The endoplasmic reticulum (ER) is a central subcellular organelle for protein and lipid synthesis as well as Ca2+
regulation and maintaining ER homeostasis is critical for cell survival. For better understanding of its function, the ER has been the subject of several recent organellar proteomics studies. An early study using a 2D gel approach identified 141 proteins from purified mouse liver ER vesicles 47
. Two new members of the thioredoxin family, Erp19 and ERp46, were identified in that study. In a more recent study 48
, a protein correlation profiling strategy was applied to map proteins to ten subcellular locations in mouse liver by quantitatively comparing fractions collected from continuous density gradient centrifugation. A total of 229 proteins were clustered together with known ER markers and therefore assigned as ER proteins. In another recent effort to quantitatively define the subcellular components of the secretory pathway using individually purified RER, smooth microsomes and Golgi, over 800 proteins were assigned uniquely to the ER 28
. Since all the above studies utilized mouse or rat liver as the source of organelles, the knowledge gained about ER subproteome has been limited to a single tissue origin. Large scale proteomics studies comparing organelles across multiple organs have indicated the presence and functional importance of tissue specific organellar proteins 29, 31
In this study, we obtained the first extensive catalog of pancreatic RER proteins with very minor contamination from other organelles including mitochodria and nuclei. This extensive list includes a full coverage of the key functional categories of ER ( and Supplement table 1
). Among these proteins, the largest functional group accounting for about 30% of total identified proteins is related to protein synthesis and translocation into ER. These include the protein translational machinery, essentially all the ribosomal subunits and major translation initiation and elongation factors. It also includes the entire machinery responsible for translocation of newly synthesized protein to ER membrane or lumen: components of signal recognition particle (SRP) and its receptor subunits to bind nascent polypeptides; the heterotrimeric SEC61 complex, SEC62 and SEC63 to function as the translocase; signal sequence receptor subunits and SEC11 signal peptidases to bind and remove signal peptides in ER lumen. One of the key functions of ER is to maintain a subcellular environment critical to correct protein folding. Consistent with this important function of ER, a major category of proteins identified are ER chaperones playing essential roles in protein folding and quality control. These include eight members of protein disulfide isomerases as well as the ER chaperones calreticulin, calnexin, BiP and several heat shock proteins. Given the high protein synthesis rate in exocrine pancreas and the importance of these proteins in protein folding, it is not surprising that all of these proteins were among the most abundant proteins in identified RER proteins when the relative protein abundance was estimated by spectral counting approach49
. Since all the secretory proteins need to traffic through the ER, as expected a group of secretory proteins were detected in the ER samples including all the digestive enzymes and major zymogen granule membrane proteins such as GP2 and syncollin. Other categories of identified proteins were involved in other important functions of the ER including membrane trafficking, calcium storage, oligosaccharide, lipid & sterol biosynthesis. Many of these identified proteins are reported in pancreatic RER for the first time. One example is hypoxia up-regulated 1 which was one of the most abundant proteins found on RER in our study (Supplemental table 1
). This protein has first been identified in astrocytes 50
and more recently has been shown to be induced during hypoxia or ER stress 22
In an effort to search for the potential tissue specific ER proteins, we compared our list of pancreatic RER proteins with published results of liver ER lists 28, 48
. Protein sequences from two supplemental tables in that paper, one with 832 proteins unique to ER (Supplemental table S5A) and the other with 405 proteins (shared between ER and COPI vesicle) were retrieved from the NCBI database using batch Entrez and blasted on our local server against our pancreatic RER protein sequences. 162 proteins in the pancreatic RER were found not present in the liver ER data set (Supplemental table 1
). Not surprisingly, all the pancreatic secretory proteins including all digestive enzymes and zymogen granule membrane proteins are uniquely found in our data set which demonstrate the validity of our comparison. Our data set comprises a more comprehensive set of protein translational machinery including ribosomal proteins and translation initiation factors. This might be attributed to the higher protein synthesis rate of exocrine pancreas. There are also tissue specific ER lipid modifying enzymes and chaperones in exocrine pancreas. Endoplasmic reticulum protein 27 is of particular interest because it has been found decreased dramatically in RER of both models of acute pancreatitis.
In order to unveil the cellular mechanisms responsible for the initiation, propagation and limitation of the inflammatory response in the early phase of acute pancreatitis, several systemic profiling studies have been carried out recently using established animal models for AP. Microarray analysis was utilized to identify genes commonly induced in rat pancreatic acinar cells within 1-4 h in two in vivo
AP models, caerulein and intraductal taurocholate administration 51
. This strategy yielded 51 known genes representing a complex array of molecules as well as identifying EGR-1 and several other novel genes likely to be important in the development and severity of acute pancreatitis. In a previous proteomics study, a 2D gel electrophoresis and MS/MS analyses were performed to compare whole pancreatic tissue extracts following AP induced by caerulein with pancreas from healthy rats 37
. A total of 125 proteins were identified from both diseased and control samples on the 2D gel and 42 proteins or protein fragments were found to be differentially expressed in diseased pancreas representing potential pathobiological pathways involved in this disease. These included activated digestive enzymes, increased expression of various inflammatory markers and changes related to oxidative and cellular stress responses. Three ER proteins were present among the 42 proteins, elongation factor 1-γ (decrease), 40S ribosomal protein SA (decrease) and PDI A6 (probable PTM) 37
In this study, we conducted the first organellar proteomics study of normal and AP RER. We found 37 proteins that showed significant changes in at least one of the two experiments with ratios (treatment vs. control) ≤ 0.75 or ≥ 1.50 and p≤0.05. This corresponds to an 11% change out of the 320 proteins with 2 or more unique peptides. This is significantly above the 5% chance to detect a random change. Furthermore, many of the 37 proteins showed similar changes in duplicate experiments and a number of them were further validated by Western blotting and immunocytochemistry. The quantitative comparison was focused on isolated RER in order to enrich for the protein machinery involved in protein synthesis and protein folding. We wanted to test whether alteration of protein synthesis and induction of ER stress are initiating cellular events during early AP. Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases, such as diabetes mellitus and Alzheimer's disease, but whether they are involved in acute pancreatitis is still uncertain. It has been shown that major ER stress sensing and signaling proteins including PERK, eIF2α
, ATF6 and BiP are present in pancreatic exocrine acini and were activated early in the arginine model of experimental AP. For example, BiP was reported up-regulated within 4 h of injection of arginine and remained elevated for 24 hours 25
. We found BiP had little change in one experiment and decreased slightly in another experiment in the arginine AP model (). These observations do not support the induction of this compensatory mechanism in AP at least not in this model of pancreatitis. In terms of the other ER stress indicators such as PERK and ATF6, they were not identified with required confidence (95%) in our RER samples nor have been reported in other published proteomics studies 28, 48
. This is likely due to their very low abundance on the ER membrane. The decrease in digestive enzymes in the ER however, could be the result of the inhibition of their translation by PERK and exit from the RER as part of their maturation. An earlier study showed inhibition of global pancreatic protein synthesis during caerulein-induced pancreatitis in mice 26
. It is worth noting that Erp27, a non-catalytic ER-located protein disulfide isomerase family member 52
, was found decreased in both animal models of acute pancreatitis. The exact function of this recently discovered protein in exocrine pancreas as well as its role in AP is not clear and is worth further investigation.
Translation initiation and elongation factors are proteins necessary for the regulation of protein synthesis in cells. Some of them play an important role in the association or dissociation of the ribosomal subunits for the translation of the mRNA into protein and can be involved in cell stress mechanisms 53
. Others, as is the case for the eukaryotic initiation factor 4A (eIF4A), can be part of a functional subunit, the eukaryotic initiation factor 4F (eIF4F) complex, that recruits and unwinds the mRNA with an RNA-dependent ATPase activity. eIF4A is the most abundant initiation factor in cells and it has been shown to be involved with and increased in the response to cell stress 54-56
. Elongation factors, such as eukaryotic elongation factors 1 and 2 (eEF1 and eEF2), regulate translation elongation steps. In particular, eEF1α forms the ternary complex (eEF1α*GTP*aa-tRNA) which is then bound to the ribosomal A site in a codon-dependent manner. eEF1A has also been shown to be involved in cell stress 57, 58
and cancer processes 59
. In our studies these two translation factors, eEF1a1 and eIF4a1, increased in the RER after arginine pancreatitis, in correlation with the ER stress mechanisms that have already been demonstrated in this model of pancreatitis 25
. The fact that these molecules do not increase in the caerulein model is most likely related to the degree of cell damage which is less than that caused by arginine. Several other ER proteins, including the eukaryotic initiation factor 2α (eIF2α), the ER stress transducers PERK (PKR-like endoplasmic reticulum kinase), IRE1α (inositol requiring element-1α) and ATF6 (activating transcription factor 6), are expected to be phosphorylated rather than up-regulated in the ER stress response. Because this modification was not specifically targeted in our proteomics study of the ER, the potential changes of their phosphorylation were not observed. The reduction seen in the amount of ER chaperone molecules, such as BiP, ERp27, and P4hb in the development of arginine pancreatitis could be associated with the induction of apoptotic processes; the latest step in the ER stress and UPR (unfolded protein response) mechanisms, when chaperoning for unfolded proteins is no longer needed.
It has been demonstrated that during the induction of experimental models of acute pancreatitis the tubulin cytoskeleton is disrupted and degraded 60, 61
. In a proteome analysis of rat pancreatic acinar (AR42J) cells, tubulin β-chain was found to be differentially expressed 62
in caerulein-treated cells. In another study, the induction of two different models of acute pancreatitis to Balb/c and FVB/n mice, caused a slight increase of mRNA levels for tubulin in the pancreatitis groups 63
. In our study, the caerulein model of pancreatitis did not show any significant change in the amount of these tubulin isoforms in the ER. The arginine model, in contrast, exhibited an increase in several tubulin isoforms. Since the results (no change or decrease) found in the caerulein model are in concordance with what it has been found before in other studies 60, 61
, we hypothesize that changes in the tubulin cytoskeleton of acinar cells are model-specific and indicative of differences in pathologies between the two models. It has been speculated that microtubules are most likely involved in the transport of zymogen granules from the Golgi to the apical pole of the cell and this process is disrupted during pancreatitis 61
, but the role of microtubules in pancreatitis has not yet been fully determined. Thus, the differences between the caerulein and arginine models could likely be dependent upon the degree of cell damage in these two models of pancreatitis.
Fibrinogen, synthesized by the liver with little evidence for synthesis by other tissues, is the principal protein of vertebrate blood clotting containing two sets of three different chains (α, β, and γ) linked to each other by disulfide bonds. Inflammation and blood coagulation has been shown to be closely linked. The fact that fibrinogen is highly up-regulated in acute pancreatitis is consistent with it being a major acute response protein during the inflammation of pancreas. Our RT-PCR results indicated that fibrinogen was not synthesized within the pancreas. Whether or not the fibrinogen was absorbed into acinar cells via retrograde transport during inflammation is still an open question and requires further investigation. Interestingly, fibrinogen was found to be 3.0-fold up-regulated in human pancreatitic juice during pancreatitis compared with normal pancreatic juice in a proteomics study using ICAT reagent and LC-MS/MS 64
In this study, we isolated highly purified pancreatic RER from normal and acute pancreatitis rats. Using iTRAQ-based quantitative proteomics approach, we reported the first comprehensive protein inventory of pancreatic RER which includes 469 proteins with a full coverage of core ER functional categories and minimal contaminating organellar proteins. Furthermore, we quantified RER protein changes during the early course of acute pancreatitis using two different animal models. Significant RER protein changes were found in multiple functional categories including translational regulator, digestive enzymes, chaperones and cytoskeleton proteins. These results suggested that the early stages of acute pancreatitis involve changes of multiple aspects of RER functions including the synthesis and processing of digestive enzymes.