7. Schematic diagram of the humanin-mediated signaling pathway and its biological functions
See this image and copyright information in PMC

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