Volume 16, Issue 2, Pages 265-273 (August 2012) Thioredoxin-Interacting Protein Mediates ER Stress-Induced β Cell Death through Initiation of the Inflammasome  Christine M. Oslowski, Takashi Hara, Bryan O'Sullivan-Murphy, Kohsuke Kanekura, Simin Lu, Mariko Hara, Shinsuke Ishigaki, Lihua J. Zhu, Emiko Hayashi, Simon T. Hui, Dale Greiner, Randal J. Kaufman, Rita Bortell, Fumihiko Urano  Cell Metabolism  Volume 16, Issue 2, Pages 265-273 (August 2012) DOI: 10.1016/j.cmet.2012.07.005 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 TXNIP Is Highly Expressed in Pancreatic β Cells and Induced by ER Stress (A) Mouse pancreata were analyzed by immunohistochemistry. Merged image shows the colocalization of TXNIP and insulin (top) or TXNIP and glucagon (bottom). (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, 5 μM). (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 hr. (E) Expression of TXNIP protein in INS-1 832/13 cells treated with thapsigargin (TG, 1 μM) for 8 hr or tunicamycin (TM, 5 μg/ml) for 24 hr, and human primary islets treated with tunicamycin (TM, 5 μg/ml) for 24 hr. n = 3; values are shown as mean ± SD. Cell Metabolism 2012 16, 265-273DOI: (10.1016/j.cmet.2012.07.005) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 TXNIP Expression Is Regulated by the IRE1 and PERK-eIF2α 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 hr. (B) Expression of TXNIP mRNA (left) and protein (right) in wild-type (WT) and Ire1α−/− (KO) MEFs treated with thapsigargin (0.5 μM) for 3 hr 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 hr. (D) Expression of TXNIP mRNA (left) and protein (right) in wild-type (WT) and Perk−/− MEFs treated with thapsigargin (0.5 μM) for 3 hr or untreated. (E) Expression of TXNIP mRNA and protein in wild-type eIF2αS/S or mutant eIF2αA/A MEFs treated with thapsigargin (TG, 0.5 μM) for 3 hr 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 hr. (G) Expression of TXNIP mRNA in wild-type (WT) and Atf6α−/− MEFs treated with thapsigargin (0.5 μM) for 3 hr 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 hr. (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 hr. (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 hr. (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 hr. (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 hr. NA, nonspecific IgG antibody. (M) Expression of ChREBP mRNA in INS-1 cells transfected with control or PERK siRNA (left), and wild-type and Perk−/− MEFs (right) treated with or without thapsigargin (TG, 0.5 μM) for 6 hr. (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 hr. (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 hr. NA, nonspecific IgG antibody. (P) Expression of ATF5 mRNA in INS-1 832/13 cells transfected with control or PERK siRNA (left), and wild-type and Perk−/− mouse embryonic fibroblasts (right) treated with or without thapsigargin (TG, 0.5 μM) for 6 hr. n = 3; values are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant. See also Figures S1–S3. Cell Metabolism 2012 16, 265-273DOI: (10.1016/j.cmet.2012.07.005) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 IL1-β Is Induced by ER Stress and Regulated by TXNIP (A) IL-1β and IL-6 mRNA expression in human islets treated with thapsigargin (TG, 1 μM) for 6 hr, tunicamycin (TM, 5 μg/ml) for 6 hr, 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 hr, cells were untreated (UT) or treated with thapsigargin (TG, 0.5 μM) for 6 hr. (C) IL-1β and IL-6 mRNA expression in human islets pretreated with interleukin-1 receptor antagonist (IL1RA, 1 μg/ml) for 24 hr, and then treated with tunicamycin (TM, 5 μg/ml) for 8 hr. (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 hr, then challenged with thapsigargin (TG, 0.5 μM) for 6 hr. (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 hr. (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 hr 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 hr or untreated. (H and 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 hr 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 shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant. Cell Metabolism 2012 16, 265-273DOI: (10.1016/j.cmet.2012.07.005) Copyright © 2012 Elsevier Inc. Terms and Conditions

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 hr, then challenged with thapsigargin (TG, 0.05 μM) or tunicamycin (TM, 5 μg/ml) for 24 hr. 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 hr, then treated with thapsigargin (TG, 10 nM), tunicamycin (TM, 5 μg/ml) for 24 hr 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 hr, and then treated with thapsigargin (TG, 50 nM) for 24 hr 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 hr. Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay (triplicated, values are shown as mean ± SD). ∗p < 0.05, ∗∗p < 0.01. Cell Metabolism 2012 16, 265-273DOI: (10.1016/j.cmet.2012.07.005) Copyright © 2012 Elsevier Inc. Terms and Conditions