Novel oxidative stress-responsive gene ERS25 functions as a regulator of the heat-shock and cell death response

Abstract

Members of the yeast p24 family, including Emp24p and Erv25p, exist as heteromeric complexes that have been proposed to cycle between the endoplasmic reticulum (ER) and Golgi compartments. The specific functions and sites of action of p24 proteins are still unknown. Here we identified a human homolog of the yeast p24 family of proteins, named ERS25 (endoplasmic reticulum stress-response protein 25), and investigated its role in stress response. ERS25 is predicted to have an ER localization signal peptide, a GOLD (Golgi dynamics) domain, which is found in several eukaryotic Golgi and lipid-trafficking proteins, a coiled-coil region, and a transmembrane domain. We demonstrate that ERS25 is localized to the ER and is induced by ER-specific stress, heat shock, and oxidative stress. The selective induction of ERS25 by brefeldin A, but not tunicamycin, implicates the involvement of ERS25 in protein trafficking between the ER and the Golgi. Small interfering RNA-mediated inhibition of ERS25 results in a significant decrease in apoptosis as well as a reduction of reactive oxygen species induced by oxidative stress. Moreover, ERS25 depletion results in a significant increase in the levels of the ER chaperone HSP70 in response to heat-shock stress through increased levels of HSF-1. We also found that inhibition of ERS25 induction in response to heat shock enhanced the binding of HSP70 to Apaf-1, which is likely to interfere in stress-mediated apoptosis. Together, the data presented here demonstrate that ERS25 may play a critical role in regulation of heat-shock response and apoptosis.

FIGURE 1.

Sequence alignment of Emp24 family proteins. Homo sapiens (H) human ERS25 (NP_872353.2), Mus musculus (M) mouse Tmed10 (NP_081051.1), Saccharomyces cerevisiae (Y) yeast Emp24 (NP_011315.1), and Caenorhabditis elegans (W) worm sel-9 (NP_505145.1). Amino acids highlighted in black are 100% conserved, and those highlighted in gray are conservative substitutions. The domains conserved within the Emp24 family are labeled by dashes.

FIGURE 2.

ERS25 localizes to the ER and is induced by various stressors. A, NIH3T3 cells were stained with antibodies against ERS25 and calnexin, and the nucleus was stained with TO-PRO3 iodide. Dapi, 4′,6-diamidino-2-phenylindole. B, Western blot analysis of NIH3T3 cell fractionation. ERS25 was identified in the membrane fraction using antibodies against calnexin (CNX), poly(ADP-ribose) polymerase-2 (PARP), and α-tubulin, as markers for the membrane (Mem), nuclear (Nuc), and cytosolic (Cyt) fractions, respectively. WCL, whole cell lysates. C, NIH3T3 cells were stressed using tunicamycin, brefeldin A, 1 mm H₂O₂, and 8 h of heat shock at 42 °C. Western blot analysis was performed with antibodies against ERS25 and β-actin as a loading control (Cont.).

FIGURE 3.

ERS25 sensitizes cells to oxidative damage and cell death. NIH3T3 cells were stably transfected with either empty pBabe-shRNA vector (control) or vector containing shRNA targeting ERS25. Cells were treated with various concentrations of H₂O₂ for 16–18 h. A, Western blot analysis of H₂O₂-treated cells using antibodies against ERS25 and β-actin as a loading control. B, reactive oxygen species were measured by DCF-DA fluorescence as described under “Experimental Procedures.” Various cell death assays were performed on H₂O₂-treated cells as described under “Experimental Procedures.” Cont., control. C, apoptosis was quantified by a cell death enzyme-linked immunosorbent assay (Roche Applied Science) measuring DNA fragmentation. D, the percentage of cell survival was quantified by trypan blue exclusion. E, apoptosis was visualized by fluorescence-activated cell sorter analysis of propidium iodide-stained cells. The percentage of sub-G₁ population and error bars reflect the mean ± S.D. from duplicate experiments.

FIGURE 4.

Inhibition of ERS25 enhances HSP70 induction upon heat shock. A, NIH3T3 cells were exposed to heat shock (42 °C) for the indicated times and then harvested for analysis by Western (upper panel) and Northern (lower panel) analysis. The levels of ERS25 and HSP70 were measured with β-actin and 36B4 as protein and mRNA controls, respectively. B, NIH3T3 cells were stably transfected with either empty pBabe shRNA vector (control (Cont.)) or vector containing shRNA targeting ERS25. Two clones were analyzed for protein and mRNA as described in A.

FIGURE 5.

ERS25 knockdown induces HSF-1 expression and enhances HSP70 promoter activity. A, luciferase assay reporter gene constructs containing the HSP70 promoter were transfected with the reporter plasmids into NIH3T3 cells expressing the empty pBabe vector or vector expressing shRNA against ERS25. Cells were harvested 48 h after transfection and assayed for luciferase activity. Re-expression of ERS25 upon the transfections was confirmed by Western blot (right panel). HS, heat shock; Cont., control. B, the effect of ERS25 depletion on HSF-1 expression upon heat shock. NIH3T3 cells were transfected with ERS25-shRNA or control shRNA followed by heat shock at 42 °C, and then Western blotting was performed against ERS25, HSF-1, and β-actin.

FIGURE 6.

ERS25 reduces HSP70 binding to Apaf-1. NIH3T3 cells expressing the empty pBabe vector or vector expressing shRNA against ERS25 were exposed to heat shock (42 °C) for 8 h, and cell lysates were used to immunoprecipitate (IP) with an Apaf-1 antibody and Western blot (WB) for HSP70. Cont., control.