Pancreatic stellate cell activation is regulated by fatty acids and ER stress

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Pancreatic stellate cell activation is regulated by fatty acids and ER stress☆ ⁎

Yael Ben-Harosh, Mariana Anosov, Hagit Salem, Yekaterina Yatchenko, R. Birk Department of Nutrition, Faculty of Health Sciences, Ariel University, Israel

A R T I C L E I N F O

A BS T RAC T

Keywords: Pancreatic stellate cells (PSC) Fibrogenesis Endoplasmic reticulum (ER) stress Fatty acids Pancreatitis

Introduction: Pancreatic pathologies are characterized by a progressive fibrosis process. Pancreatic stellate cells (PSC) play a crucial role in pancreatic fibrogenesis. Endoplasmic reticulum (ER) stress emerges as an important determinant of fibrotic remodeling. Overload of fatty acids (FA), typical to obesity, may lead to lipotoxic state and cellular stress. Aim: To study the effect of different lipolytic challenges on pancreatic ER stress and PSC activation. Methods: Primary PSCs were exposed to different FAs, palmitate (pal) and oleate (ole), at pathophysiological concentrations typical to obese state, and in acute caerulein-induced stress (cer). PSC activation and differentiation were analyzed by measuring fat accumulation (oil-red staining and quantitation), proliferation (cells count) and migration (wound- healing assay). PSC differentiation markers (α-sma, fibronectin, tgf-β and collagen secretion), ER stress unfolded protein response and immune indicators (Xbp1, CHOP, TNF-α, IL-6) were analyzed at the transcript and protein expression levels (quantitative RT-PCR and western blotting). Results: PSC exposure to pal and ole FAs (500 µM) increased significantly fat accumulation. Proliferation and migration analysis demonstrated that ole FA retained PSC activation, while exposure to pal FA significantly halted proliferation rate and delayed migration. Cer significantly augmented PSC differentiation markers αsma, fibronectin and collagen, and ER stress and inflammation markers including Xbp1, CHOP, TNF-α and IL6. The ole FA treatment significantly elevated PSC differentiation markers α-sma, fibronectin and collagen secretion. PSC ER stress was demonstrated following pal treatment with significant elevation of Xbp1 splicing and CHOP levels. Conclusion: Exposure to pal FA halted PSC activation and differentiation and elevated ER stress markers, while cer and ole exposure significantly induced activation, differentiation and fibrosis. Thus, dietary FA composition should be considered and optimized to regulate PSC activation and differentiation in pancreatic pathologies.

1. Introduction Acute pancreatitis (AP) is the second most common indication for hospitalization due to a gastrointestinal disease in the U.S [1]. Transition from acute to chronic pancreatitis (CP) is characterized by a process of pancreatic fibrosis, which might lead to exocrine pancreatic insufficiency and pancreatic cancer [2,3]. CP is a fibro- inflammatory syndrome of the pancreas. It typically involves background risk factors, both genetic and environmental, such as alcohol abuse, drugs use and smoking, partly through generation of reactive oxygen species (ROS) and altered metabolism. Those, in turn, lead to the development of persistent pathologic responses to parenchymal injury or stress, promoting deposition of extracellular matrix (ECM) protein by activated pancreatic stellate cells (PSC) [4–7]. PSCs compromise 4–7% of the total pancreas cell mass and are

located predominantly in the periacinar, periductal and perivascular spaces [8–10]. In normal physiological state, PSCs have a quiescent (non-activated) phenotype characterized by vitamin A-containing lipid droplets in the cytoplasm, and function in preservation of normal tissue architecture and regulation of ECM turnover [11,12]. Upon pancreatic injury, PSCs undergo activation, and trans-differentiate phenotypically to myofibroblast-like cells, with loss of cytoplasmic lipid droplets, enhanced proliferation and migration, as well as augmented synthesis of large amounts of ECM proteins and secretion of growth factors and inflammatory cytokines [8,13,14]. The endoplasmic reticulum (ER) is an important organelle required for cell survival and normal cellular function. Imbalance between ER capacity and protein folding load cause ER stress and increase secretory load with accumulation of misfolded proteins, causing cells to activate the unfolded protein response (UPR) [15–17]. The UPR is a

Abbreviations: PSC, Pancreatic stellate cells; ER, Endoplasmic reticulum; cer, caerulein; pal, palmitic acid; ole, oleic acid ☆ This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ⁎ Correspondence to: Nutrigenetics and Nutrigenomics lab, Nutrition Department, School of Health Sciences, Ariel University, Israel. E-mail address: [email protected] (R. Birk). http://dx.doi.org/10.1016/j.yexcr.2017.08.007 Received 19 July 2017; Accepted 3 August 2017 0014-4827/ © 2017 Published by Elsevier Inc.

Please cite this article as: Ben-Harosh, Y., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.08.007

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2.3. Cell proliferation assays

complex and coordinated adaptive signaling mechanism aimed at reestablishing homeostasis of ER function. UPR consists of 3 main signaling systems initiated by 3 ER transmembrane proteins (PERK, IRE1 and ATF6) that dissociate from Bip chaperone protein followed by activation of several transcription factors, including; CHOP and XBP1 (XBP1 is activated by mRNA splicing) [17,18]. ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in tissue fibrosis of internal organs, including lungs, liver, kidney, GI tract and heart [19,20], by enhancing the susceptibility of structural cells, in most cases the epithelium, to pro-fibrotic stimuli. Additionally, ER stress is involved in the progression of chronic disease and has been demonstrated to occur in pancreatitis; however, the mechanism through which the fibrogenic phenotype of activated PSCs induces ER stress and UPR is not fully known and might be associated with other environmental insults, such as overload of dietary fatty acids typical to obesity [21,22]. Obesity is a risk factor for many chronic diseases, including pancreatitis and pancreatic cancer. Chronic consumption of high fat diet (HFD) with augmented blood concentrations of FFAs is associated with the development of chronic diseases and pancreatic diseases [23– 25]., One of the proposed mechanisms through which obesity enhances the risk of such diseases is that overload of dietary FAs causes accumulation of fat in non- adipose tissues, resulting in lipotoxicity state, characterized by cellular and tissue stress [25–27]. In recent years, obesity-related FFAs generated from the breakdown of excess intra-pancreatic fat were shown to be associated with inflammation and pancreatic organ failure. Beyond the lipotoxic effect of the FFAs, it was suggested that the accumulating FFAs also induce a low grade chronic inflammatory process, which leads to accumulation of misfolded proteins in the ER, causing ER stress and pancreatic damage [23,25]. The most abundant dietary FAs are palmitic acid (pal) - a saturated FA, and oleic acid (ole) - a mono unsaturated FA. Little is known regarding the metabolic effects of different FAs on PSC activation and differentiation, especially in relation to ER stress [28–30]. We now demonstrate differential effects of different FAs on cellular and molecular pathways regulating PSC activation and fibrosis.

Cells were collected using Trypsin EDTA 0.05% (Biological Industries, Israel) into fresh medium and centrifuged at 300 G for 5 min. Cell pellets were re-suspended in 1 ml of medium. Cell samples (10 µl) were loaded on hemicytometer (Marienfeld, Germany) and counted using light microscopy. 2.4. Free fatty acid load and PSC treatments Fatty acid supplemented medium was prepared with slight modification of the protocol of Spector [32]. Briefly, Palmitic acid and Oleic acid (Sigma Aldrich, Israel) were dissolved in ethanol and gently mixed until completely dissolved, after which the clear FAs solution was complexed with fatty acid free BSA (Roche) at a 1:10 FA to BSA ratio (7 µg/µl). The complexed FAs solution was added to the cell culture medium to obtain the indicated final FFA concentration (500 µM). The control untreated cells were treated with the same vehicle solution without the FAs. PSCs were treated with tm (an ER stress inducer) 5 µg/ml for 24 h and cer (an AP inducer) 10nmol/L for 4 h. 2.5. Fat accumulation – oil red O staining Oil red O (ORO) staining was performed using the protocol of Koopman [33] with slight modifications. Briefly, cells were grown on 12 wells plates and subjected to different treatments. Cells were chemically fixed by 4% formaldehyde solution for 20 min and then rinsed with PBS solution. They were then rotated at room temperature in ORO solution (Sigma, Israel) for 30 min and washed (3× 30 s) in PBS. ORO staining was analyzed using light microscopy (Nikon ECLIPSE TE2000-U) and quantified by isopropyl alcohol extraction using ELISA Reader (TECAN Infinite F200, 495 nm O.D). 2.6. RNA Isolation of pancreatic stellate cells PSC were cultured in six-well plates. Following treatments, total RNA was isolated using Trizol (Rhenium, Israel) in a ratio of 1 ml per 10 cm2 of culture dish area, according to manufacturer's instructions. RNA integrity was analyzed by 1% agarose gel electrophoresis with ethidium bromide (Mercury, Israel) staining. RNA was quantified by UV absorption at 260 nm (NanoDrop ND-1000 UV–Vis; NanoDrop Technologies). Samples were stored at − 80 °C.

2. Materials and methods 2.1. Isolation of primary pancreatic stellate cells Male rats (250 gr, Sprague-Dawley, INVIVOGEN Laboratories) were housed and treated according to the institutional ethics approval and guidelines. Isolation of primary PSC was done by density gradient centrifugation according to the protocol of Minoty Apte et al. [31]. In brief, the pancreas was removed and digested by Hank's balanced salt solution (HBSS, Sigma-Aldrich), containing 0.55% Pronase, 0.13% collagenase P, 2.75% Dnase, 1.1% HEPES buffer with 18 ml of HBSS (Rocsh) at 37 °C for 12 min. The resultant suspension of pancreatic cells was separated to PSC at 1400 G for 12 min using Optiprepdensity gradient. Cell suspensions containing primary PSC were resuspended in 10 ml complete media (2% FCS, 1% glutamine, 1% penicillin and streptomycin; Biological industries, Israel). Isolated PSC were cultured at 37 °C (5% CO2).

2.7. Reverse transcription (RT) cDNA was synthesized using Tetro Reverse Transcriptase kit, according to the manufacturer's protocol (Origolab, Israel). Briefly, mRNA samples (1 µg) were subject to first strand cDNA synthesis by random hexamer using a cDNA mix. Samples were incubated at 70 °C for 5 min after which 1 µl dNTP, 4 µl Tetro RT buffer and 1 µl Tetro RT enzyme were added. The samples were then incubated at 42 °C for 60 min, followed by incubation at 70 °C for 10 min. Samples were stored at − 20 °C. 2.8. Quantitative real time PCR (qPCR) Transcript levels were determined by qPCR using SYBR® Green PCR Master Mix (Rhenium, Israel). Reactions were carried out using the MxPro3000 apparatus (Stratagene, Santa Clara, CA) according to the instructions of the manufacturer. Gene-specific primers were designed using the primer 3 online software. The qPCR primer pairs were designed across exons to avoid false positive signals from potentially contaminating genomic DNA. Primer and cDNA concentrations were optimized following the guidelines of the supplier. Each 20 µl reaction contained 2 µl cDNA, 10 µl PCR Master Mix (Rhenium, Israel), primers at 100–500 nM concentration of each forward and

2.2. Cell culture Medium was replaced every 2 days (complete medium: Dulbecco's Modified Eagle Medium, 1.5% HEPES 1 M buffer (Rhenium, Israel), 20% Fetal Bovine Serum (Biological industries, Israel) 1% (v/v) Penicillin-Streptomycin, 2% L-glutamine (Biological industries, Israel). Experiments were performed using culture activated cells (passages 2–4). 2

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reverse primer and water. Gene expression was normalized to housekeeping gene HPRT and presented as Ct values. 2.9. XBP1 splicing Amplification of XBP1 transcripts was done using PCR kit MyTaq DNA Polymerase (Origolab, Israel). PCR products were subjected to Pst1 restriction enzyme (Thermo Scientific, Israel) according to the manufacturer's protocol. Products were run on 2% agar-gel. The nonspliced (non-stressed) variant of Xbp1 (289 bp) results in two smaller fragments of 173 bp and 116 bp (Xbp1 n), whereas the spliced (stressed) form size is 266 bp (Xbp1s). Fig. 1. PSC fat accumulation following exposure to different FAs during differentiation. PSC were grown, differentiated and treated as described in material and methods section. PSC were treated with FAs: pal and ole (500 µM) throughout differentiation (day 1 and 6 days) and subjected to ORO staining. Results show quantification analysis of fat accumulation (mean ± SD). **** p < 0.0001, ** p < 0.01 * p < 0.05.

2.10. Western blot analysis Proteins were extracted from cells using standard methods. In brief, cell pellets were suspended in 50 µl RIPA lysis buffer (Biological industries, Israel) containing 2% protease inhibitor cocktail (Sigma Aldrich, Israel). Total proteins (30 µg) underwent polyacrylamide gel electrophoresis followed by protein transfer and western blotting using standard protocol. Blots were analyzed using EZ-ECL chemiluminescence detection kit (Biological industries, Israel). Bands were detected by ImageQuant LAS 4000 Mini (GE Healthcare, Life Science, Israel). The specific bands were subjected to densitometry analysis using the image J software 1.46j version. All proteins were quantified relative to housekeeping gene.

performed in triplicate. Data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 7.0 and comparisons were made using One-way or Two-way ANOVA. Statistical significance of differences between treatment groups is presented graphically by (*) at p < 0.05, (**) at p < 0.01 and (***) at p < 0.001. 3. Results

2.11. Immunofluorescence staining

3.1. PSC fat accumulation during differentiation

Cells were seeded and grown on a coverslip and fixed with 4% paraformaldehyde (Sigma- Aldrich, Israel) at room temperature for 20 min. Following wash with PBS, cells were perforated by permeabilization solution (Tritonx100 2.5% diluted in PBSX1) for 5 min. Blocking solution was added (iNGS 3%, Tritonx100 1%, PBSX1) (Biological Industries, Israel) for 1-h at room temperature, followed by additional incubation for overnight at 4 oC. After washing with PBS, the cells were incubated with anti-rabbit Alexa fluora 488 antibody for 1 h, washed and analyzed using fluorescence confocal microscope (LSM 700 Zeiss).

PSC fat accumulation following fat challenge (pal and ole, 500 µM) was analyzed during differentiation (1 and 6 days). Fat accumulation in control (con) cells decreased significantly during differentiation by 4 fold, reaching the lowest amount at day 6. Those findings indicate a normal and expected differentiation process of PSC. Pal treatment significantly increased PSC fat accumulation at day 1 (p < 0.05) and at day 6 (p < 0.0001) in comparison to control and ole treatments. The decrease of fat accumulation during differentiation, evident in control cells, was not seen following ole treatment, where no difference in fat accumulation was detected throughout the 6 days of differentiation (Fig. 1).

2.12. Migration test 3.2. Proliferation analysis: PSC proliferation during differentiation following FAs treatments

The scratch wound assay was used to measure cell migration. The procedure described by Rodriguez, L.G [34] was followed. Briefly, PSC were seeded on 24 wells plates and grown to confluence in DMEM containing 15% (FBS). Before the beginning of experiment, medium was replaced by serum free medium (0.05% FBS) for overnight incubation. A linear wound "scratch" was created using 200 µl pipette tip. After washing the cultures twice with DMEM, the medium was changed to 5% FBS. Cell migration was evaluated at 0 and 24 h and photos were taken by light microscopy and analyzed using image J.

Cell proliferation during differentiation (1,2,3 and 6 days) was studied following different treatments, with pal and ole (500 µM). PSC proliferation rate of the control group was significantly higher at day 6 in comparision to days 1 (p < 0.01), 2 and 3 (p < 0.001), by 3,5 and 5fold, respectively. No difference in proliferation rate was detected between days 1,2 and 3 of the control group. PSC ole (500 µM) treated cells exhibited significant elevation in proliferation rate at day 6 of differentiation (p < 0.0001) in comparison to days 1,2 and 3, similar to the proliferation rate of PSC control cells. PSC pal treated cells (500 µM) exhibited significant decreased proliferation rate throughout differentiation (Fig. 2)

2.13. Collagen staining (Sirius red) Cells were seeded (25 × 104) in DMEM with 15%FBS. Cells were grown to 70–80% confluence, fixated with 4% formaldehyde (Sigma Aldrich, Israel)and stained according to the protocol of TullbergReinert H [35] with 0.1% Sirius red dye solution in saturated aqueous solution of picric acid (Sigma Aldrich, Israel) for 1 h. Then, cells were washed (X 2) with acidified water and dehydrated by 100% ethanol wash (× 3). Stained cells were analyzed and photographed using bright field microscope) Nikon ECLIPSE TE2000-U).

3.3. Migration analysis: effects of FAs treatments and ER stress induction on PSC migration PSC migration is a hallmark of differentiation and activation. PSC were treated with different FAs (pal and ole at 500 µM) and tunicamycin (tm) for 24 h. As shown in Fig. 3, PSC migration rate of control untreated cells was significantly higher (p < 0.01) by 10.7 and 2.7 fold compared to pal and tm-treated cells, respectively. PSC migration rate following ole treatment was significantly higher (p < 0.01) by 12 and 3fold compared to tm and pal treated cells, respectively. No difference

2.14. Statistical analysis Results were collected from three independent experiments, each 3

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splicing. Xbp1 splicing (% of total) increased significantly (p < 0.0001) by 47 and 60-fold following pal and tm treatments, respectively compared to control untreated and ole-treated cells, which exhibited comparable XBP1 splicing levels. PSC pal-treated cells exhibited significant (p < 0.01) increase in % of XBP1 splicing levels by 1.2-fold compared to PSC tm treated group. Percent of Xbp1 splicing in certreated cells increased significantly (p < 0.001) by 6-fold compared to control untreated and ole-treated cells. 3.5. PSC differentiation markers following exposure to FAs and ER stressors Fig. 2. Effect of FAs treatments on PSC cell proliferation: PSC were treated with pal and ole (500 µM) throughout differentiation. Cells were counted using haemocytometer at days 1, 2, 3 and 6 of in-vitro differentiation (mean ± SD). Values are given as mean ± SD. **** p < 0.0001, *** p < 0.001.

PSC were treated with different FAs (pal, ole, 500 µM), tm and cer. α-sma and fibronectin are major markers for PSC differentiation and activation. As seen in Fig. 5, PSC cer-treated cells exhibited significant elevation (p < 0.05) in α-sma by 1.5-fold compared to PSC con untreated group. α-sma expression increased significantly (p < 0.001) by 4.5 in the PSC con untreated group in comparison to PSC tm, pal and ole treated groups. No significant differences in α-sma expression levels were found between the two FAs and tm treatments. Fibronectin expression in cer PSCs increased significantly (p < 0. 01) by 2.85, 2.45,3. 33 and 1.45 compared to control, tm, pal and ole-treated cells, respectively. Fibronectin levels in PSC ole treated cells were significantly higher by 1.9-fold compared to pal treated cells. tgf-β levels were elevated following PSC ole treatment by 1.- fold compared to PSC con but the differences did not reach statistical significance.(Fig. 6) 3.6. PSC ER stress markers following exposure to FAs and ER stressors Xbp1 and CHOP are important markers for ER stress and activation of UPR. PSC tm-treated cells showed significant (p < 0.05) elevation in XBP1 expression by 4.27-fold compared to PSC pal-treated cells. CHOP levels were augmented significantly (p < 0.01) in PSC tmtreated cells by 3.98, 3.5, and 3.8-fold compared to PSC control ole and cer treated cells, respectively. CHOP expression in pal treated PSCs increased significantly by 3.22, 2.83 and 3-fold compared to control ole and cer-treatments, respectively.

Fig. 3. PSC migration rate following FAs and stressor treatments. Cell migration was assessed by wound- healing assay in PSC cells. PSC were treated with different FAs as described in materials and methods. Photos were taken by light microscopy (x10 magnification, bar = 100 µm) at time points 0 and 24 h. Migration was measured by Image software. Results are expressed as mean ± SD. ** p < 0.01, * p < 0.05.

3.7. Immune stress markers following exposure of PSCs to FAs and ER stressors TNF-α and IL-6 are markers for inflammation processes and are synthesized endogenously in response to PSC activation. As shown in Fig. 7, TNF-α expression increased by 1.6-fold in the PSC pal and ole treated cells in comparison to tm-treated and control untreated PSCs, but this difference did not reach significance. IL-6 expression increased significantly (p < 0.05) by 2.2-fold in the PSC cer-treated cells in comparison to tm and ole -treated PSCs. In addition, IL-6 expression was higher in cer treated PSCs than in the pal treated group and control PSCs. However, this difference did not reach statistically significant levels.(Fig. 8) 3.8. PSC differentiation markers following FAs and ER stressors treatments: transcript levels

Fig. 4. PSC XBP1 splicing following treatments of various FAs and stressors. Xbp1 splicing at the mRNA level was analyzed following PSC treatment with different FAs and stressors. % Xbp1 splicing was analyzed as described in materials and methods. Results represent two independent experiments (n = 3). Results were analyzed by ImageJ and are presented as % Xbp1 splicing (means ± SD). ** p < 0.01. ***p < 0.001. **** 0.0001.

PSC were treated with different treatments: pal, ole (500 µM), tm and cer. Transcript levels of α-sma were augmented significantly (p < 0.01) in PSC cer treated group compared to con, tm, pal and ole groups by 4.12 and 2.2 fold in con and ole PSC treatments. Differential effects of FAs exposure on α-sma levels were found; PSC ole treatment augmented significantly (p < 0.05) α-sma transcript levels by 2-fold compared to PSC con untreated group, while PSC pal and tm treated group did not show expression of α-sma transcript levels (A). tgf-β expression levels of PSC cer treated were augmented significantly by 26, 8.1 and 8 compared to con, pal and ole treated groups, respectively.

was found in migration rate between control and ole groups.(Fig. 4) 3.4. Xbp1 splicing: quantities measurement of spliced Xbp1 mRNA One of the important markers of ER stress-associated UPR activation is XBP1 mRNA splicing. PSC were treated with different FAs - pal and ole (500 µM), tm and caerulein (cer), and were analyzed for XBP1 4

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Fig. 5. PSC differentiation markers (protein expression) following exposure to FAs and ER stressors. PSC were treated with different FAs and ER stressors and analyzed by western blot analysis as described in materials and methods. Protein levels of α-sma (A) Fibronectin (B) and tgf-β (C) were normalized to expression levels of the housekeeping gene actin ( mean ± SD). *** p < 0.0001, ** p < 0.001, * p < 0.05.

PSC treatment with pal and ole resulted in a significant increase (p < 0.05) in IL-6 transcript levels by 45.6 and 40-fold compared to PSC control untreated cells. No significant difference was seen between treatments with the two FAs.(Fig. 10)

3.9. ER stress markers following FAs and stressors treatments of PSCs As seen in Fig. 9, analysis of ER stress markers demonstrated significant 128-fold elevation (p < 0.01) in Xbp1 transcript levels after treatment with cer as compared to the con group. Levels of Xbp1 in PSC-pal treated cells were higher than in control cells but the differences did not reach significance. PSC CHOP levels were augmented significantly 8-fold (p < 0.05) following tm treatment compared to PSC control cells, indicating that tm functions as an ER stress agonist in PSC. Differential effect of ER induction was shown upon exposure to FAs pal and ole; pal treatment of PSCs significantly increased transcript levels of CHOP by 9-fold compared to the control untreated group. In contrast, ole treatment of PSCs did not alter ER stress markers xbp1 and CHOP expression.

3.11. Collagen analysis (Sirius red staining)- effect of FAs and stressors on PSC collagen levels One of the hallmarks of PSC differentiation is collagen synthesis. Quantification of collagen levels was analyzed through spectrophotometry. PSC were treated with different FAs (pal and ole at 500 µM), tm and cer. As shown in Fig. 11, collagen levels increased significantly (p < 0.01) in cer treated PSCs by 2.1-fold compared to tm-treated PSCs. Collagen levels were significantly augmented (p < 0.05) by 2-fold in control untreated, pal and ole treated PSCs compared to tm-treated PSCs.

3.10. PSC Transcript levels of immune stress markers in PSCs following exposure to FAs and stressors

3.12. Immunofluorescence assay: PSC α-SMA and XBP1 expression following FAs and stressors treatments

Cer treatment of PSCs significantly (p < 0.0001) elevated expression of TNF-α by 64-fold compared to control untreated cells. Exposure of PSCs to ole treatment augmented TNF-α transcript levels by 14, 10 and 3-fold compared to PSC control, tm and pal treated cells, respectively.

PSC were treated with different FAs (pal and ole at 500 µM), tm and cer. As shown in Fig. 12, α-SMA protein was evident in control PSCs. Cer treated PSCs showed strong α-SMA immunoreactivity; tm-treated

Fig. 6. Protein expression levels of ER stress markers following treatment of PSCs with FAs and ER stressors. PSC were treated with different FAs and stressors and analyzed by western blot analysis as described in materials and methods. Levels of A. xbp1 B. CHOP were normalized to housekeeping gene actin (mean ± SD). ** p < 0.001, * p < 0.05.

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Fig. 7. Protein levels of immune markers in PSCs following exposure to FAs and ER stressors. PSC were treated with different FAs and ER stressors and analyzed by western blot analysis as described in materials and methods Protein levels of TNF-α (A) and IL-6 (B) were normalized to expression levels of housekeeping gene actin. Results shown mean ± SD, * p < 0.05.

PSCs showed weak α-SMA immunoreactivity. Following the two different FAs treatments no difference was shown in α-SMA immunoreactivity. TM, Cer and pal treated PSC showed strong immunoreactivity in the nucleus unlike the cytosol immunoreactivity in the control and in the ole treated PSC.

lose their fat content as they differentiate. The established and expected phenomenon of gradual loss of fat content throughout differentiation of PSC and HSC [9] was clearly demonstrated in our PSC control untreated group. In other relevant pancreatic models, we have previously shown that acinar exocrine pancreas cells demonstrate significant fat accumulation following exposure to high levels of both FAs, pal and ole, compared to control cells [36]. Different results have been reported in HSC model, which might be due to difference in FA concentration and treatment duration; HSC lipid droplet concentration was higher following ole FA treatment compared to pal treatment when exposed to acute (six hours) mega-dose (1 mM) concentration e [37]. However, 24 h exposure of HSC to 400 µM of pal and ole FAs induced fat accumulation with no difference in fat accumulation between the two different FAs [38]. Interestingly, exposure of rat hepatoma cell line to SFA (palmitic acid and stearic acid) resulted in a significant increase in expression of ER stress response genes, in contrast with the effects seen in MUFA (oleic acid and linoleic acid) treated cells. Further in vivo studies using dietary models of NAFLD support this concept [39]. Thus, our data suggest for the first time that PSC respond differently than HSC to FAs challenge, with significant upregulation

4. Discussion We investigated the cellular and molecular processes involved in pancreatic stress following challenges with different stressors. We focused on two different FAs, pal (saturated) and ole (unsaturated), as well as chemical stressors: tm, known to induce ER stress, and cer, an analogue of CCK which induces AP state. First, we demonstrated that exposure of primary PSCs to FAs at high concentration (500 µM), typical to the levels found in obese humans, results in fat accumulation. Exposure to the saturated FA pal during PSC differentiation increased fat accumulation significantly. No significant changes were shown in fat accumulation following ole FA treatment throughout differentiation days and fat accumulation was consistent. These findings are indicative of differential response of PSCs in the differentiation process, where stellate cells are expected to

Fig. 8. Gene expression levels of differentiation markers following FAs and stressors treatments. PSC were treated with different FAs and stressors and cDNA was subjected to RT-PCR as described in materials and methods. Results were normalized to housekeeping HPRT gene. A. α-sma B. tgf-β. Results are expressed as mean ± SD, ** p < 0.001, * p < 0.05.

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Fig. 9. Gene expression of ER stress markers levels in PSCs following exposure to FAs and stressors. PSC were treated with different FAs and stressors and cDNA was subjected to RT-PCR as described in materials and methods. Results were normalized to housekeeping HPRT gene. A. Xbp1 B. CHOP. Results are expressed as mean ± SD, ** p < 0.001, * p < 0.05.

Fig. 10. PSC gene expression levels of immune stress markers following FAs and stressors treatments. PSC were treated with different FAs and stressors and RT-PCR was done as described in materials and methods. Results were normalized to housekeeping HPRT gene. A. TNF-α B. IL-6. Results are expressed as mean ± SD, **** p < 0.0001 **** p < 0.001 ** p < 0. 01, * p < 0.05.

hagy and increases apoptosis [30,38]. Rapid proliferation of PSC in culture can either promote apoptosis or encourage differentiation. Enhanced proliferation rate is one of the functions of PSCs that is critical to the fibrotic process [12]. Activated PSC are known to be resistant to apoptosis [40]. We demonstrate that exposure to high levels (500 µM) of pal and ole FAs result in differential proliferative responses; while ole treatment retained proliferation, pal treatment halted proliferation rate. The significant decrease in proliferation with significant increase in fat accumulation

of fat accumulation following exposure to saturated pal FA as compared to fat accumulation following ole exposure. Few mechanisms of fat accumulation were suggested in relation to the induction of lipotoxicity and hepatic injuries by FAs. One possible mechanism, shown in hepatocytes, is that ole FA can promote the TG enriching lipid droplet formation followed by induction of hepatocyte autophagy with a slight effect on apoptosis. In contrast, it has been shown in some experimental models that pal FA is a poor inducer of formation of TG- enriched lipid droplets and thus suppresses autop-

Fig. 11. PSC collagen levels following FAs and stressors treatments. PSC were stained by Picro Sirius red staining and observed under light microscopy (× 4 and × 20 magnification) (A). PSC were treated with different FAs (pal and ole at 500 µM), tm [5 µg/ml] for 24 h and with cer for 4 h [10 nmol/L]. Bar = 100 µm (magnification × 4), 50 µm (magnification × 20). PSC collagen stain was extracted by 0.1 NaOH. Quantification of collagen levels was analyzed through spectrophotometry as described in material and methods. Results are expressed as mean ± SD,** p < 0.01. * p < 0.05.

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Fig. 12. Immunofluorescence staining of α-sma (A) and XBP1 (B) protein following FAs and stressors treatments. PSC were treated with different fatty acids (pal and ole at500µM), tm [5 µg/ml] for 24 h and with cer for 4 h [10 nmol/L]. Following treatments, immunofluorescence staining was performed using direct fluorescent antibody to α-sma protein (green) and dapi staining (blue) as described in materials and methods. Photos were taken using Olympus fluorescent microscope at × 20 magnification (100 µm scale). Photos represent fields of cells (n = 5000-1000).

We demonstrated a significant elevation in PSC α-sma following cer treatment at the transcript level. In addition, cer-treated PSCs were positive to α-sma by immunofluorescence. Expression of fibronectin, one of the major ECM components and another parameter of PSC activation, was significantly elevated in cer treated PSCs compared to control untreated PSCs. Those findings reinforce the notion that PSC activation and fibrosis strongly occurs by caerulein-induced CP [48]. PSC may be a major source of ECM deposition characterized by colocalization of α-sma and collagen synthesis during injury. Here, we demonstrated the presence of collagen in control untreated as well as pal, ole and cer-treated PSCs. Consistent with our observation, mesenchymal cells, in particular fibroblasts which contain α-sma protein, were reported to produce ECM component proteins after pancreatitis, and are thought to be the principle source for ECM in pancreatic fibrosis [49]. Further research is needed to discriminate between different types of ECM components, which apparently have diverse effects in different types of pancreatitis models [50]. Our study demonstrates differences in PSC activation markers after exposure to FAs, pal and ole: significant elevation of α-sma and fibronectin levels in ole treated PSCs compared to control untreated PSCs. In addition, tgf-β levels following FA exposure were higher than in control PSCs, but this difference did not reach significance. This is in contrast with pal treated PSCs, which did not show increased expression of the different differentiation markers. Previous studies found no difference in HSC expression of α-sma and ECM components following exposure to pal and ole FAs in different concentrations [37]. Exposure to pal FA only, induced a significant decrease in α-sma [41]. In fact, injection of ole FA is one of the experimental models used to induce pancreatitis. This model is characterized by marked pancreatic fibrosis through elevation of α-sma and ECM components, leading to irreversible pancreatitis along with atrophy of the exocrine pancreas yet preservation of the endocrine pancreas [49]. In addition, injection of ole FA affects the development of renal damage and fibrosis in an experimental protein overload nephropathy [51]. Similarly, our in vitro model demonstrates that exposure to ole FA augments activation of PSC and the severity of pancreatic disease. This, together with other models, provides additional avenues to evaluate potential therapeutic interventions for CP in the future. Identification of factors activating PSC in diseased states have been

following pal treatment during PSC differentiation, strongly suggests that pal FA plays an important role in maintaining PSC at a quiescent state. The biological effect of lipid related growth arrest was already demonstrated in the PAV-1 HSC model [41]. One of the suggested mechanisms is activation of peroxisome proliferator activator receptor γ (PPARγ). Transfection experiments showed that PPAR-γ overexpression enhanced proliferation and increased rate of apoptotic cell death [42]. Importantly, PPAR-γ is a major transcription factor of both lipid accumulation and PSC regulation. However, neither pal or ole are preferred ligands for PPAR-γ; thus, this pathway is not necessarily related to the accumulation of fat following pal treatment. It is generally accepted that mono-unsaturated FA, such as ole, have protective effects against pal induced lipotoxicity in many cell types, such as exocrine pancreatic AR42J cells, β cells and granulosa cells [36,43,44]. We hypothesized that in PSC treated by pal FA, cells might change their activated destiny due to the increase in fat accumulation and the involvement of suggested apoptotic factors that are associated to pal FA [37,41]. Activated PSC migration is a key event in the progression of pancreatic fibrosis and is regulated by precise interaction with the ECM microenvironment [45]. We now demonstrated PSC motility inhibition following pal and tm exposure (24 h) compared to control untreated and ole treated PSCs that exhibited high migration rate. Increase in PSC number in injured pancreatic areas has been demonstrated in in-vivo studies and experimental models of pancreatitis, such as infusion of TNBC to pancreatic duct and alcohol consumption. This is a result of increased local proliferation as well as migration of PSC to injured areas from surrounding areas [46]. The present study provides novel and significant evidence that PSC retained their capacity to migrate after PSC exposure to ole FA, similarly to PSC control cells. This is in contrast to significant delayed migration as well as significant inhibition in proliferation rate following pal FA and tm exposure. Altogether, our data suggest that pal exposure halts PSC proliferation and migration in parallel to accelerating accumulation of fat, a hallmark of PSC un-differentiation state. Activation of quiescent PSC, followed by a differentiation process, occurs in the pancreas as a consequence of pancreatic injury. This is associated with several morphologic changes expressed in synthesis of specific proteins, such as α-sma, fibronectin, tgf-β and collagen [40,47]. 8

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did not induce ER stress and activation of UPR and elevation in ER stress markers. Previous studies demonstrated the presence of ER stress during fibrosis. One of the major concerns during continuous ER stress is ongoing exposure to cytokines, chemokines and other fibrogenic factors, which lead to activation accompanied by inflammation, as shown in HSC models [19,20]. A variety of other causes such as chronic infections, chronic exposure to alcohol, other toxins and autoimmune response, were also shown to lead to fibrosis with manifestations of HSC activation [61]. ER stress, activation of PSC and inflammation processes, all lead to pancreatic injury. When inflammation and injury are sustained or repeated, development of pancreatic fibrosis occurs with ongoing PSC activation [52]. TNF- α, IL-6 and other inflammatory mediators are known to be upregulated early in the course of AP necro-inflammation and are released simultaneously within the gland [48,62]. Our study is the first to demonstrate endogenous production and expression of IL-6 in PSC at transcription and protein levels. We demonstrated that acute exposure to high levels of FAs, pal and ole, has a pro-inflammatory effect in PSC. Pal-treated PSCs showed elevation in IL-6 transcript and TNF-α protein levels. Ole-treated PSCs demonstrated increased expression of IL-6 and TNF-α. Similar results were demonstrated after exposure of PSC to cer, indicative of increase in cytokine levels in response to AP state. TNF- α is a highly pleiotropic cytokine, inducing many biological effects. IL-6, an acute phase protein, in addition to its pro-inflammatory effects, is also known to have many anti- inflammatory effects. It has been shown in-vivo in hepatocytes that IL-6 is produced in response to increased TNF-α and IL-1 secretion [63] and also in HSC which synthesize IL-6 when activated by TNF- α, IL-1 or bacterial endotoxin [64]. ER stress could also exacerbate AP through signaling inflammation in liver cells, in intestinal epithelial cells and in white adipose tissue [65–67]. Although molecular signaling and pathways linking ER stress and inflammatory response require future investigation, our results clearly show a correlation between the ER stress markers Xbp1 and CHOP and elevated expression of the inflammatory marker TNF-α and IL-6 elevation following cer treatment of PSCs. In conclusion, our research demonstrates the differential effects of FAs on induction of PSC activation and fibrosis. While ole FA accelerate PSC activation by increasing proliferation and migration rate as well as augmented expression of PSC differentiation markers, Pal FA might reverse theses effects driving PSCs back to a quiescent state by increasing fat accumulation with growth arrest and inhibition in motility. Furthermore, pal FA induced ER stress response by activation of Xbp1 through IRE1, Xbp1 splicing and CHOP pathway. Our findings should be taken into consideration in treatment of progressive pancreatic diseases based on the differential effects of pal and ole FAs on PSC. In addition, the findings emphasize that cer leads to a significant upregulation of all examined stress markers, indicating cer as an AP inducer in PSC. This study enhances our understanding of two important processes in pancreatic biology: first, it sheds light on the mechanism PSC use to cope with induction of ER stress; secondly, it contributes to better understanding of molecular pathways through which FA exposure affects pancreatic damage.

largely based on the knowledge of mediators up-regulated during pancreatic necro-inflammation and of known agents inducing pancreatic injuries such as alcohol [52]. Our data demonstrate a significant increase in all activation parameters examined here following cer treatment of PSCs, indicating that cer has the most powerful effect on PSC activation. These findings are consistent with previous studies of pancreatic cells, suggesting that cer causes significant pancreatic damage to acinar and duct cell secretion [53]. One of the associated mechanisms for PSC activation and synthesis of ECM proteins is through TGF-β1, which contributes to production of ECM components and plays a major role in stimulating PSC proliferation and differentiation. Overexpression of TGF- β1 was found to elevate expression of PSC activation markers following ole treatment [49]. Here we confirm that the bioactive FAs, pal and ole, are involved in modulating the activity of PSCs. Our data expands existing knowledge regarding involvement of FAs in modulation of many cells types in the pancreas and liver during overload of FAs, and in the development of obesity and fibrosis [54]. ER stress has been implicated in a variety of diseases. Research concerning the link between ER stress and PSC activation is scarce [20,30]. Our study is the first to demonstrate that the ER stress inducer tm significantly increases both transcript and protein levels of both spliced Xbp1 and CHOP in PSCs. Those findings indicate that t, induces ER stress in PSC, as it has been previously shown to do in other mammalian cells, such as mouse embryonic fibroblasts, pancreatic β cells and plasma cells [16]. Exposure to different FAs demonstrated differential effects on ER stress: treatment of PSCs with pal saturated FA induced elevation of Xbp1 splicing levels and CHOP transcription and protein levels, compared to control cells. Our results are in line with a recent report by Lingaku Lee et al. [55], showing that saturated fatty acids can inhibit fibrogenesis in CP via ER stress response through the PERK pathway. We further showed that expression of Xbp1 transcripts and protein levels were significantly elevated following cer treatment of PSCs, with the strongest effect on ER stress. Cer treatment of PSCs induced an ER stress response, with enhanced Xbp1 transcript and protein levels and CHOP protein levels. In addition, there was significantly enhanced expression of Xbp1 splicing in cer treated PSCs compared to control PSCs. Increased expression of ER stress-associated genes has been previously reported in cer mouse models of AP [30] and CP [56]. In this model of cer induced AP mice activated PERK- a key ER stress indicator and protein synthesis [57,58]. Our findings demonstrate that cer-treated PSCs show significant elevation in Xbp1 splicing levels, although the levels were low in comparison to tm and pal treated PSCs. CHOP is a key transcription factor induced by ER stress, whose overexpression can lead to growth arrest and apoptosis. The IRE1 branch that leads to activation of Xbp1, governs the most conserved UPR signaling pathway which has acquired additional functions, such as promotion of apoptosis in response to ER stress [59]. CHOP is known to be regulated by all three ER stress sensors: PERK, IRE1 and ATF6. Our study is the first to show that the CHOP-mediated ER stress apoptotic pathway is activated by exposure of PSCs to high concentrations of FAs, pal and ole. We showed that this induction of ER stress in PSCs is mediated through the IRE branch in UPR. We observed significant induction of ER stress in pal treated PSC evident through elevation of Xbp1 splicing and CHOP transcript and protein levels, in correlation with significant increased levels of tm treated group. These observations are consistent with findings in many other cell types exposed to FAs, including hepatocytes, hepatoma cells, mesenchymal stem cells and pancreatic acinar cells [30,60]. High concentration of saturated FAs at the ER membrane can lead to disturbance in cell homeostasis by damaging membrane receptors and transporters promoting ER stress through UPR sensors: Xbp1, CHOP, GRP78 and ATF4. We have recently shown that saturated FA such as pal promote ER stress in models of acinar cells [36]; similar findings have been demonstrated also in models of liver injury [21,37]. Ole treated PSCs

Acknowledgements We thank Dr. Eva Vaquero and Irene Sangrador from the Dept. of Gastroenterology, Hospital Clinic of Barcelona, Barcelona, Spain for providing help in the PSC isolation protocol. References [1] P.G. Lankisch, M. Apte, P.A. Banks, Acute pancreatitis, Lancet 386 (2015) 85–96. [2] T. Kolodecik, C. Shugrue, M. Ashat, E.C. Thrower, Risk factors for pancreatic cancer: underlying mechanisms and potential targets, Front. Physiol. 4 (2014).

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