UPR-mediated TRIB3 expression correlates with reduced AKT phosphorylation and inability of interleukin 6 to overcome palmitate-induced apoptosis in RINm5F cells

Abstract

Unfolded protein response (UPR)-mediated pancreatic b-cell death has been described as a common mechanism by which palmitate (PA) and pro-inflammatory cytokines contribute to the development of diabetes. There are evidences that interleukin 6 (IL6) has a protective action against b-cell death induced by pro-inflammatory cytokines; the effects of IL6 on PA-induced apoptosis have not been investigated yet. In the present study, we have demonstrated that PA selectively disrupts IL6-induced RAC-alpha serine/threonine-protein kinase (AKT) activation without interfering with signal transducer and activator of transcription 3 phosphorylation in RINm5F cells. The inability of IL6 to activate AKT in the presence of PA correlated with an inefficient protection against PA-induced apoptosis. In contrast to PA, IL6 efficiently reduced apoptosis induced by proinflammatory cytokines. In addition, we have demonstrated that IL6 is unable to overcome PA-stimulated UPR, as assessed by activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, X-box binding protein-1 gene mRNA splicing, and pancreatic eukaryotic initiation factor-2a kinase phosphorylation, whereas no significant induction of UPR by pro-inflammatory cytokines was detected. This unconditional stimulation of UPR and apoptosis by PA was accompanied by the stimulation of CHOP and tribble3 (TRIB3) expression, irrespective of the presence of IL6. These findings suggest that IL6 is unable to protect pancreatic b-cells from PA-induced apoptosis because it does not repress UPR activation. In this way, CHOP and ATF4 might mediate PA-induced TRIB3 expression and, by extension, the suppression of IL6 activation of pro-survival kinase AKT.

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression

“…Ten thousand events were analyzed per experiment using the Cell Quest software (Becton Dickinson). DNA fragmentation was analyzed according to the method previously described (25).…”

“…Total RNA was extracted from ϳ200 freshly isolated islets using TriZOL reagent (Invitrogen), and TRB3 expression was assessed by real-time PCR using SYBR Green master mix (Applied Biosystems, Foster City, CA), as previously described (25). The sequences of primers and amplification conditions were TRB3 sense 5=-GTTGGTGCTGGAGAACCTGGAG-3= and antisense 5=-AGAGTCCTGGAACGGGTATCGG-3=, annealing temperature of 59°C; ␤-actin sense 5=-TGGCATTGTGATGGACTCCG-3= and antisense 5=-TTAATGTCACGCACGATTTCCC-3=, annealing temperature of 58°C.…”

Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation

“…11,12 In addition, administration of different anticancer agents promotes cancer cell death via TRIB3 upregulation and the subsequent inhibition of Akt. [13][14][15][16][17][18][19] However, the precise molecular basis of the regulation of Akt by TRIB3 and whether loss of this pseudokinase may contribute to cancer initiation and progression remains to be clarified.In this study, we investigated the effect of the genetic inactivation of TRIB3 in several cellular and animal models of cancer. Our findings indicate that genetic inhibition of TRIB3 enhances tumorigenesis and that this effect is due -at least primarily -to a selective inactivation of the transcription factor FOXO by the mammalian target of rapamycin complex 2 (mTORC2)/AKT axis.…”

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic β-cell proliferation in islets from early lactating mothers

“…Transfection of MIN6 cells with small interference RNA (siRNA) targeted to MKP-1 (si-MKP-1) or scrambled control siRNA were performed as described previously (3,35). Briefly, cells were washed twice with serum-and glucose-free medium (Opti-MEM) and then incubated with 1 ml of the same medium containing the siRNA (final concentration of 80 nM) previously mixed with 2 l of Lipofectamine 2000.…”

“…no. 2750; Millipore), as described previously (35). Briefly, 4 ϫ 10 4 MIN6 or RINm5F cells were seeded in 96-well plates and allowed to attach overnight in growth medium.…”