We investigated the impact of alcohol on the MDDC proteome using 2D electrophoresis with subsequent protein identification by mass spectrometry. Identified changes in DC protein expression were then compared with RT-PCR analyses of DC gene expression. Based on our criteria of a two-fold or greater change in expression, the results indicate that alcohol significantly regulates key components of the UPR stress-inducing pathways that include chaperones, ER stress antioxidant enzymes, proteolytic enzymes, as well as enzymes related to alcohol metabolism, cytoskeletal proteins, and proteins involved in apoptosis.
Among the de-regulated proteins in MDDC induced by alcohol are noticeable ER stress markers such as GRP78, PDI and ER-60. These proteins are involved in sensing and responding to the accumulation of unfolded or misfolded proteins in the ER. Since these three proteins are part of the UPR, our data support our hypothesis that alcohol activates this compensatory cellular defense mechanism. Another important up-regulated protein as identified by peptide mass fingerprinting is EIF2a, which is a key protein involved in the initiation of the UPR (Wek and Cavener, 2007
Upon the aggregation of unfolded proteins, GRP78 dissociates from the three ER stress receptors allowing them to dimerize, auto-phosphorylate, and thus become activated leading to repression of translation of other proteins relieving the accumulation of unfolded proteins (Boyce et al, 2005
; Scheuner et al, 2001
Among the proteins that play a crucial role in compensating for the stress of misfolded proteins, are chaperone proteins such as the heat shock protein, Hsp60. In the present study, this protein was up-regulated by alcohol treatment.
Several cytoskeletal proteins were found to be de-regulated by alcohol treatment in the MDDC proteome (actin, vimentin, tropomyosin and cofilin). Cytoskeletal changes after exposure to alcohol have been described in a number of cell types in adult rats and humans. Effects of alcohol on astrocytes induced a striking disorganization of the cytoskeleton. Since cytoskeletal integrity is a prerequisite for many cell functions, several studies have reported interactions between alcohol and cytoskeletal proteins (Tomás et al, 2003
). It has been reported that alcohol-induced changes in the actin cytoskeleton is mainly mediated by acetaldehyde, the main metabolite of alcohol oxidation. In addition, actin dynamics can also affect the cellular and behavioral responses of flies and mice in response to alcohol (Offenhäuser et al., 2006
). Our results demonstrated that cofilin, was found de-regulated in MDDC treated with alcohol.
We also observed glycolytic changes in the MDDC proteome following alcohol treatment. Pyruvate kinase, phosphoglycerate kinase, and alpha-enolase were up-regulated respectively 6.23, 2.66 and 2.37 fold. It has been reported that the targeting of Type II fibers by alcohol implicate glycolytic pathway lesions in myopathy and glycolytic insufficiency was attributed to the fact that Type II fibers undergo specific atrophy (Fernandez-Sola et al, 2007
). It was also noticed that in human muscle biopsies from chronic alcoholics there was a reduction in the pyruvate kinase activity before the first sign of myopathy appeared. Unlike the study mentioned above (investigating chronic ethanol effect) that lead to the down-regulation of pyruvate kinase, our data suggest that alcohol-induced up-regulation of pyruvate kinase and the glycolytic enzymes phosphoglycerate kinase and alpha-enolase might be the result of a protective cellular response triggered by the UPR mechanism to improve the glycolysis and energetic metabolism of DC status as a response to alcohol's deleterious effects.
Our results also showed a down-regulation of cyclophilin A expression following alcohol treatment in the MDDC proteome. Cyclophilin A also merits some discussion since this protein has a role in apoptosis (Bukrinsky 2002
; Ivery 2000
). Cyclophilin A is found in cell compartments where protein folding takes place, and complexes of cyclosporin A with cyclophilin A have been shown to inhibit calcineurin, a serine/threonine phosphatase inducing apoptosis (Ivery 2000
). Cyclophilin A has also been shown to participate in the activation of the caspase cascade in neuronal cells (Capano et al, 2002
Galectin 1 (Gal-1), a potent anti-inflammatory and immunoregulatory protein was triggered by alcohol treatment in our study as well. Evidence points to Gal-1 and its ligands as one of the master regulators of such immune responses as T cell homeostasis and survival, T cell immune disorders, inflammation and allergies as well as host–pathogen interactions (Camby et al, 2006
). Several lines of evidence indicate that Gal-1 may function as a pro-apoptotic factor inducing cell death of immature thymocytes and activated, but not resting, mature T cells (Rabinovich et al, 2000
) thus preserving homeostasis after the completion of an immune response. Gal-1 in its oxidized form plays a number of important roles in the regeneration of the CNS after injury (Horie et al, 2004
Alpha enolase was up-regulated by the alcohol treatment of MDDC. Alpha enolase activity with both isoenzyme forms: non-neuronal enolase (NNE) and neuron specific enolase (NSE) have been shown to be modified in many injuries related to the glycolytic pathways. Increased activity of SOD (superoxide dismutase), also found up-regulated in our study, and NSE in blood cells may be related to liver injury mainly in alcoholism while increased NNE activity may also be a marker of alcohol abuse without liver injury. Significant increase of SOD activity in alcoholic patients with liver injury and mainly in cirrhotic patients with ascitis have been reported. While we speculate in our study that SOD activates ER stress as shown in several studies, alpha enolase product likely reflects increased glycolysis in alcohol-induced cells.
Cathepsin-D, active in intracellular protein breakdown, is another key protein that was up-regulated in MDDC treated with alcohol (Benes et al, 2008
, Minarowska et al, 2008
). Cathepsin-D is known to be involved in the antigen processing capacity of MDDC (Mohamadzadeh et al, 2004
). As a protease, Cathepsin-D digests protein antigens that have been internalized by DC.
Cathepsin-B protease was up-regulated in MDDC induced with alcohol treatment. Macrophages and B cells have been reported to employ Cathepsins B, D, and/or E for digesting protein Ags.
We also observed that a protease known as cystatin B was up-regulated by alcohol treatment in DCs. Cystatin B (CSTB, also known as stefin B) may have an anti-apoptotic function in the cerebellum (Di Giaimo et al, 2002
). While the molecular basis of such function is not clear, CSTB may possibly protect the cells against inappropriate cellular degradation by proteases. It was also demonstrated that oxidative stress induces the expression of CSTB in cerebellar granule neurons and that the EPM1 (progressive myoclonus epilepsy type 1) patient-linked mutation of the cystatin B gene promoter impairs oxidative stress induction of CSTB transcription. CSTB knockout or knockdown sensitizes cerebellar granule neurons to oxidative stress-induced cell death. Interestingly, this CSTB deficiency-induced predisposition to oxidative stress in neurons is mediated by the lysosomal protease, cathepsin B (Lehtinen et al, 2009
). Since our findings reveal an oxidative stress response, reflected by differential expression of antioxidants such as superoxide dismutase and peroredoxin, as well as cathepsin B, with an induced UPR response triggered by alcohol in DC, we speculate that CSTB could modulate this specific pathway as a protective mechanism for DCs against oxidant injury and protein misfolding.
To summarize our discussion, we propose the model highlighted in . This model encompasses the alcohol-induced MDDC differentially expressed proteins detected in this study. After alcohol exposure, the MDDC becomes stressed due to the generation of high levels of reactive oxygen species (ROS), which interfere with proper protein folding in the ER, producing cellular stress and the synthesis of non-functional proteins. Alcohol oxidation, through aldehyde dehydrogenase (found up-regulated in our study), produces high levels of acetaldehyde, which is known to induce changes on the cytoskeleton. The stress generated by alcohol exposure promotes the increased expression of glycolytic enzymes (alpha enolase, pyruvate kinase, and phosphoglycerate kinase), proteolytic enzymes (cathepsin D and cathepsin B) and chaperones (Hsp60) to be able to generate energy and attenuate the accumulation of misfolded proteins under the UPR. One of the mechanisms activated by the cell to cope with the generated stress is to stop protein synthesis. The cell accomplishes this with the dissociation of GRP78 from stress receptors which results in a stoppage of protein synthesis and consequently the accumulation of misfolded proteins. We speculate that under a protective mechanism the cell will try to “eliminate” the misfolded proteins to evade the unfolded protein response (UPR)-activated apoptotic pathways induced by alcohol (). To cope with this apoptotic action, the MDDC then induces an anti-apoptotic machinery by down-regulating pro-apoptotic factors Gal-1 and cyclophilin A and by up-regulating anti-apoptotic proteins such as cystatin B to promote cell survival. In our study, interestingly, cyclophilin A another pro-apoptotic factor, was also found down-regulated in alcohol-induced DCs. Furthermore, this novel finding puts forward the evidence that alcohol induces in MDDC an unfolding response mediated by ER stress apoptotic as well as anti-apoptotic signals. For future studies, it will be interesting to further elucidate the Gal-1 apoptotic role and the putative anti-apoptotic proteins triggered by alcohol induction (such as cystatin B), as revealed in our study, to determine which specific mediated apoptosis response in alcohol-induced DCs (up- or down-regulation) would be triggered in non-constitutive receptors by Gal-1.
Proposed model highlighting UPR (unfolded protein response) pathway with pro-apoptotic and anti-apoptotic protein signatures triggered by ER stress in alcohol treated monocyte derived dendritic cell.
Overall, our findings revealed for the first time a UPR mechanism consistent with an ER-driven pathway activated through key ER stress and UPR proteins following alcohol treatment in DCs. It seems that activation of a UPR by alcohol may protect the DC from oxidant injury. Collectively, these data provide a plausible mechanistic rationale for alcohol action and suggest that the alcohol-inducible changes in the DC proteome could serve as potential biomarkers of therapeutic efficacy of reduced alcohol intake. Furthermore, alcohol-induced regulation of the UPR as well as pro- and anti-apoptotic protein signatures and the level of expression in DCs provide insight into targets and mechanistic strategies to prevent or alleviate alcohol abuse and dependence.