Thioredoxin-interacting protein mediates ER stress-induced β cell death through initiation of the inflammasome

Abstract

Recent clinical and experimental evidence suggests that endoplasmic reticulum (ER) stress contributes to the life-and-death decisions of β cells during the progression of type 1 and type 2 diabetes. Although crosstalk between inflammation and ER stress has been suggested to play a significant role in β cell dysfunction and death, a key molecule connecting ER stress to inflammation has not been identified. Here we report that thioredoxin-interacting protein (TXNIP) is a critical signaling node that links ER stress and inflammation. TXNIP is induced by ER stress through the PERK and IRE1 pathways, induces IL-1β mRNA transcription, activates IL-1β production by the NLRP3 inflammasome, and mediates ER stress-mediated β cell death. Collectively, our results suggest that TXNIP is a potential therapeutic target for diabetes and ER stress-related human diseases such as Wolfram syndrome.

Figure 1. TXNIP is highly expressed in pancreatic beta cells and induced by ER stress

(A) Mouse pancreata were analyzed by immunohistochemistry. Merged image shows the co-localization of TXNIP and insulin (upper panel) or TXNIP and glucagon (lower panel). (B) Expression of TXNIP mRNA in INS-1 832/13 cells treated with thapsigargin (TG, 0.25 μM) or tunicamycin (TM, 5 μg/ml). (C) Expression of TXNIP mRNA in INS-1 832/13 cells treated with glucose (16.7 mM) or human islet polypeptide (hIAPP, 5μM). (D) Expression of TXNIP mRNA in mouse and human primary islets treated with thapsigargin (TG, 1 μM) or tunicamycin (TM, 5 μg/ml) for 6 h. (E) Expression of TXNIP protein in INS-1 832/13 cells treated with thapsigargin (TG, 1 μM) for 8hr or tunicamycin (TM, 5 μg/ml) for 24 h, and human primary islets treated with tunicamycin (TM, 5 μg/ml) for 24 h. n=3; values are mean ± SD

Figure 2. TXNIP expression is regulated by the IRE1 and PERK-eIF2 α pathways of the UPR

(A) Expression of TXNIP mRNA in INS-1 832/13 cells transfected with control or IRE1α siRNA, and then treated with thapsigargin (TG, 0.5 μM) for 6 h. (B) Expression of TXNIP mRNA (left panel) and protein (right panel) in wild-type (WT) and Ire1α^−/− (KO) MEFs treated with thapsigargin (0.5 μM) for 3 h or untreated. (C) Expression of TXNIP mRNA in INS-1 832/13 cells transfected with control or PERK siRNA, and then treated with thapsigargin (TG, 0.5 μM) for 6 h. (D) Expression of TXNIP mRNA (left panel) and protein (right panel) in wild-type (WT) and Perk^−/− MEFs treated with thapsigargin (0.5 μM) for 3 h or untreated. (E) Expression of TXNIP mRNA and protein in wild-type eIF2α^S/S or mutant eIF2α^A/A MEFs treated with thapsigargin (TG, 0.5μM) for 3 h or untreated. (F) Expression of TXNIP mRNA and protein in INS-1 832/13 cells transfected with control or ATF6α siRNA, and then treated with thapsigargin (TG, 0.5 μM) for 6 h. (G) Expression of TXNIP mRNA in wild-type (WT) and Atf6α^−/− MEFs treated with thapsigargin (0.5 μM) for 3 h or untreated. (H) Luciferase reporter assays in INS-1 832/13 cells transiently transfected with TXNIP promoter reporter constructs. Cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 h. (I) Luciferase reporter assays in INS-1 832/13 cells transfected with TXNIP promoter reporter construct carrying 1.5 Kb of TXNIP promoter together with control, PERK expression, or GADD34 expression vector. Cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 h. (J) Luciferase reporter assay in INS-1 832/13 cells transfected with a TXNIP promoter reporter construct carrying 1.5 Kb of TXNIP promoter. Cells were untreated (UT) or treated with Salubrinal (25 μM) for 24 h. (K) Luciferase reporter assays in INS-1 832/13 cells transfected with a TXNIP promoter reporter construct carrying 1.5 Kb of TXNIP promoter together with control or ChREBP siRNA. Cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 h. (L) ChIP assays monitoring binding of ChREBP and Mlx to the TXNIP promoter in INS-1 832/13 cells treated with or without thapsigargin (TG, 0.5 μM) for 6 h. NA, Non-specific IgG Antibody. (M) Expression of ChREBP mRNA in INS-1 cells transfected with control or PERK siRNA (left panel), and wild-type and Perk^−/− MEFs (right panel) treated with or without thapsigargin (TG, 0.5 μM) for 6 h. (N) Luciferase reporter assays in INS-1 832/13 cells transfected with a TXNIP promoter reporter construct carrying 1.5 Kb of TXNIP promoter together with control or ATF5 siRNA. Cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 h. (O) ChIP assay monitoring binding of ATF5 to the TXNIP promoter in INS-1 832/13 cells treated with or without thapsigargin (TG, 0.5 μM) for 6 h. NA, Non-specific IgG Antibody. (P) Expression of ATF5 mRNA in INS-1 832/13 cells transfected with control or PERK siRNA (left panel), and wild-type and Perk^−/− mouse embryonic fibroblasts (right panel) treated with or without thapsigargin (TG, 0.5 μM) for 6 h. n=3; values are mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. n.s., not significant

Figure 3. IL1-β is induced by ER stress and regulated by TXNIP

(A) IL-1βb and IL-6 mRNA expression in human islets treated with thapsigargin (TG, 1 μM) for 6h, tunicamycin (TM, 5 μg/ml) for 6 h or untreated. (B) IL-1β and IL-6 mRNA expression in INS-1 832/13 cells transfected with control GFP or GADD34 expression plasmid. After 36 h, cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 h. (C) IL-1b and IL-6 mRNA expression in human islets pretreated with interleukin-1 receptor antagonist (IL1RA, 1 ug/ml) for 24 h, and then treated with tunicamycin (TM, 5 ug/ml) for 8 h. (D) IL-1β and IL-6 mRNA expression in INS-1 832/13 cells stably transduced with pLenti-TO/shTXNIP, inducible lentivirus expressing shTXNIP. Cells were cultured with doxycycline (2 μg/ml) to induce shTXNIP or without doxycycline for 48 h, then challenged with thapsigargin (TG, 0.5 μM) for 6 h. (E) IL-1β and IL-6 mRNA expression in primary islets from Txnip^−/− and control (WT) mice treated with thapsigargin (TG, 0.5 μM) for 6 h. (F) IL-1β and IL-6 mRNA expression in INS-1 832/13 cells transfected with control or NLRP3 siRNA, and then treated with thapsigargin (TG, 0.5 μM) for 6 h or untreated. (G) TXNIP, IL-1β and IL-6 mRNA expression in THP-1 cells transformed with phorbol 12-myristate 13-acetate (0.5 μM), and then treated with thapsigargin (TG, 1 μM), tunicamycin (TM, 20 μg/ml) for 6 h or untreated. (H)(I) THP-1 cells transformed with phorbol 12-myristate 13-acetate (0.5 μM) were treated with thapsigargin (TG, 1 μM), tunicamycin (TM, 20 μg/ml), ATP (5 mM) for 6 h or untreated. Secreted IL-1β was measured by ELISA (H) and caspase-1 cleavage was measured by immunoblot. P20, cleaved caspase-1 (I). n=3; values are mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. n.s., not significant.

Figure 4. TXNIP is an apoptotic component of the unfolded protein response

(A) TXNIP, caspase-3, and actin protein expression in INS-1 832/13 cells stably transduced with pLenti-TO/shTXNIP, inducible lentivirus expressing shTXNIP. Cells were cultured with doxycycline (Dox, 2 μg/ml) to induce shTXNIP or without doxycycline for 48 h, then challenged with thapsigargin (TG, 0.05 μM) or tunicamycin (TM, 5 μg/ml) for 24 h. Single and double asterisks indicate uncleaved and cleaved caspase-3, respectively. (B) INS-1 832/13-TetR-shTXNIP cells were incubated with doxycycline (Dox, 2 μg/ml) for 48 h, then treated with thapsigargin (TG, 10 nM), tunicamycin (TM, 5 μg/ml) for 24 h or untreated. Cells were stained with propidium iodide solution (PI) followed by flow cytometry analysis. (TG, n=6 and TM, n=9; values are mean ± SD). (C) Viability of INS-1 832/13-TetR-shTXNIP cells incubated with doxycycline (Dox, 2 μg/ml) for 48 h, and then treated with thapsigargin (TG, 50 nM) for 24 h or untreated (n=3; values are mean ± SD). (D) Human primary islets (28 year old male, BMI 21, HbA1C 5.4) were treated with thapsigargin (TG, 1 μM) in the presence of BSA(−, 1 μg/ml) or interleukin-1 receptor antagonist (+, 1 μg/ml) for 24 h. Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay (Triplicated, values are mean ± SD). * p<0.05, ** p<0.01.