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Reg4 protects against acinar cell necrosis in experimental pancreatitis
  1. Guoyong Hu1,
  2. Jiaqing Shen2,
  3. Li Cheng2,
  4. Chuanyong Guo1,
  5. Xuanfu Xu1,
  6. Feng Wang2,
  7. Li Huang1,
  8. Lijuan Yang2,
  9. Miao He3,
  10. Di Xiang3,
  11. Shunying Zhu3,
  12. Mingyuan Wu3,
  13. Yan Yu4,
  14. Wei Han3,
  15. Xingpeng Wang1
  1. 1Department of Gastroenterology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China
  2. 2Department of Gastroenterology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
  3. 3Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
  4. 4Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai, China
  1. Correspondence to Professor Dr X P Wang, Department of Gastroenterology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301Yanchang Road, Shanghai, 200072, China; wangxp1965{at}yahoo.com.cn

Abstract

Background and aims Reg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. To date, the physiological function of Reg4 is poorly understood. Previously, the authors found that Reg4 was markedly upregulated during acute pancreatitis (AP). The aim of this study was to investigate the role of Reg4 in experimental pancreatitis.

Methods AP was induced in C57BL/6 mice by administration of either l-arginine or caerulein, and Reg4 expression was assessed by immunofluorescence, reverse transcriptase (RT)-PCR and western blot analyses. Recombinant human Reg4 protein (rReg4), heat-inactivated Reg4, neutralising antibody and vehicle were also administered to mice by subcutaneous injection. The severity of AP was determined by measuring amylase and lipase activities in the serum and histological grading. The effect of rReg4 on cell death was examined and epidermal growth factor receptor (EGFR), p-EGFR, Akt, p-Akt, Bcl-2 and Bcl-xL expression were assessed by western blot analysis of isolated murine acinar cells treated with l-arginine.

Results Reg4 mRNA and protein were markedly upregulated during arginine-induced pancreatitis. Reg4 was widely expressed in residual acinar cells around the islets and regenerating metaplastic epithelium. rReg4 could protect against arginine-induced necrosis of acinar cells both in vivo and in vitro. This protective effect was also confirmed in the caerulein-induced murine model of AP. It was shown that arginine induced expression of Bcl-2 and Bcl-xL, while rReg4 upregulated Bcl-2 and Bcl-xL expression by activating the EGFR/Akt pathway. The upregulation of Bcl-xL correlated inversely with cell necrosis in isolated pancreatic acinar cells.

Conclusions The data suggest that Reg4 may protect against acinar cell necrosis in experimental pancreatitis by enhancing the expression of Bcl-2 and Bcl-xL via activation of the EGFR/Akt signalling pathway.

  • Pancreatitis
  • Reg4
  • necrosis
  • Bcl-2
  • Bcl-xl
  • family proteins
  • cell death
  • pancreatic damage
  • pancreatic pathology
  • pancreatitis

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Significance of this study

What is already known about this subject?

  • Reg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. However, the physiological function of Reg4 is poorly understood.

  • Recent research indicates that the extent of Bcl-xl upregulation correlates inversely with pancreatic necrosis in models of acute pancreatitis (AP) containing arginine, and Bcl-xl and Bcl-2 can protect acinar cells from necrosis in pancreatitis by stabilising mitochondria against death signals.

  • Several studies have shown that Reg4 can induce expression of Bcl-2 and Bcl-xl in HCT116 and intestinal crypt cells.

What are the new findings?

  • Reg4 was widely expressed in residual acinar cells around the islets and regenerating metaplastic epithelium, suggesting that it may be involved in the pancreatic repair process after AP. The observation that Reg4 upregulation follows exocrine pancreatic injury may provide a mechanistic link between chronic pancreatic injury and subsequent neoplasia.

  • It was shown that Reg4 can protect against necrosis in experimental pancreatitis, and a new biological function for Reg4 was identified: regulation of cell death responses in AP.

  • Reg4 can induce expression of Bcl-2 and Bcl-xl in pancreatic acinar cells. These data enhance the current understanding of cell death mechanisms regulating AP and provide a plausible explanation for the protective effect of Reg4 on AP.

How might it impact on clinical practice in the foreseeable future?

  • Characterisation of the molecular basis of cell death in pancreatitis will provide a novel strategy to prevent or attenuate necrosis in pancreatitis.

  • Reg4 may serve as a potential therapeutic agent for treating patients with AP.

Introduction

Regenerating gene (Reg) 4, a recently discovered member of the regenerating gene family, belongs to the calcium-dependent (C-type) lectin superfamily, and was first isolated from a cDNA library of ulcerative colitis tissues.1 Reg4 has a highly restricted tissue expression pattern. It is predominantly expressed in gastrointestinal tract tissues including the colon, small intestine, stomach and pancreas.1 2 Reg4 expression is markedly upregulated in diseases such as colon cancer,3 4 gastric cancer,5–8 gallbladder carcinoma9, pancreatic cancer,10–12 prostate cancer13 14 and inflammatory bowel disease (Crohn disease and ulcerative colitis).1–3 15 Despite these associations, the physiological role of Reg4 remains poorly understood. Recently, several studies have suggested that Reg4 may be involved in tissue repair, cell proliferation and migration. Furthermore, overexpression of Reg4 has been reported to increase resistance to apoptosis induced by chemoradiotherapy.1–3 6 7 16–20 However, the role of Reg4 in the course of acute pancreatitis (AP) has not been reported to date.

In the normal pancreas, Reg4 is only expressed in the β cells of the endocrine pancreas,5 and is not found in other types of pancreatic cells. In patients with AP and pancreatic cancer, the serum level of Reg4 has been found to be significantly raised, suggesting that Reg4 may play an important role in the progression of these pancreatic diseases.11 We have previously used a gene chip to examine multiple gene differential expression patterns in the pancreas and found that Reg4 mRNA was markedly upregulated during arginine-induced AP. In addition, upregulated Reg4 expression was detected during the entire course of pancreatitis, suggesting that it may be a mediator of the progression of this disease, serve as a protective regulator of pancreatitis, or a simple epiphenomenon. Thus, we first hypothesised that Reg4 is an intrinsic defence factor during AP. To test this hypothesis, we used a recombinant human Reg4 protein (rReg4) to investigate the role of Reg4 during experimental pancreatitis in vivo and in vitro. We found that rReg4 may protect against acinar cell necrosis in experimental pancreatitis by enhancing the expression of Bcl-2 and Bcl-xL via activation of the epidermal growth factor receptor (EGFR)/Akt signalling pathway.

Materials and methods

Animals and reagents

Male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co Ltd (Shanghai, China). Animals were maintained on a 12 h light/12 h dark cycle at 22°C, given water ad libitum, fed standard laboratory chow, and allowed to acclimatise for a minimum of 1 week. Mice were randomly assigned to control or experimental groups. All experiments were conducted with the approval of the Animal Research Committee at Shanghai Jiaotong University. l-Arginine hydrochloride, caerulein, Hoechst 33258 and propidium iodide (PI) were purchased from Sigma Chemical (Sigma-Aldrich, St. Louis, Missouri, USA). Antibodies against EGFR, p-EGFR, Akt, p-Akt, Bcl-2 and Bcl-xl were from Cell Signalling (CST, Danvers, Massachusetts, USA). Antibodies against Reg4, amylase, insulin and β-actin were from Santa Cruz Biotechnology (Santa Cruz, California, USA). Other reagents were from Sigma Chemical. rReg4 was produced in Escherichia coli and purified by fast-performance liquid chromatography as previously described.21

Induction of experimental pancreatitis

AP was induced in C57BL/6 mice (25–30 g) by administration of two intraperitoneal injections of l-arginine, each at concentrations of 4 g/kg body weight, with a 1 h interval between injections.22 Controls received similar injections of normal saline (NS). The second NS injection is defined as day 0. rReg4 (300 μg/kg body weight), heat-inactivated Reg4, neutralising antibody (200 μg/mice) or vehicle (non-immune rabbit serum) was administered to the mice as twice daily subcutaneous injections. Mice were killed at several time intervals from 2 to 4 days after the second intraperitoneal injection of l-arginine.

A second model of AP was induced in C57BL/6 mice weighing 20–25 g. After the mice had been starved for 18 h with access to water ad libitum, caerulein was administered as seven intraperitoneal injections of 50 μg/kg body weight at hourly intervals, as previously described.23 NS-injected animals served as controls. The first NS injection is defined as hour 0. rReg4 (300 μg/kg body weight), heat-inactivated Reg4, neutralising antibody (200 μg/mice) or vehicle (non-immune rabbit serum) was administered to mice as subcutaneous injections at time 0. Mice were killed at several time intervals from 9 to 21 h after the first intraperitoneal injection of caerulein.

Whole-blood samples were kept at room temperature for 2 h before centrifugation for 20 min at ∼2000×g, and serum was stored at −80°C for further studies. The pancreas was removed, placed on ice, weighed, immediately frozen in liquid nitrogen, and stored at −80°C.

Isolation of pancreatic acinar cells

Pancreatic acinar cells were isolated from mice using a collagenase digestion procedure as described previously.24–26 For treatments, the isolated acinar cells were incubated at 37°C in Dulbecco's modified Eagle's medium/Ham F-12 medium containing 10% fetal bovine serum with or without l-arginine and other agents as described in the relevant figures.

Serum amylase and lipase assay

The serum activities of amylase and lipase were measured by enzyme dynamics chemistry using commercial kits according to the manufacturer's protocols in a Roche/Hitachi modular analytics system (Roche, Mannheim, Germany).

Histological examination of the pancreas

Fresh specimens of murine pancreas were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). Tissues were embedded in paraffin, and 5 μm sections were processed for H&E staining by standard procedures.

Quantification of apoptosis

Apoptosis was quantified on pancreatic tissue or isolated acinar cell samples by the TUNEL assay. Briefly, tissues or cell smears were fixed in 4% buffered formaldehyde, embedded in paraffin, and 5 μm-thick sections were adhered to glass slides. Sections were stained using terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-labelled dUTP (TUNEL) according to the manufacturer's protocol (Roche). The numbers of apoptotic cells were counted in 10 fields at ×200 magnification, as described previously.27

Apoptosis in mouse pancreatic acinar cells was quantified using Hoechst 33258 or PI staining to visualise the nuclear chromatin morphology, as described previously.25 Briefly, cells were stained with 8 μg/ml Hoechst 33258 or 1 μg/ml PI, and were examined directly by fluorescence microscopy. Cells with nuclei containing condensed and/or fragmented chromatin were considered to be apoptotic. For quantification of apoptosis, a total of at least 3000 acinar cells were counted on cell smears for each treatment group.

Quantification of necrosis

Necrosis of murine pancreatic acinar cells was determined by the release of lactate dehydrogenase (LDH) into the incubation medium, as previously described.24–26 Cell viability was analysed using a Cell Counting Kit (CCK-8) (Dojindo, Kumamoto, Japan). Necrosis in pancreatic tissue was quantified on sections stained with H&E. The extent of acinar cell injury or necrosis was scored by two experienced morphologists, as previously described.25 28 Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into the interstitium were considered to be necrotic.

Reverse transcriptase-PCR (RT-PCR)

Total RNA was extracted from each tissue or from isolated acinar cells using the acid guanidinium/phenol/chloroform method.29 RNA was reverse-transcribed using the SuperScript II preamplification kit (Fermentas, Maryland, USA) and subjected to either real-time or conventional semiquantitative RT-PCR using gene-specific, intron-spanning primers that had been designed with software (table 1). Negative controls were performed by omitting the RT step or cDNA template from the PCR amplification. In semiquantitative RT-PCR, the target β-actin and Reg4 sequences were amplified at an annealing temperature of 52°C during 20 or 30 cycles, respectively, to yield visible products within the linear amplification range. In these experiments, cDNA derived from 400 ng total RNA was used in each sample. Resulting RT-PCR products were separated on an agarose gel and visualised by staining with ethidium bromide. The band intensities of the RT-PCR products were quantified using the imaging software. Real-time RT-PCR was performed using the 7900HT Fast Real-Time PCR System (ABI, Palo Alto, California, USA) using SYBR Green chemistry. mRNA expression was quantified by the ΔΔCt method relative to that of the β-actin used as a reference (housekeeping) control.

Table 1

Reverse transcriptase-PCR primers used for analysis of Reg4, Bcl-2, Bcl-xl and β-actin gene expression

Western blot analysis

For western blot analyses, murine pancreas was rapidly ground in liquid nitrogen or acinar cells were isolated. The resulting powder or isolated acinar cells was reconstituted in ice-cold RIPA buffer containing 1 mmol/l phenylmethanesulfonyl fluoride and a cocktail of protease inhibitors (1:100 dilution) (Sigma-Aldrich). Samples were centrifuged at 4°C for 15 min at 10 000 ×g. Supernatants were recovered, and total amounts of protein were determined using the BCA method (Pierce, Rockford, Illinois, USA). A 50 μg portion of protein was subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and then blotted following standard methods. Non-specific binding to the membrane was blocked by 5% (w/v) dry non-fat milk in Tris-buffered saline/0.05% Tween-20 (TBST) at 4°C overnight in a covered container. Blots were incubated for 1 h at room temperature with goat polyclonal anti-Reg4 antibody (1:200 dilution), rabbit polyclonal anti-EGFR (1:1000 dilution) and p-EGFR (1:1000 dilution), rabbit monoclonal anti-Akt (1:1000 dilution) and p-Akt (1:1000 dilution), rabbit monoclonal anti-Bcl-2 (1:1000 dilution), anti-Bcl-xL (1:1000 dilution) or a mouse monoclonal anti-β-actin (1:1000 dilution) diluted in 5% bovine serum albumin. Membranes were washed with TBST and incubated with a secondary donkey anti-goat IgG–horseradish peroxidase (HRP) antibody (1:2000 dilution), goat anti-rabbit IgG–HRP antibody (1:2000 dilution) or goat anti-mouse IgG–HRP antibody (1:2000 dilution) obtained from Santa Cruz Biotechnology diluted in 5% (w/v) dry non-fat milk in TBST for 1 h at room temperature. Finally, membranes were washed with TBST, developed using the ECL-detection system (Santa Cruz), quickly dried, and exposed to ECL film.

Immunofluorescence staining

Paraffin-embedded pancreas samples were deparaffinised and hydrated. Sections were microwave treated (3×5 min) in 0.01 mol/l sodium citrate buffer (pH 6), allowed to cool for 30 min, washed in PBS (3×5 min), and blocked for 20 min with 5% bovine serum albumin. Sections were then incubated for 2 h at room temperature (or overnight at 4°C) with a polyclonal antibody against Reg4 (1:100 dilution) and monoclonal antibodies against amylase (1:400 dilution) or insulin (1:400 dilution). After being rinsed in PBS, sections were incubated with a mixture of DyLight549 conjugated donkey anti-goat antibody (1:200 dilution) and DyLight488 donkey anti-mouse antibody (1:200 dilution) or DyLight488 donkey anti-rabbit antibody (1:100 dilution) for 45 min in the dark. After being rinsed in PBS, sections were incubated with 1 μg/ml 4',6-diamidino-2-phenylindole (DAPI) for 1 min. Immunofluorescence staining was analysed by fluorescence microscopy.

Statistical analysis

Results are presented as mean±SE. Statistical analysis was performed using the Mann–Whitney non-parametric U test. p<0.05 was considered to be significant.

Results

Expression of Reg4 during arginine-induced pancreatitis

RT-PCR and western blot analyses showed that, whereas Reg4 mRNA and protein expression were virtually undetectable in normal mouse pancreas, they were markedly increased during arginine-induced pancreatitis (figure 1A,B). Expression was strongly increased 2 days after induction of pancreatitis, with maximal expression observed on day 4, which decreased progressively to normal levels by day 7 (data not shown). Double immunofluorescent staining for Reg4 and amylase or insulin revealed only detectable expression of Reg4 in the pancreatic islets of control mice (figure 1C). In contrast, Reg4 was widely expressed after arginine-induced injury, with expression noted in the residual acinar cells as well as in the regenerating metaplastic epithelium (figure 1C). At day 2, only low levels of Reg4 expression were observed in residual acinar cells around the islets. This is in contrast with day 4, when significant expression of Reg4 in the regenerating metaplastic epithelium was observed. Reg4 expression at day 4 was persistent, but had abated significantly by day 7 (data not shown).

Figure 1

Expression of Reg4 during arginine-induced pancreatitis. The pancreatic tissues were harvested at 0, 2, 3 and 4 days after the second arginine injection. (A) Assessment of mRNA levels of Reg4 by semiquantitative reverse transcriptase-PCR analysis of pancreatic total RNA. β-actin mRNA was used as a housekeeping control. (B) Expression of Reg4 protein in the mouse pancreas was analysed by western blot, using a specific polyclonal antibody. (C) Double immunofluorescent staining of Reg4 (red) and amylase (green) or insulin (green) in the pancreas. 4',6-Diamidino-2-phenylindole (DAPI; blue) was used to counterstain nuclei. A general overlap of Reg4 and insulin in the normal pancreas and in areas of arginine-induced pancreatic injury was observed. At day 2, only low levels of Reg4 expression were observed in residual acinar cells around the islets. This is in contrast with day 4, when Reg4 was significantly expressed in the regenerating metaplastic epithelium. Results from one representative mouse for each group are included.

Acinar cell death pattern in arginine-induced pancreatitis

Histopathological evaluation of the H&E-stained sections of pancreas from the arginine-treated mice revealed significant accumulation of fluid, disruption of histoarchitecture, extensive acinar cell necrosis, neutrophil infiltration, and formation of tubular complexes (figure 2A–D). We measured the time-dependent changes in the extent of apoptosis and necrosis in the pancreas. At day 4, most acinar cells were lost due to acinar cell necrosis (figure 2D,M), suggesting that necrosis is the predominant form of cell death in the murine model of arginine-induced pancreatitis. Amylase immunostaining also revealed a dramatic loss of exocrine cells (figure 2E–H). We performed morphometry of the exocrine cell density, based on computer-aided area measurement of amylase protein expression, to quantitatively assess the extent of exocrine cell loss. In NS-treated mice, the exocrine density was 70.3±8.7%. At day 4, the total exocrine area was reduced to approximately 19.6% of the total pancreatic area (figure 2N). Few apoptotic cells were observed at day 2 (figure 2J,O). Although the total amount of apoptotic cells was significantly increased at days 3 and 4 (figure 2K,L), this was due primarily to extensive inflammatory cell apoptosis. Therefore, acinar cell apoptosis was less pronounced during arginine-induced pancreatitis.

Figure 2

Acinar cell death in arginine-induced pancreatitis. (A–D) H&E staining. (A) Control pancreas. (C–D) At days 3 and 4 after treatment, most acinar tissue was destroyed. (E–H) Immunofluorescence staining of amylase. (I–L) Pancreatic sections were analysed using terminal deoxynucleotidyltransferase-mediated dUTP–biotin nick-end labelling (TUNEL). (M) Percentage of necrotic cells was determined as described in Materials and Methods. (N) Morphometric analysis of amylase density compared with total area. (O) Number of apoptotic cells in 10 fields counted at ×200 magnification. Results are expressed as mean±SE (n=4 per group). DAPI, 4',6-diamidino-2-phenylindole.

rReg4 reduces acinar cell necrosis in experimental pancreatitis in vivo

Histological analysis suggested that necrosis was the predominant form of cell death during arginine-induced pancreatitis. To determine the role of Reg4 in the cell-death response of pancreatitis, we first measured the effects of rReg4 protein on acinar cell necrosis during arginine-induced pancreatitis. The severity of arginine-induced pancreatitis was evaluated by measuring the levels of amylase and lipase in the serum. Morphological evidence of the extent of acinar cell necrosis was obtained by standard histological examination. As shown in figure 3, markedly decreased amylase and lipase activities were observed in the sera of rReg4-treated mice at days 2 and 3 after the second injection of arginine (figure 3A). In addition, histological examination of the pancreas showed that the extent of acinar cell necrosis in rReg4-treated mice was less severe than that in mice treated with heat-inactivated Reg4 (figure 3C,E).

Figure 3

rReg4 reduces acinar cell necrosis in arginine- and caerulein-induced pancreatitis in vivo. Serum amylase and lipase activity were measured in control mice and mice treated with rReg4, heat-inactivated Reg4, neutralising antibody (Ab) or vehicle at 2, 3 and 4 days after the second arginine injection (A) or at 9, 15 and 21 h after the first caerulein injection (B). Representative images after 2, 3 and 4 days of arginine-induced pancreatitis are shown in (C) and at 9, 15 and 21 h after the first caerulein injection in (D). Necrosis scores of histological sections after 2, 3 and 4 days of arginine-induced pancreatitis are shown in (E) and at 9, 15 and 21 h after the first caerulein injection in (F). Results are expressed as mean±SE (n=7–9 per group in the arginine model; n=6 per group in the caerulein model). *p<0.05 vs heat-inactivated Reg4-treated group; #p<0.05 vs vehicle-treated group. Bars=50 μm.

To evaluate whether the protective effect of rReg4 against pancreatitis is a general phenomenon rather than associated with an arginine-specific mechanism, we used the caerulein-induced pancreatitis model. Again, the increase in serum amylase and lipase activity in rReg4-treated mice during caerulein-induced pancreatitis at 9, 15 and 21 h was significantly less pronounced than in mice treated with heat-inactivated Reg4 (figure 3B). Histologically (figure 3D), less tissue necrosis was observed in pancreas from rReg4-treated mice compared with pancreas from mice treated with heat-inactivated Reg4. Furthermore, histological scoring revealed that the increase in acinar necrosis (figure 3F) was significantly less prominent in pancreas from rReg4-treated mice than in pancreas from mice treated with heat-inactivated Reg4.

In mice treated with anti-Reg4 neutralising antibodies, the severity of arginine- or caerulein-induced pancreatitis was worsened, as demonstrated by increased serum amylase and lipase activity and tissue necrosis compared with non-immune serum treatment (figure 3). These findings demonstrate that Reg4 is a protective factor during AP.

Arginine induces acinar cell death in vitro

In the murine model of arginine-induced pancreatitis, we found that there was extensive acinar cell necrosis but less acinar cell apoptosis. We further assessed the pattern of cell death responses in arginine-induced acinar cell injury in vitro. The cell viability of isolated acinar cells incubated with increasing doses of arginine (0–10 mg/ml) for 0, 6, 12 and 24 h was analysed using the Cell Counting Kit-8. As expected, arginine induced death in isolated acinar cells in a time- and dose-dependent manner (figure 4A). Furthermore, by measuring LDH release, we found that arginine induced necrosis in isolated acinar cells (figure 4B). After incubation with supplemental arginine at 5 mg/ml for 12 h, acinar cells were stained with Hoechst33258/PI, and apoptosis was analysed by TUNEL. As shown in figure 4C,D, we found that arginine induced more necrosis but less apoptosis in acinar cells, suggesting that necrosis was also the predominant mode of cell death in the in vitro model of arginine-induced cell death.

Figure 4

Arginine induces acinar cell death in vitro. (A) Isolated acinar cells were incubated with increasing doses of arginine (0–10 mg/ml) for 0, 6, 12 and 24 h, and cell viability was assessed using the Cell Counting Kit-8 (CCK-8). (B) Necrosis was measured by the percentage of total cellular lactate dehydrogenase (LDH) released into the extracellular medium. Treatment with arginine resulted in a time- and dose-dependent decrease in the number of acinar cells. The data represent mean±SE from three independent experiments. (C) Acinar cells were incubated with or without 5 mg/ml arginine for 12 h. Representative photomicrographs of isolated pancreatic acinar cells stained with Hoechst 33258 and propidium iodide (PI) were obtained by fluorescent microscopy. Arrowheads indicate apoptotic cells, arrows indicate necrotic cells (original magnification ×400). (D) Acinar cell sections were analysed using terminal deoxynucleotidyltransferase-mediated dUTP–fluorescein isothiocyanate nick-end labelling (TUNEL).

rReg4 reduces arginine-induced acinar cell death by inhibiting necrosis but not apoptosis in vitro

To further elucidate the role of Reg4 in the regulation of cell-death responses, we examined the effects of rReg4 on arginine-induced acinar cell death in vitro. Interestingly, addition of rReg4 to the culture medium inhibited arginine-induced acinar cell death in a dose-dependent manner: the minimum concentration having a significant effect was 4 μg/ml (figure 5B). To further confirm whether rReg4 protects against acinar cell necrosis or apoptosis in vitro after arginine treatment, we quantified acinar cell necrosis and apoptosis by LDH release (figure 5A) and Hoechst33258/PI staining (figure 5C), respectively. As shown in figure 5, rReg4 inhibited arginine-induced acinar cell necrosis but not apoptosis.

Figure 5

rReg4 inhibits pancreatic acinar cell death induced by arginine in vitro. (A) Acinar cells were incubated with 5 mg/ml arginine and increasing doses of rReg4 (0-32 μg/ml) for 12 h, and necrosis was measured by the percentage of total cellular lactate dehydrogenase (LDH) released into the extracellular medium. (B) The cell viability of acinar cells was analysed using the Cell Counting Kit (CCK-8). The data represent mean±SE from three independent experiments. (C) Acinar cells incubated with 5 mg/ml arginine and 16 μg/ml rReg4 or phosphate-buffered saline (PBS) for 12 h were stained with Hoechst 33258 and propidium iodide (PI), and examined by fluorescent microscopy. (D) Number of necrotic and apoptotic cells in 10 fields counted at ×400 magnification. Values are given as mean±SE. *p<0.05 vs control group; #p<0.05 vs PBS-treated group.

rReg4 induces EGFR and Akt phosphorylation

The EGFR signalling pathway plays a key role in protecting against caerulein-evoked pancreatic damage.30 31 EGF family ligands can bind to the extracellular domain of EGFR and induce phosphorylation at specific tyrosine residues within the receptor cytoplasmic domain. Akt is one of the best characterised pathways triggered by growth factor receptors including EGFR.32 It has been reported that rReg4 can activate the EGFR/Akt signalling pathway in HCT116 and HT29 cells.16 To examine whether rReg4 can activate the EGFR/Akt signalling pathway in isolated acinar cells, acinar cells were incubated in the presence of increasing doses of rReg4 for 6 h. First, western blot analysis with phospho-EGFR antibodies specific to Tyr1068 was performed to assess tyrosine residue phosphorylation associated with EGFR activation. A dose-dependent increase in EGFR phosphorylation at Tyr1068 was observed after rReg4 treatment (figure 6A,B). Second, to demonstrate the involvement of EGFR signalling in Reg4-induced activation of Akt phosphorylation, phosphorylated Akt was detected by western blot analysis using an antibody specific to activated phospho-Akt Thr308. Although rReg4 treatment resulted in a significant increase in phosphorylated Akt (figure 6C,D), the total expression level of Akt did not change. These results suggest that Reg4 activates the EGFR/Akt signalling pathway in acinar cells. We also found that arginine partially induces EGFR/Akt phosphorylation (figure 6), suggesting that the EGFR/Akt signalling pathway is an important regulator of acinar cell survival.

Figure 6

rReg4 induces EGFR and Akt phosphorylation. Isolated acinar cells were incubated with 5 mg/ml arginine and 0, 4, 8, 16 and 32 μg/ml rReg4 for 6 h. Lysates were analysed by western blot using rabbit anti-phospho-EGFR antibodies specific for phospho-Tyr1068 and anti-phospho-Akt antibodies specific for phospho-Thr308. (A and B) Reg4 induces EGFR phosphorylation. Isolated acinar cells exhibit a dose-dependent induction in EGFR phosphorylation following treatment with rReg4. (C and D) EGF induces Akt phosphorylation. Isolated acinar cells exhibit a dose-dependent induction in Akt phosphorylation after treatment with rReg4. (A) and (C) show the western blots, and (B) and (D) are graphs of the band intensities determined by densitometry scanning from three representative experiments and are depicted as relative to buffer-treated controls.

rReg4 upregulates Bcl-2 and Bcl-xL expression in acinar cells

Compared with apoptosis, the signalling pathways mediating necrosis are less well understood.33–36 Recent research has indicated that the extent of Bcl-xL upregulation correlates inversely with pancreatic necrosis, but not apoptosis, in models of AP, and that Bcl-xL and Bcl-2 can protect acinar cells from necrosis in pancreatitis by stabilising mitochondria against death signals.25 We examined whether Reg4 can upregulate Bcl-2 and Bcl-xL to protect acinar cells from necrosis. Western blot analysis revealed that arginine upregulates Bcl-2 and Bcl-xL protein expression in acinar cells in vitro (figure 7A). Of note, rReg4 further increased Bcl-2 and Bcl-xL protein expression (figure 7A). Since pancreatic Bcl-2 and Bcl-xL protein levels were significantly increased in rReg4-treated acinar cells, we next examined whether upregulation occurred at the mRNA level. Quantitative analysis, using real time RT-PCR, showed that the levels of Bcl-2 and Bcl-xL transcript increased more than twofold in rReg4-treated acinar cells (figure 7B). Thus, Bcl-2 and Bcl-xL upregulation is mediated at least in part through transcriptional activation. In addition, we found that the extent of Bcl-xL upregulation correlated inversely with acinar cell necrosis in isolated acinar cells (figure 7C).

Figure 7

rReg4 upregulates expression of Bcl-2 and Bcl-xL. Isolated acinar cells were incubated with 5 mg/ml arginine and 0, 4, 8, 16 and 32 μg/ml rReg4 for 12 h. (A) Assessment of Bcl-2 and Bcl-xL proteins levels by western blot from control, buffer-treated and rReg4-treated acinar cells. β-actin was used as the loading control. The intensity of bands was measured, and the represented values correspond to the Bcl-2 and Bcl-xL/β-actin ratio. (B) Expression of Bcl-2 and Bcl-xL mRNA were quantified by real time RT-PCR and normalised to that in the control group. (C) The extent of Bcl-xL upregulation correlated inversely with acinar cell necrosis in isolated acinar cells. Values are given as mean±SE from three independent experiments. *p<0.05 vs control group; #p <0.05 vs PBS-treated group.

Discussion

In normal pancreas, Reg4 is expressed in the β cells of the endocrine pancreas at barely detectable levels.5 Here, we show for the first time that both Reg4 mRNA and protein expression were markedly upregulated in the pancreas during arginine-induced pancreatitis. Furthermore, we found that Reg4 was widely expressed in the pancreas after arginine-induced injury, with notable expression observed in the residual acinar cells around the islets. In addition, we showed that arginine could induce the expression of Bcl-2 and Bcl-xL, while rReg4 upregulated Bcl-2 and Bcl-xL expression by activating the EGFR/Akt signalling pathway. Bcl-xL expression correlated inversely with cell necrosis in isolated pancreatic acinar cells. Thus, our study suggests that, in experimental pancreatitis, Reg4 might protect against acinar cell necrosis by enhancing Bcl-2 and Bcl-xL expression via activation of the EGFR/Akt signalling pathway.

Recent data have shown that other members of the Reg family such as Reg1 and pancreatitis-associated proteins (PAP or Reg3) have protective effects against experimental pancreatitis. For example, Zhang and colleagues37 found that targeted inhibition of gene expression of PAPs by antisense oligodeoxyribonucleotides exacerbates the severity of AP in rats. Furthermore, they also reported that both small interfering RNA (siRNA)-mediated gene knockdown of PAP and administration of anti-Reg1 and anti-PAP2 antibodies worsens sodium taurocholate-induced pancreatitis.38 39 Using PAP/HIP (hepatocarcinoma intestine pancreas) knock-out mice, Gironella and colleagues23 found that PAP/HIP has anti-apoptotic and anti-inflammatory roles in vivo during caerulein-induced pancreatitis. To date, the function of Reg4, another important member of the Reg family, is poorly understood. Here, we show that it was upregulated in the murine model of arginine-induced pancreatitis. Using pharmacological approaches, we found that rReg4 can protect against acinar cell necrosis in vitro as well as in vivo during arginine- and caerulein-induced pancreatitis. Taking into account the fact that the Reg family is a group of highly conserved proteins, we propose that Reg4 acts as an intrinsic defence factor during AP.

To date, the functional signalling pathway(s) activated by Reg4 remain poorly understood. Bishnupuri and colleagues16 reported that Reg4 protein activates the EGFR/Akt/AP-1 signalling pathway in human colon cancer cell lines. Interestingly, Nanakin and colleagues15 found that EGF and transforming growth factor α enhance Reg4 gene expression via the ERK signalling pathway in the SW403 cell line, suggesting that a positive expression feedback loop may exist between EGF and Reg4 signalling pathways. Compared with apoptosis, the signalling pathways mediating necrosis are less well understood.29–32 The extent of Bcl-xL upregulation has recently been shown to correlate inversely with pancreatic necrosis, but not apoptosis, in arginine- and caerulein-induced AP models. Furthermore, Bcl-xL and Bcl-2, anti-apoptotic molecules, have been shown to protect acinar cells from necrosis in pancreatitis by stabilising mitochondria against death signals.26 These data suggest that there are some common pathways in the regulation of apoptosis and necrosis. Reg4 has been shown to induce expression of Bcl-2, Bcl-xL, survivin and matrilysin through EGFR/Akt signalling in HCT116 cells. Furthermore, rReg4 protein protected normal intestinal crypt cells from irradiation-induced apoptosis by increasing the expression of the antiapoptotic genes, Bcl-2, Bcl-xL and survivin.16 19 These results strongly suggest that Reg4 upregulates Bcl-2 and Bcl-xL expression in other cell types. In our study, we found that arginine can also upregulate Bcl-2 and Bcl-xL protein expression in acinar cells in vitro. Of note, rReg4 further increased Bcl-2 and Bcl-xL protein expression in acinar cells, and such upregulation was at the mRNA level and correlated inversely with acinar cell necrosis. Furthermore, we found that Reg4 also activated the EGFR/Akt signalling pathway in isolated acinar cells. These data enhance the current understanding of cell death mechanisms regulating AP and provide a plausible explanation for the protective effect of Reg4 on AP. In addition, previous studies have shown that Reg4 is markedly upregulated in pancreatic cancer,10 11 and that the Reg4 gene is amplified in the early stages of pancreatic cancer development,12 suggesting that Reg4 may be involved in pancreatic carcinogenesis. Based on the fact that patients with chronic pancreatitis have a high risk of pancreatic cancer,40 our observation that Reg4 upregulation follows exocrine pancreatic injury may provide a mechanistic link between chronic pancreatic injury and subsequent neoplasia.

As a result of the release of cell debris, the necrosis of pancreatic cells generates inflammation, leading to a vicious cycle of inflammation and necrosis. Several inflammatory mediators have been shown to be increased in AP, such as tumour necrosis factor (TNF)α and interleukin (IL)-1β.41 To further support the protective effect of Reg4 in AP, we examined serum levels of TNFα and IL-1β and their mRNAs in the pancreas. We found that rReg4 reduced the levels of inflammatory cytokines in the pancreas (online figure S1). In addition, acinar cell proliferation is an important factor during pancreatic regeneration after AP. Here, we found that rReg4 can also increase acinar cell proliferation (online figure S2). Interestingly, EGF, as a ligand of EGFR, can reduce the severity of caerulein-induced pancreatitis and accelerate tissue repair by improving pancreatic blood flow, increasing pancreatic cell growth and limiting the activation of cytokine release.31 Together, the beneficial effects of Reg4 appear to depend, at least in part, on inhibition of necrosis, as well as an increase in pancreatic cell growth and reduction in cytokine release.

In summary, our results strongly suggest that Reg4 protects against acinar cell necrosis at least in part by enhancing the expression of Bcl-2 and Bcl-xL via activation of the EGFR/Akt signalling pathway in experimental pancreatitis. Therefore, therapeutic approaches that enhance Reg4 expression may represent a novel strategy for preventing or attenuating necrosis in pancreatitis.

Acknowledgments

We thank Dr Aiwu Ke for language polishing and useful discussions. This work was supported in part by grant 09JC1412200 from The Science and Technology Commission of Shanghai Municipality and grant 20090072110022 from The Research Fund for the Doctoral Program of Higher Education.

References

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Supplementary materials

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    Footnotes

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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