IRE1 signaling affects cell fate during the unfolded protein response

Abstract

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.

Fig. 1

Kinetics of IRE1 signaling with persistent ER stress. (A) WT Ire1α^+/+ or Ire1α^−/− mouse embryo fibroblasts were treated with tunicamycin (tm) (5 μg/ml), and Xbp-1 mRNA splicing was determined by RT-PCR. Unspliced (u) and spliced (s) Xbp-1 mRNA products are indicated. The asterisk indicates the position of a hybrid amplicon (27). XBP-1^s was detected by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels served as a protein loading control. (B) HEK293 cells were treated with tm (5 μg/ml) or thapsigargin (tg) (500 nM) for the indicated times. tm, red bars; tg, blue bars. Results are representative of five independent experiments. (C) HEK293 cells were treated with agents for the indicated times. At 24 hours, media containing drug were transferred to fresh cells. After 4 additional hours, Xbp-1 mRNA splicing was determined by RT-PCR. (D) HEK293 cells, transfected with VCAM-1, were treated with tm (5 μg/ml) for the indicated times. Xbp-1 mRNA splicing was determined by RT-PCR. Mature and deglycosylated (dg) VCAM-1 species were determined by immunoblotting. (E) HEK293 cells, transfected with VCAM-1, were treated with tm for the indicated times and were pulse-labeled, and radiolabeled VCAM-1 was detected after immunoprecipitation. The double asterisk indicates the position of a nonspecific band used as a loading control. (F) HEK293 cells were treated with tg for the indicated times, and phospho-JNK protein levels were assessed by immunoblotting. Total JNK protein levels served as a loading control.

Fig. 2

Kinetics of ATF6 and PERK with persistent ER stress. (A) HEK293 cells, expressing FLAG-tagged ATF6α, were treated with tg (500 nM) for the indicated times, and ATF6f was detected by immunoblotting. GAPDH levels served as a protein loading control. (B) HEK293 cells were treated with tg, and phospho-PERK, phospho-eIF2α, and ATF-4 levels were determined by immunoblotting. Total eIF2α levels served as a protein loading control. In the bottom panels, cells were treated with drug for the indicated times and were pulse-labeled, and radioisotope incorporation was measured via phosphoimaging. ³⁵S-Met/Cys, ³⁵S-labeled methionine/cysteine. (C) Cells were treated with agents (tm, red bars; tg, blue bars) for the indicated hours, and normalized BiP (top panel) and Chop (bottom panel) mRNA levels were measured by quantitative PCR and shown relative to levels in untreated cells. Error bars represent SDs from five independent experiments.

Fig. 3

Chemical-genetic control of human IRE1. (A) Alignments of a portion of the ATP-binding domains of yeast and human IRE1 are shown. The residue mutated to glycine is shown in color (28). The structure of the ATP analog, 1NM-PP1, is shown below. (B) Parental WT and transgenic HEK293 cells expressing 1NM-PP1–sensitized IRE1 were treated for the indicated times with 1NM-PP1 (5 μM), and Xbp-1 mRNA splicing was determined by RT-PCR. (C) Transgenic HEK293 cells were treated with tm (5 μg/ml) ± 1NM-PP1 (5 μM). Xbp-1 mRNA splicing was assessed by RT-PCR and quantified. Results are representative of five independent experiments. (D) Transgenic HEK293 cells were treated with tg (300 nM) ± 1NM-PP1 (5 μM). Xbp-1 mRNA splicing was assessed by RT-PCR and quantified. Results are representative of five independent experiments.

Fig. 4

IRE1 signaling enhances cell viability. Parental WT and transgenic HEK293 cells were treated with the indicated agents, and adherent, cresyl violet–stained positive cells were counted at the indicated times and are shown relative to counts of mock-treated cells. Error bars represent SDs from three independent experiments. The arrows indicate P value < 0.01 when the corresponding samples ± 1NM-PP1 were compared (Student’s t test).

Fig. 5

BiP and Chop expression in animals expressing P23H rhodopsin. (A) HEK293 cells were transfected as indicated, and normalized BiP mRNA levels were measured by quantitative PCR and are shown relative to levels in untreated cells. Values represent the means ± SDs from five independent experiments. (B) Normalized BiP and Chop mRNA levels were measured in retinas [from WT Sprague-Dawley or transgenic rats expressing P23H rhodopsin at high (P23H-1 Rho Tg) or low (P23H-3 Rho Tg) levels] by quantitative PCR and are shown relative to levels at PND 6 (P6): a time when >95% of the mature complement of retinal photoreceptors has been generated. Error bars represent SDs from three animals at each age. *, P = 0.003; **, P < 0.001 [as compared with age-matched WT animals (Student’s t test)]. (C) Light micrographs of representative retinal sections from the inferior posterior retinas of WT, P23H-1, and P23H-3 rats at the indicated ages. The outer nuclear layer (ONL), which is proportional to photoreceptor nuclei numbers, thins as photoreceptors degenerate, and rhodopsin-containing outer segments (arrowheads) shorten. Scale bar, 25 μm. (D) The mean ONL thickness was measured in retinal cross sections from transgenic and WT rats at the indicated ages. Error bars represent SDs of ONL thickness from three to six animals at each age. The onset of ONL thinning in both transgenes was at P12 (P < 0.05), with progressive thinning at later ages (P < 0.0001) (Student’s t test).

The unfolded protein response. Accumulation of misfolded proteins (ER stress) triggers both cell protective and cell death responses, but with different time courses.