Sephin1 suppresses ER stress-induced cell death by inhibiting the formation of PP2A holoenzyme

Abstract

Sephin1 was discovered as a protein phosphatase inhibitor, and its efficacy against neurodegenerative diseases has been confirmed. There are conflicting reports on whether inhibition of eIF2α dephosphorylation by PP1 holoenzyme with the protein phosphatase 1 regulatory subunit 15 A is the mechanism of action of Sephin1. In the present study, we found that Sephin1 significantly suppressed renal tubular cell death in an animal model of ER stress administered with tunicamycin. CHOP, which plays a central role in the ER stress-induced cell death pathway, requires nuclear translocation to act as a transcription factor to increase the expression of cell death-related genes. Sephin1 markedly suppressed this nuclear translocation of CHOP. To elucidate the molecular mechanism underlying the cell death suppressive effect of Sephin1, we used human renal tubular epithelial cells under ER stress with tunicamycin. Sephin1 reduced intracellular CHOP levels by promoting CHOP phosphorylation at Ser30, which led to protein degradation in UPS. Phosphorylated CHOP is generated by Thr172-phosphorylated activated AMPK, and Sephin1 increased phosphorylated AMPK. Phosphorylated AMPK is inactivated by PP2A through dephosphorylation of its Thr172, and Sephin1 inhibits the formation of the PP2A holoenzyme with the PP2A subunit B isoform delta. These results indicate that inhibition of PP2A holoenzyme formation is the molecular target of Sephin1 in this experimental system.

Fig. 1. Sephin1 rescues tunicamycin-induced ER stress.

A, B Kaplan–Meier survival curves of mice receiving tunicamycin intraperitoneal injections at the following concentrations without or with Sephin1; A 1 mg/kg, n = 9, B 2 mg/kg, n = 12. C Body, kidney weight, and BUN were measured on Day 3 (n = 4). D Representative images of PAS staining of the kidney of mice on Day 3 and quantification of kidney injuries (n = 4). E Representative images of Megalin staining and Kim-1 staining of the kidney of mice on Day 3 and the Kim-1 positivity as damaged tubular epithelium (n = 4). The error bars indicate the SDs. The number of asterisks can be obtained by indicating a range of p values as follows; * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 2. Expression of CHOP induced by ER stress is suppressed by Sephin1.

A The mRNA expression of murine kidney on Day 3 (n = 9). The error bars indicate the SDs. B Western blotting analysis of CHOP in murine kidneys on Day 3. The graph shows the expression ratio of each protein corrected by α-Tubulin protein expression level. C Representative images of CHOP staining of murine kidney on Day 3 and the CHOP-positive rate in the nucleus in either the cortex or medulla (n = 3). All bars in the images are 100 μm. The number of asterisks can be obtained by indicating a range of p values as follows; * <0.05, ** < 0.01, *** <0.001, **** <0.0001.

Fig. 3. In vitro analysis of CHOP regulation by Sephin1.

A Number of HK-2 cells exposed to 20 μg/ml tunicamycin without or with 2 μM Sephin1 for 24 h (n = 3). B Apoptosis analysis using PI and annexin V of HK-2 cells at 24 h following the administration of 1 mg/ml tunicamycin without or with 1 μM Sephin1 by FACS (n = 3). C The mRNA expression of HK-2 cells at 24 h following the administration of 20 μg/ml tunicamycin without or with 1 μM Sephin1 (n = 3). D Western blotting analysis of CHOP and eIF2α of HK-2 cells at 24 h following the administration of 20 μg/ml tunicamycin without or with 1 μM Sephin1. The graph shows the expression ratio of each protein corrected by α-Tubulin protein expression level (n = 3). E Western blotting analysis of CHOP in the nuclear fraction of HK-2 cells at 24 h following the administration of 20 μg/ml tunicamycin without or with 1 μM Sephin1 (n = 3). F Immunocytochemistry using anti-CHOP antibody to HK-2 cells over time. The error bars indicate the SDs. The error bars indicate the SDs. The number of asterisks can be obtained by indicating a range of p values as follows; * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 4. Effects of mitochondrial functional by Sephin1.

A The mitochondria-related mRNA expression of HK-2 cells at 24 h following the administration of 20 μg/ml tunicamycin without or with 1 μM Sephin1 (n = 3). B Oxygen consumption rates and glycolysis analysis using Seahorse in HK-2 cells under ER stress model (N = 7). C Mitochondrial network analysis in HK-2 cells following TMRM staining (N = 4). D Mitochondrial membrane potential in HK-2 cells by FACS analysis of TMRM staining (N = 3). E Mitophagy analysis using mKeima expressing HK-2 cells by FACS analysis (N = 3). The number of asterisks can be obtained by indicating a range of p values as follows; * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 5. CHOP degradation by Sephin1 in HK-2 cells under ER stress.

A Number of CHOP-KD HK-2 cells at 24 h following the administration of 20 μg/ml tunicamycin without or with 1 μM Sephin1 (n = 6). B Number of HK-2 cells in the ER stress model under 1 μM of compound C, an AMPK inhibitor (n = 3). C Number of HK-2 cells in the ER stress model to examine the effects of FTY720 at the concentration of 1 μg/ml. D Instead of Sephin1, LB-100, an antagonist for PP2A, was used to treat the ER stress model of HK-2 cells at the concentration of 0.01 μg/ml. E Western blotting analysis for AMPK and phosphorylated AMPK on Thr172 of HK-2 at 24 h following drug administration. The graph shows the expression ratio of each protein corrected by GAPDH protein expression level (n = 3). F Co-immunoprecipitation assay for AMPK and PP2ABδ. Following immunoprecipitation for AMPK, PP2ABδ was blotted for their binding status. Relative values of PP2ABδ to AMPK in tunicamycin-exposed group and Sephin1-treated group were calculated (n = 3). G Co-immunoprecipitation assay for PP2ABδ and PP2AC. Following immunoprecipitation for PP2ABδ, PP2AC was blotted for their binding status. Relative values of PP2AC to PP2ABδ in tunicamycin exposed group and Sephin1 treated group were calculated (n = 3). H Co-immunoprecipitation assay for PP2AC and PP2ABδ. Following immunoprecipitation for PP2AC, PP2ABδ was blotted for their binding status. Relative values of PP2ABδ to PP2AC in tunicamycin-exposed group and Sephin1-treated group were calculated (n = 3). The error bars indicate the SDs. The number of asterisks can be obtained by indicating a range of p values as follows; * <0.05, ** <0.01, *** <0.001, **** <0.0001.

Fig. 6. A schema showing the mechanism of action of Sephin1.

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