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Palmitic acid aggravates inflammation of pancreatic acinar cells by enhancing unfolded protein response induced CCAAT-enhancer-binding protein β–CCAAT-enhancer-binding protein α activation
The International Journal of Biochemistry & Cell Biology 79 (2016) 181–193
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Palmitic acid aggravates inflammation of pancreatic acinar cells by enhancing unfolded protein response induced CCAAT-enhancer-binding protein –CCAAT-enhancer-binding protein ␣ activation Jianghong Wu 1 , Guoyong Hu 1 , Yingying Lu 1 , Junyuan Zheng, Jing Chen, Xingpeng Wang ∗ , Yue Zeng ∗ Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China
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Article history: Received 12 March 2016 Received in revised form 10 August 2016 Accepted 29 August 2016 Available online 31 August 2016 Keywords: Acute pancreatitis Pancreatic acinar cells ER stress NF-B Inflammatory responses C/EBP
a b s t r a c t Hypertriglyceridemia is an independent risk factor for acute pancreatitis, in which the pathological mechanisms are not fully illustrated. Intracellular inflammatory response is a key pathological response in acute pancreatitis and endoplasmic reticulum stress has been suggested to induce inflammation and CCAAT-enhancer-binding protein expression. Therefore, the current study aims to elucidate the possible relationship between endoplasmic reticulum stress and inflammation in hypertriglyceridemia associated pancreatitis and the possible involvement of CCAAT-enhancer-binding protein. In cholecystokinin-8 stimulated rat primary acinar cells, incubation with palmitic acid caused the activation of endoplasmic reticulum stress and inflammatory responses. Pre-incubation with the chemical chaperone 4-phenylbutyric acid inhibited inflammatory responses induced by palmitic acid, whereas stimulation with the endoplasmic reticulum stress inducer thapsigargin alone induced inflammatory responses. Meanwhile we found that the transcription factors CCAAT-enhancer-binding protein ␣ and CCAATenhancer-binding protein  were also induced in the palmitic acid-stimulated pancreatic acinar cells, and were similarly inhibited by 4-phenylbutyric acid pre-incubation and induced by thapsigargin stimulation alone, indicating that endoplasmic reticulum stress was responsible for CCAAT-enhancer-binding protein ␣ and CCAAT-enhancer-binding protein  induction in the pancreatic acinar cells. Knockdown of CCAAT-enhancer-binding protein  by siRNA transfection inhibited inflammatory responses and CCAAT-enhancer-binding protein ␣ induction but did not affect endoplasmic reticulum stress. Our study provides strong evidence that in response to palmitic acid stimulation, endoplasmic reticulum stress induces inflammatory responses in pancreatic acinar cells through induction of the CCAATenhancer-binding protein family, wherein CCAAT-enhancer-binding protein  activation is responsible for CCAAT-enhancer-binding protein ␣ activation. © 2016 Elsevier Ltd. All rights reserved.
Abbreviations: AP, acute pancreatitis; ATF6, activating transcription factor 6; BSA, bovine serum albumin; CCK-8, cholecystokinin-8; C/EBP, CCAAT-enhancerbinding protein; CHOP, CCAAT/enhancer binding protein (C/EBP) homologous protein; eIF2, ␣eukaryotic translation initiation factor 2␣; EMSA, electrophoretic mobility shift assays; ER, endoplasmic reticulum; GRP78, glucose-related peptide 78; H&E, hematoxylin and eosin; HFD, high fat diet; HTG, hypertriglyceridemia; IB, I kappa B; IL-6, interleukin-6; IRE1, inositol-requiring ER-to-nucleus signal kinase 1; NFB, nuclear factor kappa B; PA, palmitic acid; PAC, pancreatic acinar cell; PERK, protein kinase–like ER kinase; 4-PBA, 4-phenylbutyric acid; SD, Sprague-Dawley; siRNA, small interfering RNA; TC, total cholesterol; TG, triglycerides; TNF-␣, tumor necrosis factor-alpha; UPR, unfolded protein response; WB, western blotting; XBP-1, X-box–binding protein 1. ∗ Corresponding authors. E-mail addresses: [email protected] (X. Wang), [email protected] (Y. Zeng). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.biocel.2016.08.035 1357-2725/© 2016 Elsevier Ltd. All rights reserved.
1. Introduction Acute pancreatitis (AP) is a sudden inflammatory disease of the pancreas, in which intracellular inflammatory response is a key pathological response. Hypertriglyceridemia (HTG) is an independent risk factor for acute pancreatitis and tends to lead to a more severe form of pancreatitis (Valdivielso et al., 2014). It is commonly accepted that free fatty acids released from excess triglycerides (TG) hydrolyzed by high levels of pancreatic lipase can damage pancreatic acinar cells (PACs) (Durgampudi et al., 2014; Noel et al., 2016). However, the mechanism by which HTG aggravates AP is poorly understood, which causes major problems in clinical treatment of the disease (Valdivielso et al., 2014). ER stress activation
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has also been observed in all models of experimentally induced pancreatitis and inhibition of ER stress with chemical chaperones, including 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid, has shown protective effects against pancreatic injury (Malo et al., 2013; Malo et al., 2010). Our previous in vivo results have shown that endoplasmic reticulum (ER) stress occurs in PACs in HTG-aggravated AP (Zeng et al., 2012). A major feature of the ER stress response is the unfolded protein response (UPR), which relies on a highly coordinated response involving three parallel signaling branches using the transmembrane proteins protein kinase–like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) (Bernales et al., 2006). In response to ER stress, these three transmembrane proteins are activated, in which activation of PERK pathway leads to phosphorylation of eukaryotic translation initiation factor 2␣ (eIF2␣), ATF6 (an active transcription factor) activation leads to expression of target genes including ER chaperone glucose-related peptide 78 (GRP78), and IRE1 activation catalyzes removal of a small intron from X-box–binding protein 1 (XBP1) mRNA (Danino et al., 2015). ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune diseases, infection, neurodegenerative disease, and metabolic disorders. All three signaling pathways of UPR have been shown to induce inflammation through nuclear factor-kappa B (NF-B) activation through different mechanisms (Hummasti and Hotamisligil, 2010). The crosstalk between inflammation and ER stress has been suggested to play a significant role in pancreatic  cell dysfunction (Oslowski et al., 2012; Zhang and Kaufman, 2008). However, the interaction between ER stress and inflammation in HTG-aggravated AP and acinar cell injury remains elusive. Further understanding of those issues may enable the development of novel therapies of HTG-aggravated AP. The CCAAT enhancer-binding protein (C/EBP) family of basic leucine-zipper transcription factors includes C/EBP␣, -, -␥, -␦, and -, and the ER stress gene C/EBP homology protein (CHOP), which must heterodimerize with other members of this family, most notably C/EBP, in order to function (Rahman et al., 2012; Ron and Habener, 1992). C/EBP performs diverse functions, participating in the regulation of genes that contribute to the acute phase response, glucose metabolism, tissue differentiation and inflammation (Matsuda et al., 2010; Matsuda et al., 2015; Ramji and Foka, 2002). Previous studies have shown that C/EBP expression is upregulated by stimuli that induce ERstress, such as exposure to thapsigargin, and inhibition of C/EBP expression reduced ER stress-associated cell failure and diseases (Matsuda et al., 2010; Matsuda et al., 2015). These findings suggest that C/EBP is central to the pathogenesis of metabolism-associated diseases. While C/EBP is a key regulator of inflammation, its pivotal role in the pathogenesis of AP remains relatively unexplored. This study was conducted to illustrate the relationship between ER stress and inflammation in HTG-aggravated AP and to identify the underlying mechanism.
phosphorylated eIF2␣ (#3398) were from Cell Signaling Technology (MA, USA). Antibodies against NF-B (#ab16502) and histone H3 (#ab8580) were from Abcam (Abcam, UK). Antibodies against eIF2␣ (#11233-1-AP), GRP78 (#11587-1-AP), TNF-␣ (#602911-Ig), IL-6 (#21865-1-AP) and IL-1 (#26048-1-AP) were from Proteintech Biotechnology (Wuhan, China). Fatty-acid-supplemented medium was prepared with slight modification of the protocol of Spector (Spector, 1986). Briefly, PA was dissolved in ethanol and then gently mixed until completely dissolved, after which the clear fatty acids solution was complexed with fatty-acid-free BSA at a fatty acid to BSA ratio of 1:10. The complex fatty acid solution was added to the serum-containing cell culture medium to obtain the indicated final PA concentration. The control (untreated) cells received the same vehicle solution but without PA.
2.2. Experimental animals and protocols All animal experiments were preformed according to the guidelines of Animal Care and Use Committee of Shanghai Jiaotong University. Male Sprague-Dawley rats (weighing 100–110 g), normal chow and the high-fat diet (HFD) chow (rodent regular chow diets supplemented with 20% lard and 3% cholesterol), were all purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Rats allocated to the HTG group were fed HFD for 2 weeks, whereas rats used as controls were fed with normal chow. AP model was induced by intraperitoneal injection of caerulein, at a dose of 50 g/kg body weight, two times with an interval of 1 h between injections. The control rats were given the same volume of vehicle solution at the same time point. All rats were sacrificed 9 h after AP induction. Blood samples were collected via the abdominal aorta. A portion of each pancreas was fixed in 10% formaldehyde in phosphate-buffered saline (PBS; pH7.4) and embedded in paraffin, and part of the pancreatic tissues were quickly frozen with liquid nitrogen and stored at −80 ◦ C until use.
2.3. Pancreatic acinar cells isolation and treatment PACs were isolated from mice by a collagenase digestion procedure as described previously (Hu et al., 2011). For treatments, the isolated PACs were incubated at 37 ◦ C in Dulbecco’s modified Eagle’s medium/Ham F-12 medium containing 20% fetal bovine serum and other agents as described below. In the PA-stimulated group, PACs were incubated with 0.1 mM PA for 1, 3, 6, 9, or 12 h prior to incubation with 20 pM CCK-8 (30 min); vehicle solution was used for the control group. In the 4-PBA group, PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently incubated with 0.1 mM PA or vehicle solution (control) for 6 h prior to incubation with 20 pM CCK-8 (30 min). In the thapsigargin-stimulated group, PACs were incubated with thapsigargin (0.1, 0.3, 1, or 2 M) for 6 h. In the C/EBP small interfering RNA (siRNA) transfection groups, PACs were incubated with 0.1 mM PA for 6 h after transfection.
2. Material and methods 2.1. Materials
2.4. Small interfering RNA transfection
Caerulein, palmitic acid (PA), cholecystokinin-8 (CCK-8), 4phenylbutyric acid (4-PBA) and thapsigargin were purchased from Sigma-Aldrich Chemical (MO, USA). Bovine serum albumin (BSA) was purchased from Roche (Basel, Switzerland). Antibodies against sXBP-1 (#sc-7160), C/EBP␣ (#sc-61), C/EBP (#sc-150) and -actin (#sc-81178) were from Santa Cruz Biotechnology (TX, USA). Antibodies against IB␣ (#4812), IB (#9248), CHOP (#2895) and
For siRNA transfections, primary rat PACs were transfected with 150 nM siRNA oligo-nucleotides using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. Transfected cells were tested for their response to PA at 24 h after the initiation of siRNA transfection. The siRNA sequences used for transfection are given in Supplementary Table S1 in the online version at DOI: 10.1016/j.biocel.2016.08.035.
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2.5. Histological examination of the pancreas Formaldehyde-embedded pancreas tissue was sectioned into 4-m thick sections and stained with hematoxylin and eosin. Histopathological evaluation was performed under a light microscope by two investigators who were blinded to the experimental treatment. Edema, necrosis, hemorrhage and inflammatory infiltration were evaluated in accordance with the scoring scale reported by Schmidt et al. (Schmidt et al., 1992).
2.6. Serum and plasma biochemistry Levels of serum triglycerides and total cholesterol, and levels of plasma amylase were determined using an automated biochemical analyzer according to the manufacturer’s protocols (Advia 1650; Bayer, Leverkusen, Germany).
2.7. Cell survival assay The cell survival assay was performed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, WI, USA) according to the manufacturer’s instructions. Briefly, approximately 100 l of cell suspension was added to the wells of a 96-well culture plate. The Cell Titer-Glo® Reagent was prepared by combining the Cell Titer Glo® Buffer with the Cell Titer Glo® Substrate. After adding 50 l of the Cell Titer-Glo® Reagent to each well of the 96-well culture plate and sitting the plate at room temperature for 10 min to stabilize the luminescence, ATP level was determined by detecting the luminescence with Synergy 2 Multi-Mode Reader (BioTek, USA).
2.8. Western blotting analysis Nuclear and cytoplasmic extracts were prepared using Nuclear and Cytoplasmic Extraction Reagents from Beyotime Biotechnology (Shanghai, China). Whole-cell extracts were obtained from PACs reconstituted in ice-cold RIPA buffer containing 1 mM phenylmethanesulfonyl fluoride and a cocktail of protease inhibitors. Samples were centrifuged at 10,000g for 15 min at 4 ◦ C. The supernatants were collected, and total protein content was determined using the BCA method (Beyotime Biotechnology, China). The total protein (50-g per sample) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, following which the protein bands were transferred to a nitrocellulose membrane that was then blotted using standard procedures. Tris-buffered saline containing 5% BSA was used to block non-specific binding. The membranes were then incubated overnight at 4 ◦ C with a primary antibody. Thereafter, the membranes were blocked with 5% fat-free milk and incubated overnight at 4 ◦ C with the following antibodies: sXBP-1 (1:200), C/EBP␣ (1:200), C/EBP (1:200), -actin (1:1000), NF-B (1:500), IB␣ (1:500), IB (1:500), phosphorylated eIF2␣ (1:500), eIF2␣ (1:500), GRP78 (1:500), CHOP (1:500), TNF-␣ (1:500), histone H3 (1:500), IL-6 (1:500) and IL-1 (1:500). Subsequently, membranes were washed with Tris-buffered saline containing 0.1% Triton X-100 and incubated with horseradishperoxidase-conjugated secondary anti-rabbit or anti-mouse IgG (1:2000) for 1 h at room temperature. After washing with TBST, the membranes were developed and exposed to ECL film (Santa Cruz). Finally, the membranes were washed three times and scanned using the Odyssey two-color infrared laser imaging system (fluorescence detection). Representative blots were presented from three independent experiments. Quantitative densitometric analyses of immunoblotting images were performed using ImageJ 1.44p software. The relative expression of proteins was expressed as fold
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changes compared to the control in the blot after normalized to -actin, histone H3, or the total proteins in some experiments.
2.9. Electrophoretic mobility shift assays Nuclear extracts were prepared with the Nuclear and Cytoplasmic Extraction Reagents from Pierce (IL, USA). The single-stranded 3 3end biotin-labeled probe containing the NF-B consensus site was purchased from Beyotime Biotechnology. The EMSA binding reactions were performed by utilizing a Light Shift chemiluminescent EMSA kit (Pierce).Specifically, 5 g of nuclear extracts was incubated in 1 × binding buffer containing 2.5% glycerol, 0.05% NP40, 50 mM KCl, 5 mM MgCl2, 50 ng of poly(dI–dC), and biotinylated probe with or without protein extracts for 30 min at room temperature. The complexes were separated on a 5% polyacrylamide-0.5× Tris-borate-EDTA gel and transferred to a positively charged nylon membrane. After the transfer was completed, the membrane was crosslinked and the biotin-labeled DNA was detected by using a chemiluminescent detection kit (Pierce).
2.10. Real time RT-PCR Total RNA was extracted from mice pancreas following the acid guanidinium/phenol/chloroform method in the presence of liquid nitrogen. After the purity of the RNA products was determined according to the 260/280 ratio to be between 1.6 and 2.0, reverse transcription was performed using the commercial SuperScript II preamplification kit (Fermentas, MD, USA). The synthesized cDNA was then subjected to RT-PCR. All the procedures were performed using ABI Prism 7900HT Sequence Detection System (Applied Biosystems, CA, USA). The mRNA expressions levels of target genes, including sXBP1, GRP78, CHOP, TNF-␣, IL-6, IL-1, C/EBP␣ and C/EBP, were normalized to the housekeeping gene -actin. Each target gene was analyzed in triplicate under each triplicate experiment. Fold changes of gene expression were then calculated and expressed as relative transcript levels (2). The primer sequences are listed in Table S2 in the online version at DOI: 10.1016/j.biocel.2016.08.035.
2.11. Statistics Data are presented as the mean ± SEM, representing replicates within an experiment. Statistical significance was determined by Student’s t-test. A P-value of less than 0.05 was considered significant.
3. Results 3.1. HTG aggravated pancreatic injury in AP Rats were fed a HFD for 2 weeks to establish the HTG model. Compared with that in the normal-diet group, serum TG and total cholesterol (TC) levels were significantly increased in the HFD group confirming the establishment of this HTG model (P < 0.05, Fig. 1A). Next, we established a caerulein AP model in HTG and normal-diet rats. Compared with that in the normal-diet group, the plasma amylase level and histological scores in the pancreatic tissue were significantly increased in the HTG group (P < 0.05, Fig. 1B–D). The inflammatory responses of pancreatitis tissue were more severe in the HTG group than in the normal-diet group, as indicated by western blot analysis of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ (Fig. 1E, F).
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Fig. 1. HTG aggravated AP in SD rats. (A) Rats were fed with HFD or normal chow for 2 weeks. TG and TC levels in the serum were determined to confirm the establishment of the HTG model. (B–E) HTG rats and control rats were given intraperitoneal injections of 50 g/kg caerulein or saline once every hour for 2 h. Nine hours after the last injection, rats were sacrificed and the samples of pancreatic tissues and plasma were harvested. Levels of plasma amylase were determined (B). The pancreatic injury was determined by hematoxylin and eosin staining in pancreatic tissue (C). Histopathological evaluation including edema, necrosis and inflammatory infiltration were scored (D). WB analysis of inflammatory cytokines in the pancreas tissue was determined (E, F). Normal diet vs. HFD, *P < 0.05; non-AP vs. AP, #P < 0.05.
3.2. ER stress and inflammatory signaling were increased in PA-stimulated PACs To confirm the induction of ER stress in PA-stimulated PACs and illustrate the activation of the three branches of UPR, rat primary PACs were isolated and cultured in the presence of PA to induce an in vitro model of HTG, and markers of ER stress were examined at the indicated time points. We observed activation of all three branches of UPR and ER stress, as indicated by the increase in splicing and activation of the transcription factor XBP1 in the IRE1 pathway, eIF2˛ phosphorylation in the PERK pathway, GRP78 transcriptional induction in the ATF6 pathway and CHOP induc-
tion (Fig. 2A–C). These data confirmed the induction of ER stress and UPR in PA-incubated primary PACs. In addition to ER stress, inflammatory signaling was also apparent during the same time course. Compared with that in the control group, PA treatment induced IB degradation as IB˛ and IBˇ protein levels in the cytoplasmic extract were reduced (Fig. 2D, E), NF-B activation evidenced by elevated levels of NF-B in the nuclear extract (Fig. 2F, G), and transcriptional induction of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ evidenced by increases in their mRNA and protein levels (Fig. 3A–C). The PA-induced NF-B activity was confirmed by electrophoretic mobility shift assay (EMSA), wherein PA treatment led to enhanced NF-B binding activity (Fig. 3D). As it is commonly
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Fig. 2. ER stress and inflammation were present in PA-stimulated rat primary PACs. Rat primary PACs were isolated and were stimulated with PA for indicated time (1/3/6/9/12 h), afterwards CCK-8 was added for 30 min for all groups. Rat primary PACs were collected at the indicated time-points and analyzed by WB analysis or qRTPCRanalysis. (A, B) WB analysis with anti-spliced-XBP-1, phosphorylated eIF2␣, total eIF2␣, GRP78, CHOP or -actin antibodies on the whole-cell extract from rat primary PACs lysates showed that PA induced ER stress. (C) qRT-PCR data of mRNA expression showed that the PA induced ER stress. (D-G) WB analysis with anti-IB␣ and anti-IB antibodies on the cytoplasm extract (D, E), and anti-NF-B and anti-histone H3 (histone) antibodies on the nuclear extract (F, G) showed that PA induced inflammatory responses. PA vs. control, *P < 0.05.
accepted that necrosis is responsible for inflammatory responses in AP, we next examined the ability of PA to induce necrosis in rat primary PACs by determining the total ATP level in the cells.
Our results showed that addition of PA for 6 h, at which time ER stress and inflammation were induced, didn’t trigger PAC necrosis (Fig. 3E).
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Fig. 3. Inflammation was present but necrosis absent in PA-stimulated rat primary PACs. Rat primary PACs were isolated and were stimulated with PA for indicated time (1/3/6/9/12 h), afterwards CCK-8 was added for 30 min for all groups. Rat primary PACs were collected at the indicated time-points and analyzed by WB analysis, qRT-PCR or cell survival assay. (A–C) WB analysis of TNF-␣, IL-6 or IL-1 expression on whole cell extracts and qRT-PCR data of mRNA expression showed that the PA induced inflammatory cytokines induction. (D) 5 g of nuclear extract were subjected to electrophoretic mobility shift assay (EMSA) using a DNA probe containing NF-B binding elements showed that PA induced NF-B binding ability. (E) Rat primary PACs were stimulated with PA for 6 h, afterwards CCK-8 was added for 30 min for all groups. Cell viability was determined by measuring ATP levels using Cell Titer-Glo kit. PA vs. control, *P < 0.05.
3.3. ER stress was responsible for inflammatory responses in PA-stimulated PACs Multiple investigations have shown that ER stress is responsible for lipid-induced inflammatory responses. To confirm the correlation between ER stress and inflammatory responses in PA-stimulated PACs, we selected an intermediate PA incubation period (6 h; when ER stress and inflammatory responses were activated) and investigated changes in inflammatory response after ER stress inhibition by pre-incubation with 4-phenylbutyric acid (4-PBA) or ER stress induction with thapsigargin alone. Pre-incubation with 4-PBA (an exogenous chemical chaperone specifically used to inhibit ER stress) effectively prevented ER stress in PA-stimulated PACs. Western blot analysis showed that 4-PBA pre-incubation inhibited PA-induced XBP-1 splicing, phosphorylation of eIF2a, GRP78 and CHOP induction (Fig. 4A, B). Next, we determined the efficacy of 4-PBA in inflammation. 4-PBA pre-incubation inhibited IB degradation, as evidenced by elevation of IB˛ and IBˇ protein levels in the cytoplasmic extract
(Fig. 4C, D); NF-B inhibition, as evidenced by reduced concentrations of NF-B in the nuclear extract (Fig. 4E, F); and inhibition of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ, as evidenced by their reduced protein levels in the whole-cell extracts (Fig. 4G, H). To further confirm the relationship between ER stress and inflammation, we stimulated PACs with thapsigargin, a known ER stress inducer. Thapsigargin stimulation alone activated all three branches of UPR, as evidenced by the increases in XBP-1 splicing, phosphorylation of eIF2a, GRP78 production and CHOP expression (Fig. 5A–C). Next, we determined the effect of thapsigargin on inflammation, where it was found to increase IB˛ and IBˇ degradation (Fig. 5D, E), NF-B activation (Fig. 5F, G), and inflammatory cytokine production including TNF-˛, IL-6 and IL-1ˇ (Fig. 5H–J).
3.4. C/EBP˛ and C/EBPˇ activation was dependent on ER stress in PA-stimulated PACs To test the possible involvement of C/EBP in ER stress-induced inflammation in PA-stimulated PACs, we first analyzed the changes
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Fig. 4. ER stress inhibition reduced inflammation in PA-stimulated PACs. Primary isolated rat PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently were incubated in the presence or absence of PA for 6 h, afterwards CCK-8 was added in all groups for 30 min. ER stress was determined by WB analysis of whole cell extracts (A, B) and inflammatory responses was determined by IBs protein expression of plasma extracts (C, D), NF-B and histone H3 (histone) protein expression of nuclear extracts (E, F), and inflammatory cytokines TNF-␣, IL-6 or IL-1 expressions of whole cell extracts (G, H). PA vs. control, *P < 0.05; 4-PBA + PA vs. PA, #P < 0.05.
of C/EBP˛ and C/EBPˇ expression. PA stimulation significantly increased C/EBP˛ and C/EBPˇ activation as higher amounts of both proteins were detected in the nuclear extract (Fig. 6A, B). The mRNA expression of C/EBP˛ and C/EBPˇ was increased in PA-stimulated rat primary PACs (Fig. 6C), indicating that the activation of C/EBP˛ and C/EBPˇ may involve transcriptional enhancement. Next, we tested whether ER stress could stimulate C/EBP˛ and C/EBPˇ activation by detecting their expression changes after inhibition or induction of ER stress. Inhibition of ER stress by 4-PBA pre-incubation led to reduced C/EBP˛ and C/EBPˇ expression in the nuclear extract of PA-stimulated PACs (Fig. 6D, E). However, induction of ER stress with thapsigargin stimulation alone led to elevated C/EBP˛ and C/EBPˇ protein levels in the whole-cell extract (Fig. 6F, G) and nuclear extract (Fig. 6H, I) and also enhanced mRNA expression (Fig. 6J).
3.5. C/EBPˇ inhibition reduced C/EBP˛ activation and inflammation but had no effect on ER stress in PA-stimulated PACs Previous reports have demonstrated that inflammation induced by HFD was driven by C/EBP signaling pathway (Rahman et al., 2012). To further evaluate the role of C/EBPˇ in PAC inflammation and ER stress, we transfected the cells with C/EBPˇ small interfering RNA (siRNA) or control siRNA. In our preliminary studies, we used three pairs of C/EBPˇ siRNA. Fluorescent microscopy showed that siRNA was successfully transfected (Fig. 7A). Western blot analysis showed that C/EBPˇ#1 siRNA had the highest efficacy of inhibition (Fig. 7B, C) and this siRNA was chosen for our later experiments. Further studies showed that C/EBPˇ siRNA transfection had effectively inhibited C/EBPˇ protein expression in whole-cell extract (Fig. 7D, E) and nuclear extract (Fig. 7F, G) and mRNA expression (Fig. 7H). In accordance with the fact that C/EBP˛ acts as a
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Fig. 5. ER stress induction aggravated inflammation in rat primary PACs. Primary isolated rat PACs were treated with thapsigargin (Tg; 0.1, 0.3, 1, or 2 M) for 6 h followed by WB and qRT-PCR analysis. ER stress was determined by WB analysis of whole-cell extract (A, B) and qRT-PCR analysis (C). Inflammatory responses was determined by IBs protein expression of plasma extracts (D, E), NF-B protein and histone H3 (histone) expression of nuclear extract (F, G), and inflammatory cytokines TNF-␣, IL-6 or IL-1 expressions of whole-cell extract (H–J). Tg vs. control, *P < 0.05.
downstream target of C/EBPˇ (Zuo et al., 2006), the inhibition of C/EBPˇ inhibited C/EBP˛ protein expression in whole-cell extract (Fig. 7D, E) and nuclear extract (Fig. 7F, G) and mRNA expression (Fig. 7H). Inhibition of C/EBPˇ exerted no effect on ER stress, as the three branches of UPR were not significantly changed, which agrees with the interpretation that C/EBPˇ acts downstream of ER stress (Fig. 8A–C). Compared with the control transfection, transfection of siRNA targeting C/EBPˇ led to decreased IB degradation
(Fig. 8D, E), NF-B activation (Fig. 8F, G), and inflammatory cytokine production (Fig. 8H–J) in PA-stimulated PACs. 4. Discussion The well-recognized inflammatory responses activated by ER stress likely represent a conserved immune response (Zuo et al., 2006), but their possible relationship with AP was confirmed for the
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Fig. 6. C/EBP activation in PA-stimulated PACs was dependent on ER stress. (A–C) Primary isolated rat PACs were cultured in the presence or absence of PA for indicated time (1/3/6/9/12 h) and afterwards CCK-8 was added for 30 min for all groups. Nuclear extract and RNA were harvested for C/EBP␣ and C/EBP detection by WB analysis (A, B) and qRT-PCR (C). (D, E) Primary isolated rat PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently were incubated in the presence or absence of PA for 6 h, afterwards CCK-8 was added in all groups for 30 min. Nuclear extracts of rat primary PACs were collected at the indicated time points and subjected to WB analysis. (F–J) Primary isolated rat PACs were treated with thapsigargin (Tg; 0.1, 0.3, 1, or 2 M) for 6 h followed by WB in the whole-cell extract (F, G) and nuclear extract (H, I) and qRT-PCR analysis (J). PA vs. control, *P < 0.05; 4-PBA + PA vs. PA, #P < 0.05; Tg vs. control, *P < 0.05.
first time in our study (Fig. 9). We also found that transcriptional factors C/EBP˛ and C/EBPˇ mediated inflammation in response to ER stress. In our study, we first confirmed the activation of ER stress and inflammatory responses in PA-induced PAC injury. Inhibition of ER stress by 4-PBA pre-incubation led to a reduction of inflam-
mation induced by PA stimulation, whereas induction of ER stress by thapsigargin stimulation alone led to an activation of inflammation, indicating that ER stress contributed to inflammation in PA-induced PAC injury. C/EBP˛ and C/EBPˇ are key transcription factors that were activated in PA-stimulated PACs and depen-
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Fig. 7. C/EBP inhibition reduced C/EBP␣ activation in PA-stimulated PACs. (A) Picture of light scope and fluorescent after C/EBP siRNA transfection. (B, C) C/EBP siRNA transfection successfully inhibited C/EBP expression. Rat PACs were transfected three pairs of C/EBP or control siRNA for 24 h. Whole cell extracts were harvested for WB analysis detecting total C/EBP level. (D-H) Rat PACs were transfected C/EBP or control siRNA for 24 h. Transfected cells were cultured in the presence or absence of PA for 6 h and afterwards CCK-8 was added for 30 min for all groups. For C/EBP activation, whole-cell extract, nuclear extract and RNA were harvested for C/EBP␣ and C/EBP detection by WB analysis in the whole cell (D, E), in the nuclear (F, G) and qRT-PCR (H). PA vs. control, *P < 0.05; C/EBP-siRNA vs. NC-siRNA, #P < 0.05.
dent on ER stress. Further studies with C/EBPˇ siRNA transfection showed that C/EBPˇ was responsible for C/EBP˛ activation and inflammation, but it exerted no effect on ER stress. Our results indicated that in PA-induced PAC injury, ER stress activation led to inflammatory responses through C/EBPˇ-C/EBP˛ axis. HTG is caused by many factors such as overeating, consumption of HFD and alcohol abuse, and clinical studies have found that HTG aggravates the episodes of severe AP. In accordance with the deleterious role of HTG in aggravating AP, our results showed that HTG increased the severity of rat caerulein-induced AP model, and that AP inflammation in pancreatitis tissues of HTG rats was more intense than that of rats with normal lipid. Inflammatory cells infiltrating the pancreas have been suggested to be the main cells that release inflammatory cytokines, but reports have found that PACs (accounting for the largest number of cells in the pancreas) are the main cells in producing inflammatory cytokines and inducing inflammation. In the present study, 0.1 mM PA activated inflammation through activation of NF-B by IB˛ and IBˇ degradation, leading to production of TNF-˛, IL-6 and IL-1ˇ.
Inflammatory responses secondary to PAC necrosis have been suggested to be the main mechanism initiating inflammation in AP (Gukovsky et al., 2013; Zheng et al., 2013). PA has been shown to induce necrosis in PACs, possibly through ATP depletion (Samad et al., 2014; Voronina et al., 2010). To test the possible involvement of necrosis in initiating inflammatory responses in PACs, we evaluated the development of necrosis in PA-stimulated PACs. However, 6-h stimulation with 0.1 mM PA, which induced inflammation in rat PACs, failed to trigger necrosis. The disparity of PA in being unable to trigger necrosis but able to induce inflammation may reflect concentration-specific effects. However, the inability of 0.1 mM PA to trigger rat PAC necrosis confirmed that inflammatory responses in PA-stimulated PACs are not secondary to necrosis. The exocrine pancreas is the organ with the highest level of protein synthesis (e.g., of digestive enzymes) with high amount and right character to match the dietary intake (Logsdon and Ji, 2013). When PACs are subjected to high levels of stress such as pathological stimulation and consumption of HFD, ER stress ensues as a result of an inability to maintain the proper and sufficient protein folding. Evidence of ER stress is present in all models of experi-
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Fig. 8. C/EBP inhibition reduced inflammation but exerted no efficacy on ER stress in PA-stimulated PACs. Rat PACs were transfected C/EBP or control siRNA for 24 h. Transfected cells were cultured in the presence or absence of PA for 6 h and afterwards CCK-8 was added for 30 min for all groups. ER stress was detected by WB analysis with anti-spliced-XBP-1, phosphorylated eIF2␣, total eIF2␣, GRP78, CHOP and -actin antibodies on the whole cell from rat primary PACs lysates (A, B) and qR-PCR data of mRNA expression (C). Inflammation was determined by WB analysis with anti-IB␣ and anti-IB antibodies on the cytoplasm extract (D, E), anti-NF-B and anti-histone H3 (histone) antibodies on the nuclear extract (F, G), and anti-TNF-␣, IL-6 or IL-1 antibodies on the whole-cell extract (H, I) and qRT-PCR data of mRNA expression (J). PA vs. control, *P < 0.05; C/EBP-siRNA vs. NC-siRNA, #P < 0.05.
mentally induced pancreatitis (Logsdon and Ji, 2013). Our previous studies confirmed the activation of ER stress in HTG pancreatitis in vivo (Zeng et al., 2012). The PA released from TG lipolysis has been suggested to be the main toxic reagents in AP (Durgampudi et al., 2014; Noel et al., 2016) and has been shown to activate ER
stress in rat PAC cell line AR42J (Danino et al., 2015). Our in vitro study showed that PA-stimulated primary isolated PACs showed ER stress. Our further experiments detailed the activation of all three branches of UPR, and found that ER stress and the UPR branches were all activated in PA-stimulated rat primary PACs.
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Fig. 9. Mechanism of hypertriglyceridemia aggravated acute pancreatitis. In HTG aggravated AP, ER stress and inflammatory responses were activated in which ER stress was responsible for inflammatory responses in PA stimulated pancreatic acinar cells. C/EBP␣ and C/EBP were involved in HTG-aggravated AP and they were responsible for inflammation induced by ER stress.
ER stress has been suggested to be a mechanism for inducing inflammation in metabolism and inflammatory diseases (Cao and Kaufman, 2013; Cao et al., 2016). All three UPR branches have the potency in activation of NF-B, the key regulator for immune and inflammatory responses, through different mechanisms (Kitamura, 2011). Under ER stress, IRE1 forms a complex with IKK through TRAF2, which mediated NF-B activation through phosphorylation of IKK and degradation of IB˛ (Kaneko et al., 2003). PERK-induced phosphorylation of eIF2˛ is essential for activation of NF-B by ER stress through the direct effect of eIF2˛ in global translational suppression (Jiang et al., 2003). Activation of ATF6 pathway induces Akt phosphorylation and NF-B activation (Yamazaki et al., 2009). To evaluate the possible relationship between ER stress and inflammation, we detected inflammation changes after ER stress inhibition or induction. Pre-incubation with 4-PBA addition effectively inhibited NF-B activation, IB degradation, and cytokine production, whereas thapsigargin activated the inflammatory response, confirming that ER stress induces inflammation in PA-induced PAC injury. C/EBP˛ and C/EBPˇ, key factors in inducing inflammation, were activated by ER stress (Rahman et al., 2012). The first clue that suggested a possible role for C/EBP˛ and C/EBPˇ in pancreatic injury derived from in vitro studies showing increased activity of these transcription factors during the induction of inflammation and ER stress in PACs. The present in vitro studies showed that PA was capable of increasing the mRNA and nuclear protein expression of C/EBP˛ and C/EBPˇ in rat primary PACs. These findings are consistent with previous studies indicating that C/EBP˛ and C/EBPˇ are induced under lipid stimulation (Rahman et al., 2012; van der Krieken et al., 2015).
In vitro studies have shown that C/EBPˇ expression is upregulated by stimuli that induce ER stress, such as exposure to thapsigargin (Matsuda et al., 2010; Matsuda et al., 2015). We have shown that the expression of C/EBP˛ and C/EBPˇ in rat primary acinar cells was also induced by thapsigargin. In addition, we have demonstrated that the activation of C/EBP˛ and C/EBPˇ resulted from ER stress, as 4-PBA reduced the PA-stimulated activation of C/EBP˛ and C/EBPˇ. Inhibition of C/EBPˇ with C/EBPˇ siRNA transfection exerted no effect on ER stress activation and UPR branches, which further supports that C/EBPˇ acts as downstream targets of ER stress. C/EBPˇ has been previously shown to regulate inflammation in inflammatory cells and adipose tissue (Rahman et al., 2012; van der Krieken et al., 2015). We have shown here that excessive accumulation of C/EBPˇ aggravated inflammatory responses of PA-stimulated PACs. Activation of the inflammatory responses was markedly inhibited by inhibition of C/EBPˇ via C/EBPˇ siRNA transfection in PA-stimulated PACs. We also showed that C/EBP˛ acts as a down-stream target of C/EBPˇ, as the inhibition of C/EBPˇ with C/EBPˇ siRNA transfection led to the reduced activation of C/EBP˛ in PA-stimulated acinar cells. 5. Conclusions Our data provides a new important insight into the cellular and molecular mechanism into the pathogenesis of HTG aggravated pancreatitis. Our finding indicates that in PA-induced PAC injury, ER stress induces inflammatory responses through induction of C/EBP family in which C/EBPˇ activation is responsible for C/EBP˛ activation. The most novel finding is that we find ER stress is
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responsible for inflammatory responses in PA-stimulated pancreatic acinar cells. C/EBP˛ and C/EBPˇ are involved in HTG-related AP and are responsible for inflammation induced by ER stress. Conflict of interest No conflicts of interest were declared. Author contributions Substantial contributions to conception and design: Yue Zeng, Xingpeng Wang, Jianghong Wu, Guoyong Hu, Yingying Lu; acquisition of data: Jianghong Wu, Junyuan Zheng, Jing Chen; analysis and interpretation of data: Jianghong Wu, Junyuan Zheng, Jing Chen; drafting the article: Yue Zeng, Jianghong Wu; final approval of the version; Yue Zeng, Xingpeng Wang. Acknowledgements This work was supported by funding from Natural Science Foundation for Young Scholars of China (No. 81200322), Shanghai Natural Science Foundation (No. 12ZR1424000), and Open Project of Shanghai Key Laboratory of Pancreatic Diseases (No. SPDF2013004). References Bernales, S., Papa, F.R., Walter, P., 2006. Intracellular signaling by the unfolded protein response. Annu. Rev. Cell. Dev. Biol. 22, 487–508. Cao, S.S., Kaufman, R.J., 2013. Targeting endoplasmic reticulum stress in metabolic disease. Expert Opin. Ther. Targets 17, 437–448. Cao, S.S., Luo, K.L., Shi, L., 2016. Endoplasmic reticulum stress interacts with inflammation in human diseases. J. Cell. Physiol. 231, 288–294. Danino, H., Ben-Dror, K., Birk, R., 2015. Exocrine pancreas ER stress is differentially induced by different fatty acids. Exp. Cell Res. 339, 397–406. Durgampudi, C., Noel, P., Patel, K., Cline, R., Trivedi, R.N., DeLany, J.P., Yadav, D., Papachristou, G.I., Lee, K., Acharya, C., Jaligama, D., Navina, S., Murad, F., Singh, V.P., 2014. Acute lipotoxicity regulates severity of biliary acute pancreatitis without affecting its initiation. Am. J. Pathol. 184, 1773–1784. Gukovsky, I., Li, N., Todoric, J., Gukovskaya, A., Karin, M., 2013. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer. Gastroenterology 144, 1199–1209. Hu, G., Shen, J., Cheng, L., Guo, C., Xu, X., Wang, F., Huang, L., Yang, L., He, M., Xiang, D., Zhu, S., Wu, M., Yu, Y., Han, W., Wang, X., 2011. Reg4 protects against acinar cell necrosis in experimental pancreatitis. Gut 60, 820–828. Hummasti, S., Hotamisligil, G.S., 2010. Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circ. Res. 107, 579–591. Jiang, H.Y., Wek, S.A., McGrath, B.C., Scheuner, D., Kaufman, R.J., Cavener, D.R., Wek, R.C., 2003. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses. Mol. Cell. Biol. 23, 5651–5663. Kaneko, M., Niinuma, Y., Nomura, Y., 2003. Activation signal of nuclear factor-kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. Biol. Pharm. Bull. 26, 931–935. Kitamura, M., 2011. Control of NF-kappaB and inflammation by the unfolded protein response. Int. Rev. Immunol. 30, 4–15. Logsdon, C.D., Ji, B., 2013. The role of protein synthesis and digestive enzymes in acinar cell injury. Nat. Rev. Gastroenterol. Hepatol. 10, 362–370. Malo, A., Kruger, B., Seyhun, E., Schafer, C., Hoffmann, R.T., Goke, B., Kubisch, C.H., 2010. Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini. Am. J. Physiol. Liver Physiol. 299, G877–G886.
193
Malo, A., Kruger, B., Goke, B., Kubisch, C.H., 2013. 4-Phenylbutyric acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini. Pancreas 42, 92–101. Matsuda, T., Kido, Y., Asahara, S., Kaisho, T., Tanaka, T., Hashimoto, N., Shigeyama, Y., Takeda, A., Inoue, T., Shibutani, Y., Koyanagi, M., Hosooka, T., Matsumoto, M., Inoue, H., Uchida, T., Koike, M., Uchiyama, Y., Akira, S., Kasuga, M., 2010. Ablation of C/EBPbeta alleviates ER stress and pancreatic beta cell failure through the GRP78 chaperone in mice. J. Clin. Invest. 120, 115–126. Matsuda, T., Takahashi, H., Mieda, Y., Shimizu, S., Kawamoto, T., Matsuura, Y., Takai, T., Suzuki, E., Kanno, A., Koyanagi-Kimura, M., Asahara, S., Bartolome, A., Yokoi, N., Inoue, H., Ogawa, W., Seino, S., Kido, Y., 2015. Regulation of pancreatic beta cell mass by cross-interaction between CCAAT enhancer binding protein beta induced by endoplasmic reticulum stress and AMP-activated protein kinase activity. PLoS One 10, e130757. Noel, P., Patel, K., Durgampudi, C., Trivedi, R.N., de Oliveira, C., Crowell, M.D., Pannala, R., Lee, K., Brand, R., Chennat, J., Slivka, A., Papachristou, G.I., Khalid, A., Whitcomb, D.C., De Lany, J.P., Cline, R.A., Acharya, C., Jaligama, D., Murad, F.M., Yadav, D., Navina, S., Singh, V.P., 2016. Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections. Gut 65, 100–111. Oslowski, C.M., Hara, T., O’Sullivan-Murphy, B., Kanekura, K., Lu, S., Hara, M., Ishigaki, S., Zhu, L.J., Hayashi, E., Hui, S.T., Greiner, D., Kaufman, R.J., Bortell, R., Urano, F., 2012. Thioredoxin-interacting protein mediates ER stress-induced beta cell death through initiation of the inflammasome. Cell Metab. 16, 265–273. Rahman, S.M., Janssen, R.C., Choudhury, M., Baquero, K.C., Aikens, R.M., de la Houssaye, B.A., Friedman, J.E., 2012. CCAAT/enhancer-binding protein beta (C/EBPbeta) expression regulates dietary-induced inflammation in macrophages and adipose tissue in mice. J. Biol. Chem. 287, 34349–34360. Ramji, D.P., Foka, P., 2002. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365, 561–575. Ron, D., Habener, J.F., 1992. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 6, 439–453. Samad, A., James, A., Wong, J., Mankad, P., Whitehouse, J., Patel, W., Alves-Simoes, M., Siriwardena, A.K., Bruce, J.I., 2014. Insulin protects pancreatic acinar cells from palmitoleic acid-induced cellular injury. J. Biol. Chem. 289, 23582–23595. Schmidt, J., Rattner, D.W., Lewandrowski, K., Compton, C.C., Mandavilli, U., Knoefel, W.T., Warshaw, A.L., 1992. A better model of acute pancreatitis for evaluating therapy. Ann. Surg. 215, 44–56. Spector, A.A., 1986. Structure and lipid binding properties of serum albumin. Methods Enzymol. 128, 320–339. Valdivielso, P., Ramirez-Bueno, A., Ewald, N., 2014. Current knowledge of hypertriglyceridemic pancreatitis. Eur. J. Intern. Med. 25, 689–694. Voronina, S.G., Barrow, S.L., Simpson, A.W., Gerasimenko, O.V., Da, S.X.G., Rutter, G.A., Petersen, O.H., Tepikin, A.V., 2010. Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells. Gastroenterology 138, 1976–1987. Yamazaki, H., Hiramatsu, N., Hayakawa, K., Tagawa, Y., Okamura, M., Ogata, R., Huang, T., Nakajima, S., Yao, J., Paton, A.W., Paton, J.C., Kitamura, M., 2009. Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response. J. Immunol. 183, 1480–1487. Zeng, Y., Wang, X., Zhang, W., Wu, K., Ma, J., 2012. Hypertriglyceridemia aggravates ER stress and pathogenesis of acute pancreatitis. Hepatogastroenterology 59, 2318–2326. Zhang, K., Kaufman, R.J., 2008. From endoplasmic-reticulum stress to the inflammatory response. Nature 454, 455–462. Zheng, L., Xue, J., Jaffee, E.M., Habtezion, A., 2013. Role of immune cells and immune-based therapies in pancreatitis and pancreatic ductal adenocarcinoma. Gastroenterology 144, 1230–1240. Zuo, Y., Qiang, L., Farmer, S.R., 2006. Activation of CCAAT/enhancer-binding protein (C/EBP) alpha expression by C/EBP beta during adipogenesis requires a peroxisome proliferator-activated receptor-gamma-associated repression of HDAC1 at the C/ebp alpha gene promoter. J. Biol. Chem. 281, 7960–7967. van der Krieken, S.E., Popeijus, H.E., Mensink, R.P., Plat, J., 2015. CCAAT/Enhancer binding protein ˇ in relation to ER stress, inflammation, and metabolic disturbances. Biomed. Res. Int. 2015, 1–13.
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Palmitic acid aggravates inflammation of pancreatic acinar cells by enhancing unfolded protein response induced CCAAT-enhancer-binding protein –CCAAT-enhancer-binding protein ␣ activation Jianghong Wu 1 , Guoyong Hu 1 , Yingying Lu 1 , Junyuan Zheng, Jing Chen, Xingpeng Wang ∗ , Yue Zeng ∗ Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 12 March 2016 Received in revised form 10 August 2016 Accepted 29 August 2016 Available online 31 August 2016 Keywords: Acute pancreatitis Pancreatic acinar cells ER stress NF-B Inflammatory responses C/EBP
a b s t r a c t Hypertriglyceridemia is an independent risk factor for acute pancreatitis, in which the pathological mechanisms are not fully illustrated. Intracellular inflammatory response is a key pathological response in acute pancreatitis and endoplasmic reticulum stress has been suggested to induce inflammation and CCAAT-enhancer-binding protein expression. Therefore, the current study aims to elucidate the possible relationship between endoplasmic reticulum stress and inflammation in hypertriglyceridemia associated pancreatitis and the possible involvement of CCAAT-enhancer-binding protein. In cholecystokinin-8 stimulated rat primary acinar cells, incubation with palmitic acid caused the activation of endoplasmic reticulum stress and inflammatory responses. Pre-incubation with the chemical chaperone 4-phenylbutyric acid inhibited inflammatory responses induced by palmitic acid, whereas stimulation with the endoplasmic reticulum stress inducer thapsigargin alone induced inflammatory responses. Meanwhile we found that the transcription factors CCAAT-enhancer-binding protein ␣ and CCAATenhancer-binding protein  were also induced in the palmitic acid-stimulated pancreatic acinar cells, and were similarly inhibited by 4-phenylbutyric acid pre-incubation and induced by thapsigargin stimulation alone, indicating that endoplasmic reticulum stress was responsible for CCAAT-enhancer-binding protein ␣ and CCAAT-enhancer-binding protein  induction in the pancreatic acinar cells. Knockdown of CCAAT-enhancer-binding protein  by siRNA transfection inhibited inflammatory responses and CCAAT-enhancer-binding protein ␣ induction but did not affect endoplasmic reticulum stress. Our study provides strong evidence that in response to palmitic acid stimulation, endoplasmic reticulum stress induces inflammatory responses in pancreatic acinar cells through induction of the CCAATenhancer-binding protein family, wherein CCAAT-enhancer-binding protein  activation is responsible for CCAAT-enhancer-binding protein ␣ activation. © 2016 Elsevier Ltd. All rights reserved.
Abbreviations: AP, acute pancreatitis; ATF6, activating transcription factor 6; BSA, bovine serum albumin; CCK-8, cholecystokinin-8; C/EBP, CCAAT-enhancerbinding protein; CHOP, CCAAT/enhancer binding protein (C/EBP) homologous protein; eIF2, ␣eukaryotic translation initiation factor 2␣; EMSA, electrophoretic mobility shift assays; ER, endoplasmic reticulum; GRP78, glucose-related peptide 78; H&E, hematoxylin and eosin; HFD, high fat diet; HTG, hypertriglyceridemia; IB, I kappa B; IL-6, interleukin-6; IRE1, inositol-requiring ER-to-nucleus signal kinase 1; NFB, nuclear factor kappa B; PA, palmitic acid; PAC, pancreatic acinar cell; PERK, protein kinase–like ER kinase; 4-PBA, 4-phenylbutyric acid; SD, Sprague-Dawley; siRNA, small interfering RNA; TC, total cholesterol; TG, triglycerides; TNF-␣, tumor necrosis factor-alpha; UPR, unfolded protein response; WB, western blotting; XBP-1, X-box–binding protein 1. ∗ Corresponding authors. E-mail addresses: [email protected] (X. Wang), [email protected] (Y. Zeng). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.biocel.2016.08.035 1357-2725/© 2016 Elsevier Ltd. All rights reserved.
1. Introduction Acute pancreatitis (AP) is a sudden inflammatory disease of the pancreas, in which intracellular inflammatory response is a key pathological response. Hypertriglyceridemia (HTG) is an independent risk factor for acute pancreatitis and tends to lead to a more severe form of pancreatitis (Valdivielso et al., 2014). It is commonly accepted that free fatty acids released from excess triglycerides (TG) hydrolyzed by high levels of pancreatic lipase can damage pancreatic acinar cells (PACs) (Durgampudi et al., 2014; Noel et al., 2016). However, the mechanism by which HTG aggravates AP is poorly understood, which causes major problems in clinical treatment of the disease (Valdivielso et al., 2014). ER stress activation
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has also been observed in all models of experimentally induced pancreatitis and inhibition of ER stress with chemical chaperones, including 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid, has shown protective effects against pancreatic injury (Malo et al., 2013; Malo et al., 2010). Our previous in vivo results have shown that endoplasmic reticulum (ER) stress occurs in PACs in HTG-aggravated AP (Zeng et al., 2012). A major feature of the ER stress response is the unfolded protein response (UPR), which relies on a highly coordinated response involving three parallel signaling branches using the transmembrane proteins protein kinase–like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) (Bernales et al., 2006). In response to ER stress, these three transmembrane proteins are activated, in which activation of PERK pathway leads to phosphorylation of eukaryotic translation initiation factor 2␣ (eIF2␣), ATF6 (an active transcription factor) activation leads to expression of target genes including ER chaperone glucose-related peptide 78 (GRP78), and IRE1 activation catalyzes removal of a small intron from X-box–binding protein 1 (XBP1) mRNA (Danino et al., 2015). ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune diseases, infection, neurodegenerative disease, and metabolic disorders. All three signaling pathways of UPR have been shown to induce inflammation through nuclear factor-kappa B (NF-B) activation through different mechanisms (Hummasti and Hotamisligil, 2010). The crosstalk between inflammation and ER stress has been suggested to play a significant role in pancreatic  cell dysfunction (Oslowski et al., 2012; Zhang and Kaufman, 2008). However, the interaction between ER stress and inflammation in HTG-aggravated AP and acinar cell injury remains elusive. Further understanding of those issues may enable the development of novel therapies of HTG-aggravated AP. The CCAAT enhancer-binding protein (C/EBP) family of basic leucine-zipper transcription factors includes C/EBP␣, -, -␥, -␦, and -, and the ER stress gene C/EBP homology protein (CHOP), which must heterodimerize with other members of this family, most notably C/EBP, in order to function (Rahman et al., 2012; Ron and Habener, 1992). C/EBP performs diverse functions, participating in the regulation of genes that contribute to the acute phase response, glucose metabolism, tissue differentiation and inflammation (Matsuda et al., 2010; Matsuda et al., 2015; Ramji and Foka, 2002). Previous studies have shown that C/EBP expression is upregulated by stimuli that induce ERstress, such as exposure to thapsigargin, and inhibition of C/EBP expression reduced ER stress-associated cell failure and diseases (Matsuda et al., 2010; Matsuda et al., 2015). These findings suggest that C/EBP is central to the pathogenesis of metabolism-associated diseases. While C/EBP is a key regulator of inflammation, its pivotal role in the pathogenesis of AP remains relatively unexplored. This study was conducted to illustrate the relationship between ER stress and inflammation in HTG-aggravated AP and to identify the underlying mechanism.
phosphorylated eIF2␣ (#3398) were from Cell Signaling Technology (MA, USA). Antibodies against NF-B (#ab16502) and histone H3 (#ab8580) were from Abcam (Abcam, UK). Antibodies against eIF2␣ (#11233-1-AP), GRP78 (#11587-1-AP), TNF-␣ (#602911-Ig), IL-6 (#21865-1-AP) and IL-1 (#26048-1-AP) were from Proteintech Biotechnology (Wuhan, China). Fatty-acid-supplemented medium was prepared with slight modification of the protocol of Spector (Spector, 1986). Briefly, PA was dissolved in ethanol and then gently mixed until completely dissolved, after which the clear fatty acids solution was complexed with fatty-acid-free BSA at a fatty acid to BSA ratio of 1:10. The complex fatty acid solution was added to the serum-containing cell culture medium to obtain the indicated final PA concentration. The control (untreated) cells received the same vehicle solution but without PA.
2.2. Experimental animals and protocols All animal experiments were preformed according to the guidelines of Animal Care and Use Committee of Shanghai Jiaotong University. Male Sprague-Dawley rats (weighing 100–110 g), normal chow and the high-fat diet (HFD) chow (rodent regular chow diets supplemented with 20% lard and 3% cholesterol), were all purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Rats allocated to the HTG group were fed HFD for 2 weeks, whereas rats used as controls were fed with normal chow. AP model was induced by intraperitoneal injection of caerulein, at a dose of 50 g/kg body weight, two times with an interval of 1 h between injections. The control rats were given the same volume of vehicle solution at the same time point. All rats were sacrificed 9 h after AP induction. Blood samples were collected via the abdominal aorta. A portion of each pancreas was fixed in 10% formaldehyde in phosphate-buffered saline (PBS; pH7.4) and embedded in paraffin, and part of the pancreatic tissues were quickly frozen with liquid nitrogen and stored at −80 ◦ C until use.
2.3. Pancreatic acinar cells isolation and treatment PACs were isolated from mice by a collagenase digestion procedure as described previously (Hu et al., 2011). For treatments, the isolated PACs were incubated at 37 ◦ C in Dulbecco’s modified Eagle’s medium/Ham F-12 medium containing 20% fetal bovine serum and other agents as described below. In the PA-stimulated group, PACs were incubated with 0.1 mM PA for 1, 3, 6, 9, or 12 h prior to incubation with 20 pM CCK-8 (30 min); vehicle solution was used for the control group. In the 4-PBA group, PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently incubated with 0.1 mM PA or vehicle solution (control) for 6 h prior to incubation with 20 pM CCK-8 (30 min). In the thapsigargin-stimulated group, PACs were incubated with thapsigargin (0.1, 0.3, 1, or 2 M) for 6 h. In the C/EBP small interfering RNA (siRNA) transfection groups, PACs were incubated with 0.1 mM PA for 6 h after transfection.
2. Material and methods 2.1. Materials
2.4. Small interfering RNA transfection
Caerulein, palmitic acid (PA), cholecystokinin-8 (CCK-8), 4phenylbutyric acid (4-PBA) and thapsigargin were purchased from Sigma-Aldrich Chemical (MO, USA). Bovine serum albumin (BSA) was purchased from Roche (Basel, Switzerland). Antibodies against sXBP-1 (#sc-7160), C/EBP␣ (#sc-61), C/EBP (#sc-150) and -actin (#sc-81178) were from Santa Cruz Biotechnology (TX, USA). Antibodies against IB␣ (#4812), IB (#9248), CHOP (#2895) and
For siRNA transfections, primary rat PACs were transfected with 150 nM siRNA oligo-nucleotides using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. Transfected cells were tested for their response to PA at 24 h after the initiation of siRNA transfection. The siRNA sequences used for transfection are given in Supplementary Table S1 in the online version at DOI: 10.1016/j.biocel.2016.08.035.
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2.5. Histological examination of the pancreas Formaldehyde-embedded pancreas tissue was sectioned into 4-m thick sections and stained with hematoxylin and eosin. Histopathological evaluation was performed under a light microscope by two investigators who were blinded to the experimental treatment. Edema, necrosis, hemorrhage and inflammatory infiltration were evaluated in accordance with the scoring scale reported by Schmidt et al. (Schmidt et al., 1992).
2.6. Serum and plasma biochemistry Levels of serum triglycerides and total cholesterol, and levels of plasma amylase were determined using an automated biochemical analyzer according to the manufacturer’s protocols (Advia 1650; Bayer, Leverkusen, Germany).
2.7. Cell survival assay The cell survival assay was performed using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, WI, USA) according to the manufacturer’s instructions. Briefly, approximately 100 l of cell suspension was added to the wells of a 96-well culture plate. The Cell Titer-Glo® Reagent was prepared by combining the Cell Titer Glo® Buffer with the Cell Titer Glo® Substrate. After adding 50 l of the Cell Titer-Glo® Reagent to each well of the 96-well culture plate and sitting the plate at room temperature for 10 min to stabilize the luminescence, ATP level was determined by detecting the luminescence with Synergy 2 Multi-Mode Reader (BioTek, USA).
2.8. Western blotting analysis Nuclear and cytoplasmic extracts were prepared using Nuclear and Cytoplasmic Extraction Reagents from Beyotime Biotechnology (Shanghai, China). Whole-cell extracts were obtained from PACs reconstituted in ice-cold RIPA buffer containing 1 mM phenylmethanesulfonyl fluoride and a cocktail of protease inhibitors. Samples were centrifuged at 10,000g for 15 min at 4 ◦ C. The supernatants were collected, and total protein content was determined using the BCA method (Beyotime Biotechnology, China). The total protein (50-g per sample) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, following which the protein bands were transferred to a nitrocellulose membrane that was then blotted using standard procedures. Tris-buffered saline containing 5% BSA was used to block non-specific binding. The membranes were then incubated overnight at 4 ◦ C with a primary antibody. Thereafter, the membranes were blocked with 5% fat-free milk and incubated overnight at 4 ◦ C with the following antibodies: sXBP-1 (1:200), C/EBP␣ (1:200), C/EBP (1:200), -actin (1:1000), NF-B (1:500), IB␣ (1:500), IB (1:500), phosphorylated eIF2␣ (1:500), eIF2␣ (1:500), GRP78 (1:500), CHOP (1:500), TNF-␣ (1:500), histone H3 (1:500), IL-6 (1:500) and IL-1 (1:500). Subsequently, membranes were washed with Tris-buffered saline containing 0.1% Triton X-100 and incubated with horseradishperoxidase-conjugated secondary anti-rabbit or anti-mouse IgG (1:2000) for 1 h at room temperature. After washing with TBST, the membranes were developed and exposed to ECL film (Santa Cruz). Finally, the membranes were washed three times and scanned using the Odyssey two-color infrared laser imaging system (fluorescence detection). Representative blots were presented from three independent experiments. Quantitative densitometric analyses of immunoblotting images were performed using ImageJ 1.44p software. The relative expression of proteins was expressed as fold
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changes compared to the control in the blot after normalized to -actin, histone H3, or the total proteins in some experiments.
2.9. Electrophoretic mobility shift assays Nuclear extracts were prepared with the Nuclear and Cytoplasmic Extraction Reagents from Pierce (IL, USA). The single-stranded 3 3end biotin-labeled probe containing the NF-B consensus site was purchased from Beyotime Biotechnology. The EMSA binding reactions were performed by utilizing a Light Shift chemiluminescent EMSA kit (Pierce).Specifically, 5 g of nuclear extracts was incubated in 1 × binding buffer containing 2.5% glycerol, 0.05% NP40, 50 mM KCl, 5 mM MgCl2, 50 ng of poly(dI–dC), and biotinylated probe with or without protein extracts for 30 min at room temperature. The complexes were separated on a 5% polyacrylamide-0.5× Tris-borate-EDTA gel and transferred to a positively charged nylon membrane. After the transfer was completed, the membrane was crosslinked and the biotin-labeled DNA was detected by using a chemiluminescent detection kit (Pierce).
2.10. Real time RT-PCR Total RNA was extracted from mice pancreas following the acid guanidinium/phenol/chloroform method in the presence of liquid nitrogen. After the purity of the RNA products was determined according to the 260/280 ratio to be between 1.6 and 2.0, reverse transcription was performed using the commercial SuperScript II preamplification kit (Fermentas, MD, USA). The synthesized cDNA was then subjected to RT-PCR. All the procedures were performed using ABI Prism 7900HT Sequence Detection System (Applied Biosystems, CA, USA). The mRNA expressions levels of target genes, including sXBP1, GRP78, CHOP, TNF-␣, IL-6, IL-1, C/EBP␣ and C/EBP, were normalized to the housekeeping gene -actin. Each target gene was analyzed in triplicate under each triplicate experiment. Fold changes of gene expression were then calculated and expressed as relative transcript levels (2). The primer sequences are listed in Table S2 in the online version at DOI: 10.1016/j.biocel.2016.08.035.
2.11. Statistics Data are presented as the mean ± SEM, representing replicates within an experiment. Statistical significance was determined by Student’s t-test. A P-value of less than 0.05 was considered significant.
3. Results 3.1. HTG aggravated pancreatic injury in AP Rats were fed a HFD for 2 weeks to establish the HTG model. Compared with that in the normal-diet group, serum TG and total cholesterol (TC) levels were significantly increased in the HFD group confirming the establishment of this HTG model (P < 0.05, Fig. 1A). Next, we established a caerulein AP model in HTG and normal-diet rats. Compared with that in the normal-diet group, the plasma amylase level and histological scores in the pancreatic tissue were significantly increased in the HTG group (P < 0.05, Fig. 1B–D). The inflammatory responses of pancreatitis tissue were more severe in the HTG group than in the normal-diet group, as indicated by western blot analysis of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ (Fig. 1E, F).
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Fig. 1. HTG aggravated AP in SD rats. (A) Rats were fed with HFD or normal chow for 2 weeks. TG and TC levels in the serum were determined to confirm the establishment of the HTG model. (B–E) HTG rats and control rats were given intraperitoneal injections of 50 g/kg caerulein or saline once every hour for 2 h. Nine hours after the last injection, rats were sacrificed and the samples of pancreatic tissues and plasma were harvested. Levels of plasma amylase were determined (B). The pancreatic injury was determined by hematoxylin and eosin staining in pancreatic tissue (C). Histopathological evaluation including edema, necrosis and inflammatory infiltration were scored (D). WB analysis of inflammatory cytokines in the pancreas tissue was determined (E, F). Normal diet vs. HFD, *P < 0.05; non-AP vs. AP, #P < 0.05.
3.2. ER stress and inflammatory signaling were increased in PA-stimulated PACs To confirm the induction of ER stress in PA-stimulated PACs and illustrate the activation of the three branches of UPR, rat primary PACs were isolated and cultured in the presence of PA to induce an in vitro model of HTG, and markers of ER stress were examined at the indicated time points. We observed activation of all three branches of UPR and ER stress, as indicated by the increase in splicing and activation of the transcription factor XBP1 in the IRE1 pathway, eIF2˛ phosphorylation in the PERK pathway, GRP78 transcriptional induction in the ATF6 pathway and CHOP induc-
tion (Fig. 2A–C). These data confirmed the induction of ER stress and UPR in PA-incubated primary PACs. In addition to ER stress, inflammatory signaling was also apparent during the same time course. Compared with that in the control group, PA treatment induced IB degradation as IB˛ and IBˇ protein levels in the cytoplasmic extract were reduced (Fig. 2D, E), NF-B activation evidenced by elevated levels of NF-B in the nuclear extract (Fig. 2F, G), and transcriptional induction of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ evidenced by increases in their mRNA and protein levels (Fig. 3A–C). The PA-induced NF-B activity was confirmed by electrophoretic mobility shift assay (EMSA), wherein PA treatment led to enhanced NF-B binding activity (Fig. 3D). As it is commonly
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Fig. 2. ER stress and inflammation were present in PA-stimulated rat primary PACs. Rat primary PACs were isolated and were stimulated with PA for indicated time (1/3/6/9/12 h), afterwards CCK-8 was added for 30 min for all groups. Rat primary PACs were collected at the indicated time-points and analyzed by WB analysis or qRTPCRanalysis. (A, B) WB analysis with anti-spliced-XBP-1, phosphorylated eIF2␣, total eIF2␣, GRP78, CHOP or -actin antibodies on the whole-cell extract from rat primary PACs lysates showed that PA induced ER stress. (C) qRT-PCR data of mRNA expression showed that the PA induced ER stress. (D-G) WB analysis with anti-IB␣ and anti-IB antibodies on the cytoplasm extract (D, E), and anti-NF-B and anti-histone H3 (histone) antibodies on the nuclear extract (F, G) showed that PA induced inflammatory responses. PA vs. control, *P < 0.05.
accepted that necrosis is responsible for inflammatory responses in AP, we next examined the ability of PA to induce necrosis in rat primary PACs by determining the total ATP level in the cells.
Our results showed that addition of PA for 6 h, at which time ER stress and inflammation were induced, didn’t trigger PAC necrosis (Fig. 3E).
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Fig. 3. Inflammation was present but necrosis absent in PA-stimulated rat primary PACs. Rat primary PACs were isolated and were stimulated with PA for indicated time (1/3/6/9/12 h), afterwards CCK-8 was added for 30 min for all groups. Rat primary PACs were collected at the indicated time-points and analyzed by WB analysis, qRT-PCR or cell survival assay. (A–C) WB analysis of TNF-␣, IL-6 or IL-1 expression on whole cell extracts and qRT-PCR data of mRNA expression showed that the PA induced inflammatory cytokines induction. (D) 5 g of nuclear extract were subjected to electrophoretic mobility shift assay (EMSA) using a DNA probe containing NF-B binding elements showed that PA induced NF-B binding ability. (E) Rat primary PACs were stimulated with PA for 6 h, afterwards CCK-8 was added for 30 min for all groups. Cell viability was determined by measuring ATP levels using Cell Titer-Glo kit. PA vs. control, *P < 0.05.
3.3. ER stress was responsible for inflammatory responses in PA-stimulated PACs Multiple investigations have shown that ER stress is responsible for lipid-induced inflammatory responses. To confirm the correlation between ER stress and inflammatory responses in PA-stimulated PACs, we selected an intermediate PA incubation period (6 h; when ER stress and inflammatory responses were activated) and investigated changes in inflammatory response after ER stress inhibition by pre-incubation with 4-phenylbutyric acid (4-PBA) or ER stress induction with thapsigargin alone. Pre-incubation with 4-PBA (an exogenous chemical chaperone specifically used to inhibit ER stress) effectively prevented ER stress in PA-stimulated PACs. Western blot analysis showed that 4-PBA pre-incubation inhibited PA-induced XBP-1 splicing, phosphorylation of eIF2a, GRP78 and CHOP induction (Fig. 4A, B). Next, we determined the efficacy of 4-PBA in inflammation. 4-PBA pre-incubation inhibited IB degradation, as evidenced by elevation of IB˛ and IBˇ protein levels in the cytoplasmic extract
(Fig. 4C, D); NF-B inhibition, as evidenced by reduced concentrations of NF-B in the nuclear extract (Fig. 4E, F); and inhibition of inflammatory cytokines TNF-˛, IL-6 and IL-1ˇ, as evidenced by their reduced protein levels in the whole-cell extracts (Fig. 4G, H). To further confirm the relationship between ER stress and inflammation, we stimulated PACs with thapsigargin, a known ER stress inducer. Thapsigargin stimulation alone activated all three branches of UPR, as evidenced by the increases in XBP-1 splicing, phosphorylation of eIF2a, GRP78 production and CHOP expression (Fig. 5A–C). Next, we determined the effect of thapsigargin on inflammation, where it was found to increase IB˛ and IBˇ degradation (Fig. 5D, E), NF-B activation (Fig. 5F, G), and inflammatory cytokine production including TNF-˛, IL-6 and IL-1ˇ (Fig. 5H–J).
3.4. C/EBP˛ and C/EBPˇ activation was dependent on ER stress in PA-stimulated PACs To test the possible involvement of C/EBP in ER stress-induced inflammation in PA-stimulated PACs, we first analyzed the changes
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Fig. 4. ER stress inhibition reduced inflammation in PA-stimulated PACs. Primary isolated rat PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently were incubated in the presence or absence of PA for 6 h, afterwards CCK-8 was added in all groups for 30 min. ER stress was determined by WB analysis of whole cell extracts (A, B) and inflammatory responses was determined by IBs protein expression of plasma extracts (C, D), NF-B and histone H3 (histone) protein expression of nuclear extracts (E, F), and inflammatory cytokines TNF-␣, IL-6 or IL-1 expressions of whole cell extracts (G, H). PA vs. control, *P < 0.05; 4-PBA + PA vs. PA, #P < 0.05.
of C/EBP˛ and C/EBPˇ expression. PA stimulation significantly increased C/EBP˛ and C/EBPˇ activation as higher amounts of both proteins were detected in the nuclear extract (Fig. 6A, B). The mRNA expression of C/EBP˛ and C/EBPˇ was increased in PA-stimulated rat primary PACs (Fig. 6C), indicating that the activation of C/EBP˛ and C/EBPˇ may involve transcriptional enhancement. Next, we tested whether ER stress could stimulate C/EBP˛ and C/EBPˇ activation by detecting their expression changes after inhibition or induction of ER stress. Inhibition of ER stress by 4-PBA pre-incubation led to reduced C/EBP˛ and C/EBPˇ expression in the nuclear extract of PA-stimulated PACs (Fig. 6D, E). However, induction of ER stress with thapsigargin stimulation alone led to elevated C/EBP˛ and C/EBPˇ protein levels in the whole-cell extract (Fig. 6F, G) and nuclear extract (Fig. 6H, I) and also enhanced mRNA expression (Fig. 6J).
3.5. C/EBPˇ inhibition reduced C/EBP˛ activation and inflammation but had no effect on ER stress in PA-stimulated PACs Previous reports have demonstrated that inflammation induced by HFD was driven by C/EBP signaling pathway (Rahman et al., 2012). To further evaluate the role of C/EBPˇ in PAC inflammation and ER stress, we transfected the cells with C/EBPˇ small interfering RNA (siRNA) or control siRNA. In our preliminary studies, we used three pairs of C/EBPˇ siRNA. Fluorescent microscopy showed that siRNA was successfully transfected (Fig. 7A). Western blot analysis showed that C/EBPˇ#1 siRNA had the highest efficacy of inhibition (Fig. 7B, C) and this siRNA was chosen for our later experiments. Further studies showed that C/EBPˇ siRNA transfection had effectively inhibited C/EBPˇ protein expression in whole-cell extract (Fig. 7D, E) and nuclear extract (Fig. 7F, G) and mRNA expression (Fig. 7H). In accordance with the fact that C/EBP˛ acts as a
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Fig. 5. ER stress induction aggravated inflammation in rat primary PACs. Primary isolated rat PACs were treated with thapsigargin (Tg; 0.1, 0.3, 1, or 2 M) for 6 h followed by WB and qRT-PCR analysis. ER stress was determined by WB analysis of whole-cell extract (A, B) and qRT-PCR analysis (C). Inflammatory responses was determined by IBs protein expression of plasma extracts (D, E), NF-B protein and histone H3 (histone) expression of nuclear extract (F, G), and inflammatory cytokines TNF-␣, IL-6 or IL-1 expressions of whole-cell extract (H–J). Tg vs. control, *P < 0.05.
downstream target of C/EBPˇ (Zuo et al., 2006), the inhibition of C/EBPˇ inhibited C/EBP˛ protein expression in whole-cell extract (Fig. 7D, E) and nuclear extract (Fig. 7F, G) and mRNA expression (Fig. 7H). Inhibition of C/EBPˇ exerted no effect on ER stress, as the three branches of UPR were not significantly changed, which agrees with the interpretation that C/EBPˇ acts downstream of ER stress (Fig. 8A–C). Compared with the control transfection, transfection of siRNA targeting C/EBPˇ led to decreased IB degradation
(Fig. 8D, E), NF-B activation (Fig. 8F, G), and inflammatory cytokine production (Fig. 8H–J) in PA-stimulated PACs. 4. Discussion The well-recognized inflammatory responses activated by ER stress likely represent a conserved immune response (Zuo et al., 2006), but their possible relationship with AP was confirmed for the
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Fig. 6. C/EBP activation in PA-stimulated PACs was dependent on ER stress. (A–C) Primary isolated rat PACs were cultured in the presence or absence of PA for indicated time (1/3/6/9/12 h) and afterwards CCK-8 was added for 30 min for all groups. Nuclear extract and RNA were harvested for C/EBP␣ and C/EBP detection by WB analysis (A, B) and qRT-PCR (C). (D, E) Primary isolated rat PACs were pre-incubated with 4-PBA (2.5, 5, 10, or 20 M) for 30 min and subsequently were incubated in the presence or absence of PA for 6 h, afterwards CCK-8 was added in all groups for 30 min. Nuclear extracts of rat primary PACs were collected at the indicated time points and subjected to WB analysis. (F–J) Primary isolated rat PACs were treated with thapsigargin (Tg; 0.1, 0.3, 1, or 2 M) for 6 h followed by WB in the whole-cell extract (F, G) and nuclear extract (H, I) and qRT-PCR analysis (J). PA vs. control, *P < 0.05; 4-PBA + PA vs. PA, #P < 0.05; Tg vs. control, *P < 0.05.
first time in our study (Fig. 9). We also found that transcriptional factors C/EBP˛ and C/EBPˇ mediated inflammation in response to ER stress. In our study, we first confirmed the activation of ER stress and inflammatory responses in PA-induced PAC injury. Inhibition of ER stress by 4-PBA pre-incubation led to a reduction of inflam-
mation induced by PA stimulation, whereas induction of ER stress by thapsigargin stimulation alone led to an activation of inflammation, indicating that ER stress contributed to inflammation in PA-induced PAC injury. C/EBP˛ and C/EBPˇ are key transcription factors that were activated in PA-stimulated PACs and depen-
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Fig. 7. C/EBP inhibition reduced C/EBP␣ activation in PA-stimulated PACs. (A) Picture of light scope and fluorescent after C/EBP siRNA transfection. (B, C) C/EBP siRNA transfection successfully inhibited C/EBP expression. Rat PACs were transfected three pairs of C/EBP or control siRNA for 24 h. Whole cell extracts were harvested for WB analysis detecting total C/EBP level. (D-H) Rat PACs were transfected C/EBP or control siRNA for 24 h. Transfected cells were cultured in the presence or absence of PA for 6 h and afterwards CCK-8 was added for 30 min for all groups. For C/EBP activation, whole-cell extract, nuclear extract and RNA were harvested for C/EBP␣ and C/EBP detection by WB analysis in the whole cell (D, E), in the nuclear (F, G) and qRT-PCR (H). PA vs. control, *P < 0.05; C/EBP-siRNA vs. NC-siRNA, #P < 0.05.
dent on ER stress. Further studies with C/EBPˇ siRNA transfection showed that C/EBPˇ was responsible for C/EBP˛ activation and inflammation, but it exerted no effect on ER stress. Our results indicated that in PA-induced PAC injury, ER stress activation led to inflammatory responses through C/EBPˇ-C/EBP˛ axis. HTG is caused by many factors such as overeating, consumption of HFD and alcohol abuse, and clinical studies have found that HTG aggravates the episodes of severe AP. In accordance with the deleterious role of HTG in aggravating AP, our results showed that HTG increased the severity of rat caerulein-induced AP model, and that AP inflammation in pancreatitis tissues of HTG rats was more intense than that of rats with normal lipid. Inflammatory cells infiltrating the pancreas have been suggested to be the main cells that release inflammatory cytokines, but reports have found that PACs (accounting for the largest number of cells in the pancreas) are the main cells in producing inflammatory cytokines and inducing inflammation. In the present study, 0.1 mM PA activated inflammation through activation of NF-B by IB˛ and IBˇ degradation, leading to production of TNF-˛, IL-6 and IL-1ˇ.
Inflammatory responses secondary to PAC necrosis have been suggested to be the main mechanism initiating inflammation in AP (Gukovsky et al., 2013; Zheng et al., 2013). PA has been shown to induce necrosis in PACs, possibly through ATP depletion (Samad et al., 2014; Voronina et al., 2010). To test the possible involvement of necrosis in initiating inflammatory responses in PACs, we evaluated the development of necrosis in PA-stimulated PACs. However, 6-h stimulation with 0.1 mM PA, which induced inflammation in rat PACs, failed to trigger necrosis. The disparity of PA in being unable to trigger necrosis but able to induce inflammation may reflect concentration-specific effects. However, the inability of 0.1 mM PA to trigger rat PAC necrosis confirmed that inflammatory responses in PA-stimulated PACs are not secondary to necrosis. The exocrine pancreas is the organ with the highest level of protein synthesis (e.g., of digestive enzymes) with high amount and right character to match the dietary intake (Logsdon and Ji, 2013). When PACs are subjected to high levels of stress such as pathological stimulation and consumption of HFD, ER stress ensues as a result of an inability to maintain the proper and sufficient protein folding. Evidence of ER stress is present in all models of experi-
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Fig. 8. C/EBP inhibition reduced inflammation but exerted no efficacy on ER stress in PA-stimulated PACs. Rat PACs were transfected C/EBP or control siRNA for 24 h. Transfected cells were cultured in the presence or absence of PA for 6 h and afterwards CCK-8 was added for 30 min for all groups. ER stress was detected by WB analysis with anti-spliced-XBP-1, phosphorylated eIF2␣, total eIF2␣, GRP78, CHOP and -actin antibodies on the whole cell from rat primary PACs lysates (A, B) and qR-PCR data of mRNA expression (C). Inflammation was determined by WB analysis with anti-IB␣ and anti-IB antibodies on the cytoplasm extract (D, E), anti-NF-B and anti-histone H3 (histone) antibodies on the nuclear extract (F, G), and anti-TNF-␣, IL-6 or IL-1 antibodies on the whole-cell extract (H, I) and qRT-PCR data of mRNA expression (J). PA vs. control, *P < 0.05; C/EBP-siRNA vs. NC-siRNA, #P < 0.05.
mentally induced pancreatitis (Logsdon and Ji, 2013). Our previous studies confirmed the activation of ER stress in HTG pancreatitis in vivo (Zeng et al., 2012). The PA released from TG lipolysis has been suggested to be the main toxic reagents in AP (Durgampudi et al., 2014; Noel et al., 2016) and has been shown to activate ER
stress in rat PAC cell line AR42J (Danino et al., 2015). Our in vitro study showed that PA-stimulated primary isolated PACs showed ER stress. Our further experiments detailed the activation of all three branches of UPR, and found that ER stress and the UPR branches were all activated in PA-stimulated rat primary PACs.
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Fig. 9. Mechanism of hypertriglyceridemia aggravated acute pancreatitis. In HTG aggravated AP, ER stress and inflammatory responses were activated in which ER stress was responsible for inflammatory responses in PA stimulated pancreatic acinar cells. C/EBP␣ and C/EBP were involved in HTG-aggravated AP and they were responsible for inflammation induced by ER stress.
ER stress has been suggested to be a mechanism for inducing inflammation in metabolism and inflammatory diseases (Cao and Kaufman, 2013; Cao et al., 2016). All three UPR branches have the potency in activation of NF-B, the key regulator for immune and inflammatory responses, through different mechanisms (Kitamura, 2011). Under ER stress, IRE1 forms a complex with IKK through TRAF2, which mediated NF-B activation through phosphorylation of IKK and degradation of IB˛ (Kaneko et al., 2003). PERK-induced phosphorylation of eIF2˛ is essential for activation of NF-B by ER stress through the direct effect of eIF2˛ in global translational suppression (Jiang et al., 2003). Activation of ATF6 pathway induces Akt phosphorylation and NF-B activation (Yamazaki et al., 2009). To evaluate the possible relationship between ER stress and inflammation, we detected inflammation changes after ER stress inhibition or induction. Pre-incubation with 4-PBA addition effectively inhibited NF-B activation, IB degradation, and cytokine production, whereas thapsigargin activated the inflammatory response, confirming that ER stress induces inflammation in PA-induced PAC injury. C/EBP˛ and C/EBPˇ, key factors in inducing inflammation, were activated by ER stress (Rahman et al., 2012). The first clue that suggested a possible role for C/EBP˛ and C/EBPˇ in pancreatic injury derived from in vitro studies showing increased activity of these transcription factors during the induction of inflammation and ER stress in PACs. The present in vitro studies showed that PA was capable of increasing the mRNA and nuclear protein expression of C/EBP˛ and C/EBPˇ in rat primary PACs. These findings are consistent with previous studies indicating that C/EBP˛ and C/EBPˇ are induced under lipid stimulation (Rahman et al., 2012; van der Krieken et al., 2015).
In vitro studies have shown that C/EBPˇ expression is upregulated by stimuli that induce ER stress, such as exposure to thapsigargin (Matsuda et al., 2010; Matsuda et al., 2015). We have shown that the expression of C/EBP˛ and C/EBPˇ in rat primary acinar cells was also induced by thapsigargin. In addition, we have demonstrated that the activation of C/EBP˛ and C/EBPˇ resulted from ER stress, as 4-PBA reduced the PA-stimulated activation of C/EBP˛ and C/EBPˇ. Inhibition of C/EBPˇ with C/EBPˇ siRNA transfection exerted no effect on ER stress activation and UPR branches, which further supports that C/EBPˇ acts as downstream targets of ER stress. C/EBPˇ has been previously shown to regulate inflammation in inflammatory cells and adipose tissue (Rahman et al., 2012; van der Krieken et al., 2015). We have shown here that excessive accumulation of C/EBPˇ aggravated inflammatory responses of PA-stimulated PACs. Activation of the inflammatory responses was markedly inhibited by inhibition of C/EBPˇ via C/EBPˇ siRNA transfection in PA-stimulated PACs. We also showed that C/EBP˛ acts as a down-stream target of C/EBPˇ, as the inhibition of C/EBPˇ with C/EBPˇ siRNA transfection led to the reduced activation of C/EBP˛ in PA-stimulated acinar cells. 5. Conclusions Our data provides a new important insight into the cellular and molecular mechanism into the pathogenesis of HTG aggravated pancreatitis. Our finding indicates that in PA-induced PAC injury, ER stress induces inflammatory responses through induction of C/EBP family in which C/EBPˇ activation is responsible for C/EBP˛ activation. The most novel finding is that we find ER stress is
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responsible for inflammatory responses in PA-stimulated pancreatic acinar cells. C/EBP˛ and C/EBPˇ are involved in HTG-related AP and are responsible for inflammation induced by ER stress. Conflict of interest No conflicts of interest were declared. Author contributions Substantial contributions to conception and design: Yue Zeng, Xingpeng Wang, Jianghong Wu, Guoyong Hu, Yingying Lu; acquisition of data: Jianghong Wu, Junyuan Zheng, Jing Chen; analysis and interpretation of data: Jianghong Wu, Junyuan Zheng, Jing Chen; drafting the article: Yue Zeng, Jianghong Wu; final approval of the version; Yue Zeng, Xingpeng Wang. Acknowledgements This work was supported by funding from Natural Science Foundation for Young Scholars of China (No. 81200322), Shanghai Natural Science Foundation (No. 12ZR1424000), and Open Project of Shanghai Key Laboratory of Pancreatic Diseases (No. SPDF2013004). References Bernales, S., Papa, F.R., Walter, P., 2006. Intracellular signaling by the unfolded protein response. Annu. Rev. Cell. Dev. Biol. 22, 487–508. Cao, S.S., Kaufman, R.J., 2013. Targeting endoplasmic reticulum stress in metabolic disease. Expert Opin. Ther. Targets 17, 437–448. Cao, S.S., Luo, K.L., Shi, L., 2016. Endoplasmic reticulum stress interacts with inflammation in human diseases. J. Cell. Physiol. 231, 288–294. Danino, H., Ben-Dror, K., Birk, R., 2015. Exocrine pancreas ER stress is differentially induced by different fatty acids. Exp. Cell Res. 339, 397–406. Durgampudi, C., Noel, P., Patel, K., Cline, R., Trivedi, R.N., DeLany, J.P., Yadav, D., Papachristou, G.I., Lee, K., Acharya, C., Jaligama, D., Navina, S., Murad, F., Singh, V.P., 2014. Acute lipotoxicity regulates severity of biliary acute pancreatitis without affecting its initiation. Am. J. Pathol. 184, 1773–1784. Gukovsky, I., Li, N., Todoric, J., Gukovskaya, A., Karin, M., 2013. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer. Gastroenterology 144, 1199–1209. Hu, G., Shen, J., Cheng, L., Guo, C., Xu, X., Wang, F., Huang, L., Yang, L., He, M., Xiang, D., Zhu, S., Wu, M., Yu, Y., Han, W., Wang, X., 2011. Reg4 protects against acinar cell necrosis in experimental pancreatitis. Gut 60, 820–828. Hummasti, S., Hotamisligil, G.S., 2010. Endoplasmic reticulum stress and inflammation in obesity and diabetes. Circ. Res. 107, 579–591. Jiang, H.Y., Wek, S.A., McGrath, B.C., Scheuner, D., Kaufman, R.J., Cavener, D.R., Wek, R.C., 2003. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses. Mol. Cell. Biol. 23, 5651–5663. Kaneko, M., Niinuma, Y., Nomura, Y., 2003. Activation signal of nuclear factor-kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. Biol. Pharm. Bull. 26, 931–935. Kitamura, M., 2011. Control of NF-kappaB and inflammation by the unfolded protein response. Int. Rev. Immunol. 30, 4–15. Logsdon, C.D., Ji, B., 2013. The role of protein synthesis and digestive enzymes in acinar cell injury. Nat. Rev. Gastroenterol. Hepatol. 10, 362–370. Malo, A., Kruger, B., Seyhun, E., Schafer, C., Hoffmann, R.T., Goke, B., Kubisch, C.H., 2010. Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini. Am. J. Physiol. Liver Physiol. 299, G877–G886.
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Malo, A., Kruger, B., Goke, B., Kubisch, C.H., 2013. 4-Phenylbutyric acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini. Pancreas 42, 92–101. Matsuda, T., Kido, Y., Asahara, S., Kaisho, T., Tanaka, T., Hashimoto, N., Shigeyama, Y., Takeda, A., Inoue, T., Shibutani, Y., Koyanagi, M., Hosooka, T., Matsumoto, M., Inoue, H., Uchida, T., Koike, M., Uchiyama, Y., Akira, S., Kasuga, M., 2010. Ablation of C/EBPbeta alleviates ER stress and pancreatic beta cell failure through the GRP78 chaperone in mice. J. Clin. Invest. 120, 115–126. Matsuda, T., Takahashi, H., Mieda, Y., Shimizu, S., Kawamoto, T., Matsuura, Y., Takai, T., Suzuki, E., Kanno, A., Koyanagi-Kimura, M., Asahara, S., Bartolome, A., Yokoi, N., Inoue, H., Ogawa, W., Seino, S., Kido, Y., 2015. Regulation of pancreatic beta cell mass by cross-interaction between CCAAT enhancer binding protein beta induced by endoplasmic reticulum stress and AMP-activated protein kinase activity. PLoS One 10, e130757. Noel, P., Patel, K., Durgampudi, C., Trivedi, R.N., de Oliveira, C., Crowell, M.D., Pannala, R., Lee, K., Brand, R., Chennat, J., Slivka, A., Papachristou, G.I., Khalid, A., Whitcomb, D.C., De Lany, J.P., Cline, R.A., Acharya, C., Jaligama, D., Murad, F.M., Yadav, D., Navina, S., Singh, V.P., 2016. Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections. Gut 65, 100–111. Oslowski, C.M., Hara, T., O’Sullivan-Murphy, B., Kanekura, K., Lu, S., Hara, M., Ishigaki, S., Zhu, L.J., Hayashi, E., Hui, S.T., Greiner, D., Kaufman, R.J., Bortell, R., Urano, F., 2012. Thioredoxin-interacting protein mediates ER stress-induced beta cell death through initiation of the inflammasome. Cell Metab. 16, 265–273. Rahman, S.M., Janssen, R.C., Choudhury, M., Baquero, K.C., Aikens, R.M., de la Houssaye, B.A., Friedman, J.E., 2012. CCAAT/enhancer-binding protein beta (C/EBPbeta) expression regulates dietary-induced inflammation in macrophages and adipose tissue in mice. J. Biol. Chem. 287, 34349–34360. Ramji, D.P., Foka, P., 2002. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365, 561–575. Ron, D., Habener, J.F., 1992. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 6, 439–453. Samad, A., James, A., Wong, J., Mankad, P., Whitehouse, J., Patel, W., Alves-Simoes, M., Siriwardena, A.K., Bruce, J.I., 2014. Insulin protects pancreatic acinar cells from palmitoleic acid-induced cellular injury. J. Biol. Chem. 289, 23582–23595. Schmidt, J., Rattner, D.W., Lewandrowski, K., Compton, C.C., Mandavilli, U., Knoefel, W.T., Warshaw, A.L., 1992. A better model of acute pancreatitis for evaluating therapy. Ann. Surg. 215, 44–56. Spector, A.A., 1986. Structure and lipid binding properties of serum albumin. Methods Enzymol. 128, 320–339. Valdivielso, P., Ramirez-Bueno, A., Ewald, N., 2014. Current knowledge of hypertriglyceridemic pancreatitis. Eur. J. Intern. Med. 25, 689–694. Voronina, S.G., Barrow, S.L., Simpson, A.W., Gerasimenko, O.V., Da, S.X.G., Rutter, G.A., Petersen, O.H., Tepikin, A.V., 2010. Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells. Gastroenterology 138, 1976–1987. Yamazaki, H., Hiramatsu, N., Hayakawa, K., Tagawa, Y., Okamura, M., Ogata, R., Huang, T., Nakajima, S., Yao, J., Paton, A.W., Paton, J.C., Kitamura, M., 2009. Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response. J. Immunol. 183, 1480–1487. Zeng, Y., Wang, X., Zhang, W., Wu, K., Ma, J., 2012. Hypertriglyceridemia aggravates ER stress and pathogenesis of acute pancreatitis. Hepatogastroenterology 59, 2318–2326. Zhang, K., Kaufman, R.J., 2008. From endoplasmic-reticulum stress to the inflammatory response. Nature 454, 455–462. Zheng, L., Xue, J., Jaffee, E.M., Habtezion, A., 2013. Role of immune cells and immune-based therapies in pancreatitis and pancreatic ductal adenocarcinoma. Gastroenterology 144, 1230–1240. Zuo, Y., Qiang, L., Farmer, S.R., 2006. Activation of CCAAT/enhancer-binding protein (C/EBP) alpha expression by C/EBP beta during adipogenesis requires a peroxisome proliferator-activated receptor-gamma-associated repression of HDAC1 at the C/ebp alpha gene promoter. J. Biol. Chem. 281, 7960–7967. van der Krieken, S.E., Popeijus, H.E., Mensink, R.P., Plat, J., 2015. CCAAT/Enhancer binding protein ˇ in relation to ER stress, inflammation, and metabolic disturbances. Biomed. Res. Int. 2015, 1–13.