Signaling by IL-1β+IFN-γ and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic β-cell apoptosis

Abstract

Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic b-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1b þ IFN-c and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in b-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for b-cell apoptosis after different stimuli, and that it can act up-and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1b þ IFN-c induce b-cell death and the nature of the downstream signals by which ER stress 'convinces' b-cells to trigger apoptosis.

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

“…3,[5][6][7]13,18,32 Here, we show that cytokines, palmitate and thapsigargin, induce Bax translocation, cytochrome c release and caspase-3 cleavage, independently of changes in Bcl-2, Bcl-XL, Bax and Bak expression levels, but partly as a consequence of Mcl-1 downregulation (Figure 8). Our data are in agreement with previous studies showing that Mcl-1, in spite of its preferential mitochondrial localization, 33 is able to prevent Bax activation and translocation.…”

“…JNK has been reported to inactivate Mcl-1, 16 and cytokines induce a strong and rapid (1 h) JNK phosphorylation in INS-1E cells, 17,18 leading to a peak of c-Jun phosphorylation between 6 and 8 h. We thus exposed INS-1E cells to IL-1b þ IFN-g for 8 h in the presence of the JNK inhibitor peptide (JNKi) 19 or the chemical JNK inhibitor SP600125. 18 In other cell types, Mcl-1 stability has been shown to be modulated through phosphorylation by the ERK1,2 and the glycogen synthase kinase-3b (GSK-3b).…”

“…18 In other cell types, Mcl-1 stability has been shown to be modulated through phosphorylation by the ERK1,2 and the glycogen synthase kinase-3b (GSK-3b). 8 The role of Mcl-1 in b-cell apoptosis F Allagnat et al cells.…”

“…Mcl-1 stability in other cell types is influenced through phosphorylation of various residues by multiple kinases, including JNK, ERK and GSK-3b. 8 Cytokines induce JNK in b-cells 18,29 and phosphorylation of Mcl-1 by JNK facilitates Mcl-1 degradation in HEK293 cells. 16 We presently observed that JNK contributes to cytokine-mediated Mcl-1 downregulation.…”

“…Lysates were then resolved by SDS-PAGE and transferred to a PVDF membrane. Immunoblot analyses were performed as described previously, 13,18 using the following antibodies: polyclonal anti-rat Mcl-1 from Biovision (Gentaur, Brussels, Belgium); polyclonal anti-CHOP/GADD153, monoclonal anti-b-catenin (D-10) and polyclonal anti-Bax (P-19) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); polyclonal anti-P-GSK-3a,b, polyclonal anti-GSK-3b, polyclonal anti-P-ERK1,2, polyclonal anti-ERK1,2, polyclonal anti-P-PERK, polyclonal anti-PERK, polyclonal anti-P-eIF2a, polyclonal anti-CoxIV, …”