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  • The PERK branch of the UPR is


    The PERK branch of the UPR is strongly protective at modest levels of signaling but contributes signals leading to cell death pathways. PERK is the major protein responsible for the attenuation of mRNA translation, indirectly through the inhibition of eIF2α. Under unstressed conditions PERK is kept inactive by the ER luminal binding to BiP but under ER stress the chaperone dissociates from PERK resulting in PERK oligomerization and autophosphorylation [9]. In the present study, we used a phospho-specific PERK antibody to evaluate its activation status following cerulein administration in the presence or absence of ANP. A previous study shows that PERK is not activated in cerulein-induced AP [29]. Nevertheless, whether PERK was earlier modified by ANP, it cannot be excluded, given that downstream effectors of the PERK pathway like eIF2α and CHOP Concanamycin A were changed in AP and by ANP pretreatment. PERK phosphorylates eIF2α leading to the inhibition translation and the reduction of protein load to the ER. However, some mRNA containing short open reading frames in their 5′ untranslated regions are preferentially translated like the transcription factor ATF4 which regulates the expression of CHOP, a transcription factor that controls genes involved in apoptosis and GADD34 which leads to eIF2α dephosphorylation [10,[30], [31], [32], [33]]. In the present study, ANP reduced eIF2α phosphorylation suggesting that protein translation has been resumed. Furthermore, it also enhanced CHOP expression supporting stimulation of ER-dependent programmed cell death. CHOP inhibits the expression of the gene encoding Bcl2 thus leading to the activation of the proapoptotic proteins Bax and Bak. In line with enhanced ANP-induced CHOP expression in AP we found that ANP reduced Bcl2 expression and upregulated proapoptotic Bax and Bak. These findings correlate well with previous studies showing that ANP stimulates caspase 3 activation and expression [15]. ER-dependent apoptosis is not only mediated by CHOP but also by caspase 12 and caspase 2 activation. How the UPR switches its signaling from an adaptative reaction to the stimulation of cell death is poorly understood. Caspase 12 is the only member of the caspase family, which is in the cytoplasm of the ER and can act on both ER and mitochondria [34]. Caspase 12 is activated by calpain, a cytosolic protease which translocates to the cell membrane upon calcium increase [35]. In AP caspase 12 was activated, but not further activation was observed in the presence of ANP. However, caspase 2 activity was enhanced by ANP in rats with or without pancreatitis. It was shown that caspase 2 activates the proapoptotic protein BID which in turn activates Bax and Bak [36]. Apoptosis attenuates the severity of acute pancreatitis given that it inversely correlates with necrosis [37,38]. Switching necrosis to apoptosis results in a better outcome of the disease. In previous studies we showed that ANP stimulates caspase-3 activity and expression as well as cleaved PARP-1, an early substrate of the enzyme, which is able to switch the type of cell death from apoptosis to necrosis [15]. Furthermore, it also significantly increased the number of apoptotic nuclei as revealed by TUNEL assay [15]. In this study we show that ANP stimulates ER-dependent programmed cell death. Present results show that in AP the ATF6 branch of the UPR remains activated in the presence of ANP. ATF6 activation (involved in the reprogramming of gene expression) represents with ATF4 (upstream effector of CHOP) the second phase in the adaptative response controlled by the UPR [3]. Upon activation, ATF6 is delivered to the Golgi apparatus where it undergoes proteolytic cleavage to yield a fragment, which then moves to the nucleus to promote the transcription of genes involved in ERAD and protein folding [39]. In AP the expression of ATF6 remained further increased in the presence of ANP suggesting that the atrial peptide stimulates ERAD and the ER folding capacity. Surprisingly, ATF6 was enhanced in normal animals infused with ANP. In this sense, it was reported that selective activation of ATF6 triggers ER expansion under conditions in which there is no indication of stress likely to protect it from potential protein overload [40]. These findings suggest that ANP-induced ATF6 activation would be beneficial for the pancreatic acinar cell which is prone to protein overload.