HUPO (Human Proteome Organization) edition:7 location:Amsterdam date:16-20 August 2008
Introduction Pancreatic β-cells are the cells responsible for insulin formation. Because of the high demand on insulin processing, β-cells have a highly developed endoplasmic reticulum and are thus very sensitive to ER stress, in particular to agents that deplete the ER Ca2+, such as cyclopiazonic acid (CPA). In previous studies we have investigated, at the mRNA level, the global alterations induced by CPA, in INS-1E cells (1). We now investigated the changes induced by CPA with 2D-DIGE. The results point to a marked decrease in multiple proteins involved in the ubiquitin-proteasome pathway.
Methods INS-1E cells were treated with 25µM CPA dissolved in DMSO. Control cells were treated with DMSO alone. Cell viability was assessed by cell counting with Hoechst/PI staining. INS-1E cells were incubated for 6 or 12 hours with CPA or with DMSO alone. Quadruplicate experiments were performed, originating from 4 independent experiments. Protein separation was done in the first dimension on 24cm strips, at pH ranges pH 4-7 and pH 6-9. Second dimension was performed using 11.5% SDS/polyacrylamide gel. Scanning of the gels was performed using a Typhoon 9400 and analysis with the DeCyder V6.5 software. Spots (p<0.05) were identified using in-gel digestion and MALDI-TOF/TOF. Some proteins were confirmed using western blot. We constructed a human protein interaction network as reported (2).
Results We assessed the effects of CPA on cell viability and on the mRNA expression of the pro-apoptotic transcription factor CHOP. The percentage of apoptotic cells increased from 1,12% +/- 0.84% to 11,45% +/- 3.10% at 6 hours (n=4, p<0.01) and to 18,24% +/- 6.39 % (n=4, p<0.01 ) after 12 hours. mRNA levels for the pro-apoptotic transcription factor CHOP increased 26-fold to 31-fold after 6 and 12 hours of CPA treatment, respectively (p<0.01). For the 2D-DIGE proteomic study, 1841 ± 294 spots could be matched for the 6 and 12 hour time point in the pH 4-7 range. Of these, 48 and 90 spots were differentially expressed between control and CPA-treated cells for the 6 and 12 hour time point respectively (p<0,05). No differentially expressed spots (with p<0,05) were observed in the pH 6-9 range. We were able to identify 91 spots, resulting in 60 distinct proteins. We grouped these proteins according to their function in 9 groups. Probably the most important process in CPA induced apoptosis is the ubiquitin/proteasome pathway, of which we detected numerous players. Compared to our previous study (3), we can conclude that cytokine-induced or ER-stress-induced apoptosis has distinct mechanisms