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hydroxypyruvate reductase (GRHPR) is associated with intestinal epithelial cells apoptosis in TNBS-induced experimental colitis
Pathology – Research and Practice 212 (2016) 365–371
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Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Up regulation of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is associated with intestinal epithelial cells apoptosis in TNBS-induced experimental colitis Chunyan Zong a,1 , Xiaoke Nie b,1 , Dongmei Zhang c , Qianqian Ji a , Yongwei Qin c , Liang Wang a , Dawei Jiang a , Chen Gong a , Yifei Liu d , Guoxiong Zhou a,b,c,d,∗ a
Department of Gastroenterology, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong, Jiangsu, China Department of Nutrition and Food Hygiene, School of Public Hygiene, Nantong University, Nantong, Jiangsu, China c Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong, Jiangsu, China d Department of Pathology, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong, Jiangsu, China b
a r t i c l e
i n f o
Article history: Received 26 December 2014 Received in revised form 17 August 2015 Accepted 21 September 2015 Keywords: Crohn’s disease Glyoxylate reductase/hydroxypyruvate reductase Intestinal epithelial cells Apoptosis
a b s t r a c t Glyoxylate reductase/hydroxypyruvate reductase (GRHPR), which exists mainly in the liver, is a D-2hydroxy-acid dehydrogenase that plays a critical role in the formation of primary hyperoxaluria type 2 (PH2). Here, we investigated GRHPR expression and its potential role in both human Crohn’s disease (CD) and experimental colitis. Murine experimental colitis models were established by administration of trinitrobenzenesulphonic acid (TNBS). As shown by Western blot, significant up-regulation of GRHPR was found in TNBS-treated mice as compared with normal controls. Immunohistochemistry (IHC) also showed increased GRHPR expression, and the molecule was located in intestinal epithelial cells (IECs). This phenomenon also occurred in patients with Crohn’s disease. Besides, in an in vitro study, human IEC line HT-29 cells cultured with tumor necrosis factor ␣ (TNF-␣) were used to evaluate the changes in expression of GRHPR. Moreover, overexpression of GRHPR was accompanied by active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) accumulation. Furthermore, knock-down GRHPR could inhibit the accumulation of active caspase-3 and cleaved PARP as shown by Western blot in TNF-␣ treated HT-29 cells. Flow cytometry assay indicated that interference of GRHPR led to increasing apoptosis of IECs. These data suggested that GRHPR might exert its pro-apoptosis function in IECs. Thus, GRHPR might play an important role in regulating IECs apoptosis, and might be a potential therapeutic target for CD. © 2016 Published by Elsevier GmbH.
1. Introduction Crohn’s disease (CD) and ulcerative colitis (UC) are two forms of inflammatory bowel disease (IBD) that develop chronic and relapsing inflammation in the gastrointestinal tract [1,5,13,18]. Although both of the diseases have certain characteristics in common, they present different pathophysiological features. In UC, inflammation involves the superficial mucosal and submucosal layer. However, inflammation in Crohn’s disease is transmural, and affects any part of the gastrointestinal tract, especially ileum [4,8,22]. Although the pathogenesis of IBD remains unknown, a number of reports have demonstrated that genetic, environmental and immunological
∗ Corresponding author at: Department of Gastroenterology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu, China. E-mail address: [email protected] (G. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.prp.2015.09.019 0344-0338/© 2016 Published by Elsevier GmbH.
factors were responsible for IBD initiation and progression [4,12]. It is well known that IECs can protect the intestinal wall against luminal antigens and bacteria [23], and intestinal mucosal integrity and barrier function are disrupted in case of IECs apoptosis [11,24]. Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from the liver. Most previous studies indicated that deficiency of this enzyme was the underlying cause of primary hyperoxaluria type 2 (PH2) and could lead to increased urinary oxalate levels, formation of kidney stones and renal failure [3]. Except for its metabolic function, a recent study revealed a lower expression of GRHPR in liver tumor tissues than in non-tumoral tissues. This study demonstrates that GRHPR was negatively correlated with tumor proliferation [20]. So we presumed that the molecule expression was different in our murine models. Unexpectedly, we found that GRHPR was significantly up-regulated both in experimental colitis and CD patients, and located in IECs. Owing to this finding, we tried
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to define the role that GRHPR plays. Since abnormal apoptosis was commonly seen in CD, we then hypothesized that GRHPR is involved in the pathogenesis of colitis by mediating IECs apoptosis. This study is the first to report on GRHPR in IECs. The expression level was increased in murine experimental colitis and CD patients. In our vitro study, TNF-␣ treated human IEC line HT29 cells were used to establish an apoptosis model. Besides, GRHPR was knocked down by siRNA to identify whether it was involved in IECs apoptosis. Our research suggested that GRHPR might play an important role in promoting overactive apoptosis of IECs, and that it might be a potential therapeutic target for CD. 2. Materials and methods 2.1. Animals and induction of colitis All animal surgical interventions were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and approved by the Chinese National Committee to Use of Experimental Animals for Medical Purposes, Jiangsu Branch. Female BALB/c mice with an average weight of 17–20 g were obtained from the Animal Center, Medical College of Nantong University. Mice were randomized into 3 groups: (1) untreated animals; (2) animals treated with 50% ethanol; (3) animals receiving TNBS (Sigma Chemical Co., St. Louis, MO, USA). Experimental colitis was induced by TNBS according to a published method [12]. To induce colitis, mice were forbidden to eat overnight, and were anesthetized through an intraperitoneal injection of Sodium pentobarbital (0.6% solution). Then, 0.1 ml of a 2.5% (w/v) TNBS solution in 50% ethanol was administered into the colon lumen via a 1 ml syringe fitted to a 3.5F catheter. The catheter was then inserted carefully into the rectum, so that the tip was 4 cm proximal to the anal verge, and the mice were held in a vertical position for 1 min after instillation to ensure TNBS distributed throughout the colon. 2.2. Histological analysis of experimental colitis Body weight changes were recorded daily. According to previous studies and our recent work, the peak of inflammation is on day 3 [10,26,30]. So mice were sacrificed on day 3. The colon was removed, cleared of feces, and subsequently cut into small sections. The samples were fixed in 4% formalin, embedded in paraffin, cut into 5 m sections, and then stained with both hematoxylin and eosin (H&E). The histological score of microscopic cross-sections was as follows: 0. no signs of inflammation; 1. very low level; 2. low level of leukocyte infiltration; 3. high level of leukocyte infiltration, high vascular density, thickening of the colon wall; 4. transmural infiltrations, loss of goblet cells, high vascular density, and thickening of the colon wall [19]. 2.3. Cell culture and stimulation Human IEC line HT-29 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C, 5% CO2 . To study apoptosis, HT29 cells were seeded into 60 mm dishes and incubated overnight, then incubated in low-concentration serum (1% fetal bovine serum) with 100 ng/ml TNF-␣ (Human, Sigma). The cells were harvested after treatment. 2.4. Antibodies and reagents The antibodies used in this study were as follows: GRHPR (antirabbit, 1:1000; Santa Cruz), active caspase-3 (anti-rabbit, 1:1000;
cell-signaling), cleaved poly ADP-ribose polymerase (PARP) (antirabbit, 1:2000; cell-signaling), GAPDH (anti-rabbit, 1:3000; Santa Cruz), -actin (anti-mouse, 1:1000; Santa Cruz). 2.5. Western blot The colon tissues were cut into small sections and immediately frozen at −80 ◦ C. To prepare lysates, frozen colon tissue samples were minced with eye scissors on ice, then homogenized in lysis buffer (1% NP-4 0, 50 mmol/L Tris, pH = 7.5, 5 mmol/L EDTA, 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% TritonX 100, 1 mmol/L PMSF, 10 g/ml aprotinin, and 1 g/ml leupeptin) and centrifuged at 12,000 rpm and 4 ◦ C for 20 min to collect the supernatant. Cell cultures for Western blot were lysed in buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and protease inhibitor mixture) followed by centrifugation to discard cell debris. The protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% dried skim milk for 2 h and incubated with primary antibodies at 4 ◦ C overnight, then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The band density was measured by a computer-assisted image analysis system (Adobe Systems, San Jose, CA). 2.6. Sections and Immunohistochemistry Tissues were fixed in formalin and transferred to 20% sucrose, 30% sucrose. Mucosal biopsy specimens were collected from CD patients (n = 10) and normal ones (n = 10). Colon tissues embedded in paraffin were cut at 5 m, deparaffinized, and rehydrated. Thereafter, all sections were processed in 10 mM citrate buffer and heated to 121 ◦ C for 20 min. Endogenous peroxidase activity was blocked by soaking in 3% hydrogen peroxide. After washing in PBS (pH = 7.2), the sections were incubated with the primary antibody against GRHPR (diluted 1:100) for 4 h at room temperature. After rinsing in PBS, the peroxidase reaction was visualized by incubating the sections with DAB (0.02% diaminobenzidine tetrahydrochloride, 0.1% phosphate buffer solution, and 3% H2 O2 ). After washing in water, the sections were counterstained with hematoxylin, dehydrated, and cover slipped. 2.7. siRNA and transfection The GRHPR-targeting sequences of siRNA oligos were: 5 -GGACAAGAGGAUCCUGGAUTT-3 , 5 -GCAGUCUCCCUGCUACUUATT-3 , 5 -CCUGAGGAAGCAGCAGAAUTT-3 , named siRNA#1, #2, and #3, respectively. The control target was 5 -UUCUCCGAACGUGUCACGU-3 . HT-29 cells were seeded the day before transfection. Transient transfection was carried out using lipofectamine 2000 in accordance with the manufacturer’s instructions [27], and cells were incubated 6 h in 1640 medium with no serum or antibiotics. The media were replaced with 10% FBS-containing 1640 medium, and cells were cultured for further 48 h before use. 2.8. Flow cytometry assay In order to evaluate HT-29 cell apoptosis, flow cytometry assay was established using Annexin-V/PI staining. Annexin V-FITC Apoptosis Detection Kit (Southern Biotechnology, Birmingham, AL, USA) was used in accordance with the manufacturer’s protocol. After giving different treatment measures, cells were washed by PBS, and stained with 100 l labeling solution containing FITClabeled annexin-V and PI for 15 min at room temperature in the
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dark. Then, signals from cells were determined by flow cytometer (Becton Dickinson, San Jose, CA).
2.9. Statistical analysis All data were analyzed with Stata 7.0 statistics software. Values were expressed as mean ± SEM. The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey’s post hoc multiple comparison tests. P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition.
3. Results
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3.2. GRHPR was up-regulated in TNBS-induced colitis To identify the levels of GRHPR expression between TNBStreated, sham-operated and untreated groups, immunoblot analysis was performed. Since the peak of inflammation was on day 3, we used both typical protein and colonic sections on day 3. The level of GRHPR protein was obviously increased in the TNBS-treated groups (Fig. 2A, P < 0.05). Moreover, we applied immunohistochemical analysis to explore the location of GRHPR in murine models. The TNBS-treated groups showed stronger staining of GRHPR in IECs, and the molecule was located in cytoplasm (Fig. 2B). All these findings demonstrated that GRHPR protein was elevated in TNBS-induced experimental colitis, and located in cytoplasm of IECs.
3.1. Experimental colitis was established by TNBS
3.3. GRHPR might be involved in IECs apoptosis in vivo
Since the clinical and morphological features of TNBS induced colitis resembles human CD, this model has been used to study CD as described in previous studies [12]. Compared with control groups, the TNBS-treated BALB/c mice developed colitis with classical characteristics of diarrhea, severe weight loss (Fig. 1A, P < 0.05). Several previous studies [10,30] and our recent work [26] have proved that the peak of inflammation level was on day 3, then colons were taken on day 3. HE-staining analysis revealed that a strong infiltration of inflammatory cells, a transmural diffuse inflammation, edema, thickened colon wall, epithelial damage, and disruption of crypt structure were observed during the course of TNBS-induced colitis (Fig. 1B). The histological score was higher in experimental colitis than in control groups (Fig. 1C, P < 0.05). In addition, there were minor differences in body weight changes and the histological scores between ETOH-treated and untreated control groups. So we used ETOH-treated and TNBS-treated mice for the subsequent research.
Many studies report that increased IECs apoptosis can be tested in TNBS-induced experimental colitis [21] We performed immunoblot to examine the expression of active caspase-3 and cleaved PARP, which are biochemical markers for apoptosis. As we predicted, they were markedly increased in murine experimental colitis models. At the same time, accumulation of GRHPR could also be observed (Fig. 2A, P < 0.05). The results indicated that GRHPR might play an important role in IECs apoptosis. 3.4. GRHPR might be involved in TNF-˛-induced apoptosis in HT-29 cells Since several studies have demonstrated that increased cytokine production, such as TNF, IL-1, and interferon family members, can result in IECs abnormal apoptosis [21], we then incubated human IEC line HT-29 cells with TNF-␣ to establish an apoptosis model. To determine the most effective dose of TNF-␣ capable of
Fig. 1. Mouse model of experimental colitis was induced by administration of TNBS. BALB/c mice were used to establish experimental colitis models through intrarectal TNBS as indicated in Section 2. (A) Body weight changes after TNBS or ETOH treatment of BALB/c mice and normal ones. Data were shown as mean ± SEM (n = 3 of each group, *P < 0.05). (B) Representative light microscopy images of H&E stained colonic tissues from normal, ETOH or TNBS treated mice. Scale bars 200 m. (C) Histological scoring performed in HE-stained sections. Data were shown as mean ± SEM (n = 3 of each group, *P < 0.05).
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Fig. 2. GRHPR was up-regulated in experimental colitis, and might be involved in IECs apoptosis. (A) Representative immunoblots of GRHPR, active caspase-3 and cleaved PARP in protein extracts of colons from normal, ETOH and TNBS treated mice. They were all markedly increased after TNBS treatment. The bar graph indicated the density of GRHPR, active caspase-3 and cleaved PARP normalized to -actin. Data was shown as mean ± SEM (n = 3, *, **, ***P < 0.05). (B) Immunohistochemical analysis against GRHPR in colon sections between ETOH and TNBS treated groups. Intensive staining of GRHPR was detected in TNBS group, and located in IECs. Scale bar, left column: 100 m; right column: 50 m.
Fig. 3. GRHPR was up-regulated in TNF-␣ treated HT-29 cells, and knockdown of GRHPR decreased TNF-␣ induced apoptosis. (A) Western blot was performed to identify the protein levels of GRHPR at different concentrations of TNF-␣. GRHPR expression and apoptosis reached the peak when incubated with 100 ng/ml TNF-␣. The bar graph indicated the density of GRHPR, active caspase-3 and cleaved PARP normalized to GAPDH. Data were shown as mean ± SEM (*, **, ***P < 0.05). (B) Upon 100 ng/ml TNF-␣ stimulation, GRHPR expression was up-regulated gradually accompanied with active caspase-3 and cleaved PARP time dependently. The bar graph indicated the density of the three molecules normalized to GAPDH at each time point. Data were shown as mean ± SEM (*, **, ***P < 0.05).
inducing IECs apoptosis, we incubated these cells with 0, 0.1, 1, 10 or 100 ng/ml TNF-␣ for 24 h. Results were shown by representative Western blots, and we found that the level of active caspase-3 and cleaved PARP increased. The correlation between them and GRHPR was TNF-␣ dose-dependent (Fig. 3A, P < 0.05). Based on this observation, we continued all our following experiments with a concentration of 100 ng/ml TNF-␣. To determine the time course of TNF-␣ induced apoptosis and GRHPR expression, we treated HT-29 cells with 100 ng/ml TNF-␣ for 4, 8, 12, 24, 48 and 72 h, and discovered that GRHPR protein level was significantly up-regulated in a time-dependent way accompanied with active caspase-3, cleaved PARP accumulation (Fig. 3B, P < 0.05).
3.5. Knock-down of GRHPR attenuated IECs apoptosis To further investigate the function of GRHPR on HT-29 cells apoptosis, we transfected cells with either control siRNA or GRHPRspecific siRNA. To verify the efficiency of the siRNA knockdown, Western blot was performed. The result demonstrated that the expression of GRHPR was dramatically reduced after transfection of GRHPR-targeting siRNA oligos as compared with control siRNAtreated cells, especially in #1siRNA group (Fig. 4A, P < 0.05). Besides, Western blot analysis revealed that down-regulation of GRHPR decreased active caspase-3 and cleaved PARP expression by administrating 100 ng/ml TNF-␣ (Fig. 4B, P < 0.05). To further explore the
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Fig. 4. Knock-down of GRHPR attenuated IECs apoptosis. (A) HT-29 cells were transfected with GRHPR-specific siRNA, control siRNA, and then incubated for another 48 h. Verification of GRHPR knocking down efficiency was evaluated by Western blot. The bar graph indicated the density of GRHPR versus GAPDH. Data were shown as mean ± SEM (*P < 0.05). (B) Knocking GRHPR down led to decreased active caspase-3 and cleaved PARP accumulation in TNF-␣ treated HT-29 cells. The bar graph indicated the density of cleaved PARP and active caspase-3 normalized to GAPDH. Data were shown as mean ± SEM (*, **, ***P < 0.05). (C) Annexin V/PI staining assay indicated that depletion of GRHPR attenuated TNF-␣ induced HT-29 cell apoptosis.
role of GRHPR in TNF-␣ induced HT-29 cells apoptosis, the viability and apoptosis of cells after GRHPR depletion were evaluated by flow cytometry assay. The result showed that, as compared with the control-siRNA group, cell apoptosis was significantly reduced when GRHPR was knocked down after incubation with 100 ng/ml TNF-␣ (Fig. 4C). All the data indicated that GRHPR might promote IECs apoptosis.
3.6. GRHPR was also over-expressed in CD patients To identify the correlation of these findings with human disease, we performed immunohistochemical analysis of GRHPR in human colonic biopsy specimens from normal controls and CD patients. In normal human beings, only a low level of GRHPR protein was detectable (Fig. 5). In contrast, the tissue specimens from patients
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Fig. 5. GRHPR was strongly expressed in colonic biopsies from patients with active CD. Representative images of GRHPR staining in colonic biopsies derived from normal controls (n = 10) and patients with active CD (n = 10). The result was the same with experimental colitis. Scale bar, left column: 100 m; right column: 50 m.
with CD featured strong GRHPR staining in the epithelial layer, indicating that the protein was highly expressed in IECs in CD patients (Fig. 5). These data were in accordance with our previous results in experimental colitis, and underline the clinical significance of our experiments. 4. Discussion The inflammatory bowel disease is thought to be due to abnormal activation of the intestinal mucosal immune system and environmental factors [18]. Recently, the morbidity of IBD has increased markedly. Due to the high incidence rate of IBD, the pathogenesis of IBD has attracted great attention. In this study, we found that GRHPR played an important role in IECs apoptosis. We found that the level of GRHPR protein was up-regulated in TNBS-induced colitis as compared with controls using Western blot. Furthermore, immunohistochemical analysis showed that GRHPR were mainly located in IECs. When knocking down GRHPR expression in HT-29 cells, both Western blot and flow cytometry assay showed that cell apoptosis was significantly inhibited. To investigate the clinical significance of GRHPR up-regulation in CD, we used inflamed and normal colonic biopsy samples obtained respectively from CD patients and normal controls. It was verified that GRHPR showed higher expression in CD patients, and was mainly located in intestinal epithelial cells. The data were in accordance with those of murine experimental colitis. GRHPR, also called GlxR, was a cAMP-responsive regulator, and was confirmed as the transcriptional repressor of the aceB gene, encoding malate synthase of the glyoxylate cycle [6,14]. It not only activated gapA expression [28,29] but also participated in various physiological functions, including carbon and nitrogen metabolism, respiration, SOS and stress responses, and cell division [2,16,17,29]. Only little is known about GRHPR except for
the fact that its dysfunction could result in the formation of primary hyperoxaluria type 2 (PH2) [7,15], which was regulated by peroxisome proliferator-activated receptor ␣ (PPAR ␣) [9]. However, one recent study demonstrated that GRHPR was negatively correlated with HCC proliferation [25]. In our study, increased GRHPR expression was also observed in murine experimental colitis and CD patients. Since enterocyte apoptosis is a typical characteristic of CD, we speculated that GRHPR might participate in IECs apoptosis. Then we gave 100 ng/ml TNF-␣ to stimulate HT-29 cells to establish an IEC in vitro apoptosis model, which demonstrated a time-dependent increase in GRHPR expression with accumulation of active caspase-3 and cleaved PARP. Furthermore, Western blot and flow cytometry assay showed that knockdown of GRHPR by siRNA caused decreased apoptosis in IECs. Taken together, these results revealed that GRHPR might be involved in the intestinal epithelium aberrant apoptosis. Because GRHPR is a metabolism enzyme, it is still unknown whether the enzyme itself or its metabolites lead to activation of apoptotic pathway. In summary, we investigated GRHPR expression in colonic tissues with or without inflammation. We found that the expression of GRHPR was up-regulated in human CD and experimental colitis, and the molecule was mainly located in IECs. Furthermore, knockdown of GRHPR expression could inhibit IECs apoptosis, indicating that GRHPR might play an important role in promoting overactive apoptosis in murine experimental colitis and CD patients. However, further studies are warranted to explore the molecular mechanism of GRHPR-regulating apoptosis in intestinal homeostasis. Acknowledgments This study was supported by the Natural Science Foundation of China (No. 81201252), the Jiangsu Province’s Key Provincial Talents
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Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Up regulation of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is associated with intestinal epithelial cells apoptosis in TNBS-induced experimental colitis Chunyan Zong a,1 , Xiaoke Nie b,1 , Dongmei Zhang c , Qianqian Ji a , Yongwei Qin c , Liang Wang a , Dawei Jiang a , Chen Gong a , Yifei Liu d , Guoxiong Zhou a,b,c,d,∗ a
Department of Gastroenterology, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong, Jiangsu, China Department of Nutrition and Food Hygiene, School of Public Hygiene, Nantong University, Nantong, Jiangsu, China c Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong, Jiangsu, China d Department of Pathology, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong, Jiangsu, China b
a r t i c l e
i n f o
Article history: Received 26 December 2014 Received in revised form 17 August 2015 Accepted 21 September 2015 Keywords: Crohn’s disease Glyoxylate reductase/hydroxypyruvate reductase Intestinal epithelial cells Apoptosis
a b s t r a c t Glyoxylate reductase/hydroxypyruvate reductase (GRHPR), which exists mainly in the liver, is a D-2hydroxy-acid dehydrogenase that plays a critical role in the formation of primary hyperoxaluria type 2 (PH2). Here, we investigated GRHPR expression and its potential role in both human Crohn’s disease (CD) and experimental colitis. Murine experimental colitis models were established by administration of trinitrobenzenesulphonic acid (TNBS). As shown by Western blot, significant up-regulation of GRHPR was found in TNBS-treated mice as compared with normal controls. Immunohistochemistry (IHC) also showed increased GRHPR expression, and the molecule was located in intestinal epithelial cells (IECs). This phenomenon also occurred in patients with Crohn’s disease. Besides, in an in vitro study, human IEC line HT-29 cells cultured with tumor necrosis factor ␣ (TNF-␣) were used to evaluate the changes in expression of GRHPR. Moreover, overexpression of GRHPR was accompanied by active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) accumulation. Furthermore, knock-down GRHPR could inhibit the accumulation of active caspase-3 and cleaved PARP as shown by Western blot in TNF-␣ treated HT-29 cells. Flow cytometry assay indicated that interference of GRHPR led to increasing apoptosis of IECs. These data suggested that GRHPR might exert its pro-apoptosis function in IECs. Thus, GRHPR might play an important role in regulating IECs apoptosis, and might be a potential therapeutic target for CD. © 2016 Published by Elsevier GmbH.
1. Introduction Crohn’s disease (CD) and ulcerative colitis (UC) are two forms of inflammatory bowel disease (IBD) that develop chronic and relapsing inflammation in the gastrointestinal tract [1,5,13,18]. Although both of the diseases have certain characteristics in common, they present different pathophysiological features. In UC, inflammation involves the superficial mucosal and submucosal layer. However, inflammation in Crohn’s disease is transmural, and affects any part of the gastrointestinal tract, especially ileum [4,8,22]. Although the pathogenesis of IBD remains unknown, a number of reports have demonstrated that genetic, environmental and immunological
∗ Corresponding author at: Department of Gastroenterology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu, China. E-mail address: [email protected] (G. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.prp.2015.09.019 0344-0338/© 2016 Published by Elsevier GmbH.
factors were responsible for IBD initiation and progression [4,12]. It is well known that IECs can protect the intestinal wall against luminal antigens and bacteria [23], and intestinal mucosal integrity and barrier function are disrupted in case of IECs apoptosis [11,24]. Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from the liver. Most previous studies indicated that deficiency of this enzyme was the underlying cause of primary hyperoxaluria type 2 (PH2) and could lead to increased urinary oxalate levels, formation of kidney stones and renal failure [3]. Except for its metabolic function, a recent study revealed a lower expression of GRHPR in liver tumor tissues than in non-tumoral tissues. This study demonstrates that GRHPR was negatively correlated with tumor proliferation [20]. So we presumed that the molecule expression was different in our murine models. Unexpectedly, we found that GRHPR was significantly up-regulated both in experimental colitis and CD patients, and located in IECs. Owing to this finding, we tried
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to define the role that GRHPR plays. Since abnormal apoptosis was commonly seen in CD, we then hypothesized that GRHPR is involved in the pathogenesis of colitis by mediating IECs apoptosis. This study is the first to report on GRHPR in IECs. The expression level was increased in murine experimental colitis and CD patients. In our vitro study, TNF-␣ treated human IEC line HT29 cells were used to establish an apoptosis model. Besides, GRHPR was knocked down by siRNA to identify whether it was involved in IECs apoptosis. Our research suggested that GRHPR might play an important role in promoting overactive apoptosis of IECs, and that it might be a potential therapeutic target for CD. 2. Materials and methods 2.1. Animals and induction of colitis All animal surgical interventions were carried out in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and approved by the Chinese National Committee to Use of Experimental Animals for Medical Purposes, Jiangsu Branch. Female BALB/c mice with an average weight of 17–20 g were obtained from the Animal Center, Medical College of Nantong University. Mice were randomized into 3 groups: (1) untreated animals; (2) animals treated with 50% ethanol; (3) animals receiving TNBS (Sigma Chemical Co., St. Louis, MO, USA). Experimental colitis was induced by TNBS according to a published method [12]. To induce colitis, mice were forbidden to eat overnight, and were anesthetized through an intraperitoneal injection of Sodium pentobarbital (0.6% solution). Then, 0.1 ml of a 2.5% (w/v) TNBS solution in 50% ethanol was administered into the colon lumen via a 1 ml syringe fitted to a 3.5F catheter. The catheter was then inserted carefully into the rectum, so that the tip was 4 cm proximal to the anal verge, and the mice were held in a vertical position for 1 min after instillation to ensure TNBS distributed throughout the colon. 2.2. Histological analysis of experimental colitis Body weight changes were recorded daily. According to previous studies and our recent work, the peak of inflammation is on day 3 [10,26,30]. So mice were sacrificed on day 3. The colon was removed, cleared of feces, and subsequently cut into small sections. The samples were fixed in 4% formalin, embedded in paraffin, cut into 5 m sections, and then stained with both hematoxylin and eosin (H&E). The histological score of microscopic cross-sections was as follows: 0. no signs of inflammation; 1. very low level; 2. low level of leukocyte infiltration; 3. high level of leukocyte infiltration, high vascular density, thickening of the colon wall; 4. transmural infiltrations, loss of goblet cells, high vascular density, and thickening of the colon wall [19]. 2.3. Cell culture and stimulation Human IEC line HT-29 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C, 5% CO2 . To study apoptosis, HT29 cells were seeded into 60 mm dishes and incubated overnight, then incubated in low-concentration serum (1% fetal bovine serum) with 100 ng/ml TNF-␣ (Human, Sigma). The cells were harvested after treatment. 2.4. Antibodies and reagents The antibodies used in this study were as follows: GRHPR (antirabbit, 1:1000; Santa Cruz), active caspase-3 (anti-rabbit, 1:1000;
cell-signaling), cleaved poly ADP-ribose polymerase (PARP) (antirabbit, 1:2000; cell-signaling), GAPDH (anti-rabbit, 1:3000; Santa Cruz), -actin (anti-mouse, 1:1000; Santa Cruz). 2.5. Western blot The colon tissues were cut into small sections and immediately frozen at −80 ◦ C. To prepare lysates, frozen colon tissue samples were minced with eye scissors on ice, then homogenized in lysis buffer (1% NP-4 0, 50 mmol/L Tris, pH = 7.5, 5 mmol/L EDTA, 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% TritonX 100, 1 mmol/L PMSF, 10 g/ml aprotinin, and 1 g/ml leupeptin) and centrifuged at 12,000 rpm and 4 ◦ C for 20 min to collect the supernatant. Cell cultures for Western blot were lysed in buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and protease inhibitor mixture) followed by centrifugation to discard cell debris. The protein samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% dried skim milk for 2 h and incubated with primary antibodies at 4 ◦ C overnight, then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The band density was measured by a computer-assisted image analysis system (Adobe Systems, San Jose, CA). 2.6. Sections and Immunohistochemistry Tissues were fixed in formalin and transferred to 20% sucrose, 30% sucrose. Mucosal biopsy specimens were collected from CD patients (n = 10) and normal ones (n = 10). Colon tissues embedded in paraffin were cut at 5 m, deparaffinized, and rehydrated. Thereafter, all sections were processed in 10 mM citrate buffer and heated to 121 ◦ C for 20 min. Endogenous peroxidase activity was blocked by soaking in 3% hydrogen peroxide. After washing in PBS (pH = 7.2), the sections were incubated with the primary antibody against GRHPR (diluted 1:100) for 4 h at room temperature. After rinsing in PBS, the peroxidase reaction was visualized by incubating the sections with DAB (0.02% diaminobenzidine tetrahydrochloride, 0.1% phosphate buffer solution, and 3% H2 O2 ). After washing in water, the sections were counterstained with hematoxylin, dehydrated, and cover slipped. 2.7. siRNA and transfection The GRHPR-targeting sequences of siRNA oligos were: 5 -GGACAAGAGGAUCCUGGAUTT-3 , 5 -GCAGUCUCCCUGCUACUUATT-3 , 5 -CCUGAGGAAGCAGCAGAAUTT-3 , named siRNA#1, #2, and #3, respectively. The control target was 5 -UUCUCCGAACGUGUCACGU-3 . HT-29 cells were seeded the day before transfection. Transient transfection was carried out using lipofectamine 2000 in accordance with the manufacturer’s instructions [27], and cells were incubated 6 h in 1640 medium with no serum or antibiotics. The media were replaced with 10% FBS-containing 1640 medium, and cells were cultured for further 48 h before use. 2.8. Flow cytometry assay In order to evaluate HT-29 cell apoptosis, flow cytometry assay was established using Annexin-V/PI staining. Annexin V-FITC Apoptosis Detection Kit (Southern Biotechnology, Birmingham, AL, USA) was used in accordance with the manufacturer’s protocol. After giving different treatment measures, cells were washed by PBS, and stained with 100 l labeling solution containing FITClabeled annexin-V and PI for 15 min at room temperature in the
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dark. Then, signals from cells were determined by flow cytometer (Becton Dickinson, San Jose, CA).
2.9. Statistical analysis All data were analyzed with Stata 7.0 statistics software. Values were expressed as mean ± SEM. The statistical significance of differences between groups was determined by one-way analysis of variance followed by Tukey’s post hoc multiple comparison tests. P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition.
3. Results
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3.2. GRHPR was up-regulated in TNBS-induced colitis To identify the levels of GRHPR expression between TNBStreated, sham-operated and untreated groups, immunoblot analysis was performed. Since the peak of inflammation was on day 3, we used both typical protein and colonic sections on day 3. The level of GRHPR protein was obviously increased in the TNBS-treated groups (Fig. 2A, P < 0.05). Moreover, we applied immunohistochemical analysis to explore the location of GRHPR in murine models. The TNBS-treated groups showed stronger staining of GRHPR in IECs, and the molecule was located in cytoplasm (Fig. 2B). All these findings demonstrated that GRHPR protein was elevated in TNBS-induced experimental colitis, and located in cytoplasm of IECs.
3.1. Experimental colitis was established by TNBS
3.3. GRHPR might be involved in IECs apoptosis in vivo
Since the clinical and morphological features of TNBS induced colitis resembles human CD, this model has been used to study CD as described in previous studies [12]. Compared with control groups, the TNBS-treated BALB/c mice developed colitis with classical characteristics of diarrhea, severe weight loss (Fig. 1A, P < 0.05). Several previous studies [10,30] and our recent work [26] have proved that the peak of inflammation level was on day 3, then colons were taken on day 3. HE-staining analysis revealed that a strong infiltration of inflammatory cells, a transmural diffuse inflammation, edema, thickened colon wall, epithelial damage, and disruption of crypt structure were observed during the course of TNBS-induced colitis (Fig. 1B). The histological score was higher in experimental colitis than in control groups (Fig. 1C, P < 0.05). In addition, there were minor differences in body weight changes and the histological scores between ETOH-treated and untreated control groups. So we used ETOH-treated and TNBS-treated mice for the subsequent research.
Many studies report that increased IECs apoptosis can be tested in TNBS-induced experimental colitis [21] We performed immunoblot to examine the expression of active caspase-3 and cleaved PARP, which are biochemical markers for apoptosis. As we predicted, they were markedly increased in murine experimental colitis models. At the same time, accumulation of GRHPR could also be observed (Fig. 2A, P < 0.05). The results indicated that GRHPR might play an important role in IECs apoptosis. 3.4. GRHPR might be involved in TNF-˛-induced apoptosis in HT-29 cells Since several studies have demonstrated that increased cytokine production, such as TNF, IL-1, and interferon family members, can result in IECs abnormal apoptosis [21], we then incubated human IEC line HT-29 cells with TNF-␣ to establish an apoptosis model. To determine the most effective dose of TNF-␣ capable of
Fig. 1. Mouse model of experimental colitis was induced by administration of TNBS. BALB/c mice were used to establish experimental colitis models through intrarectal TNBS as indicated in Section 2. (A) Body weight changes after TNBS or ETOH treatment of BALB/c mice and normal ones. Data were shown as mean ± SEM (n = 3 of each group, *P < 0.05). (B) Representative light microscopy images of H&E stained colonic tissues from normal, ETOH or TNBS treated mice. Scale bars 200 m. (C) Histological scoring performed in HE-stained sections. Data were shown as mean ± SEM (n = 3 of each group, *P < 0.05).
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Fig. 2. GRHPR was up-regulated in experimental colitis, and might be involved in IECs apoptosis. (A) Representative immunoblots of GRHPR, active caspase-3 and cleaved PARP in protein extracts of colons from normal, ETOH and TNBS treated mice. They were all markedly increased after TNBS treatment. The bar graph indicated the density of GRHPR, active caspase-3 and cleaved PARP normalized to -actin. Data was shown as mean ± SEM (n = 3, *, **, ***P < 0.05). (B) Immunohistochemical analysis against GRHPR in colon sections between ETOH and TNBS treated groups. Intensive staining of GRHPR was detected in TNBS group, and located in IECs. Scale bar, left column: 100 m; right column: 50 m.
Fig. 3. GRHPR was up-regulated in TNF-␣ treated HT-29 cells, and knockdown of GRHPR decreased TNF-␣ induced apoptosis. (A) Western blot was performed to identify the protein levels of GRHPR at different concentrations of TNF-␣. GRHPR expression and apoptosis reached the peak when incubated with 100 ng/ml TNF-␣. The bar graph indicated the density of GRHPR, active caspase-3 and cleaved PARP normalized to GAPDH. Data were shown as mean ± SEM (*, **, ***P < 0.05). (B) Upon 100 ng/ml TNF-␣ stimulation, GRHPR expression was up-regulated gradually accompanied with active caspase-3 and cleaved PARP time dependently. The bar graph indicated the density of the three molecules normalized to GAPDH at each time point. Data were shown as mean ± SEM (*, **, ***P < 0.05).
inducing IECs apoptosis, we incubated these cells with 0, 0.1, 1, 10 or 100 ng/ml TNF-␣ for 24 h. Results were shown by representative Western blots, and we found that the level of active caspase-3 and cleaved PARP increased. The correlation between them and GRHPR was TNF-␣ dose-dependent (Fig. 3A, P < 0.05). Based on this observation, we continued all our following experiments with a concentration of 100 ng/ml TNF-␣. To determine the time course of TNF-␣ induced apoptosis and GRHPR expression, we treated HT-29 cells with 100 ng/ml TNF-␣ for 4, 8, 12, 24, 48 and 72 h, and discovered that GRHPR protein level was significantly up-regulated in a time-dependent way accompanied with active caspase-3, cleaved PARP accumulation (Fig. 3B, P < 0.05).
3.5. Knock-down of GRHPR attenuated IECs apoptosis To further investigate the function of GRHPR on HT-29 cells apoptosis, we transfected cells with either control siRNA or GRHPRspecific siRNA. To verify the efficiency of the siRNA knockdown, Western blot was performed. The result demonstrated that the expression of GRHPR was dramatically reduced after transfection of GRHPR-targeting siRNA oligos as compared with control siRNAtreated cells, especially in #1siRNA group (Fig. 4A, P < 0.05). Besides, Western blot analysis revealed that down-regulation of GRHPR decreased active caspase-3 and cleaved PARP expression by administrating 100 ng/ml TNF-␣ (Fig. 4B, P < 0.05). To further explore the
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Fig. 4. Knock-down of GRHPR attenuated IECs apoptosis. (A) HT-29 cells were transfected with GRHPR-specific siRNA, control siRNA, and then incubated for another 48 h. Verification of GRHPR knocking down efficiency was evaluated by Western blot. The bar graph indicated the density of GRHPR versus GAPDH. Data were shown as mean ± SEM (*P < 0.05). (B) Knocking GRHPR down led to decreased active caspase-3 and cleaved PARP accumulation in TNF-␣ treated HT-29 cells. The bar graph indicated the density of cleaved PARP and active caspase-3 normalized to GAPDH. Data were shown as mean ± SEM (*, **, ***P < 0.05). (C) Annexin V/PI staining assay indicated that depletion of GRHPR attenuated TNF-␣ induced HT-29 cell apoptosis.
role of GRHPR in TNF-␣ induced HT-29 cells apoptosis, the viability and apoptosis of cells after GRHPR depletion were evaluated by flow cytometry assay. The result showed that, as compared with the control-siRNA group, cell apoptosis was significantly reduced when GRHPR was knocked down after incubation with 100 ng/ml TNF-␣ (Fig. 4C). All the data indicated that GRHPR might promote IECs apoptosis.
3.6. GRHPR was also over-expressed in CD patients To identify the correlation of these findings with human disease, we performed immunohistochemical analysis of GRHPR in human colonic biopsy specimens from normal controls and CD patients. In normal human beings, only a low level of GRHPR protein was detectable (Fig. 5). In contrast, the tissue specimens from patients
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Fig. 5. GRHPR was strongly expressed in colonic biopsies from patients with active CD. Representative images of GRHPR staining in colonic biopsies derived from normal controls (n = 10) and patients with active CD (n = 10). The result was the same with experimental colitis. Scale bar, left column: 100 m; right column: 50 m.
with CD featured strong GRHPR staining in the epithelial layer, indicating that the protein was highly expressed in IECs in CD patients (Fig. 5). These data were in accordance with our previous results in experimental colitis, and underline the clinical significance of our experiments. 4. Discussion The inflammatory bowel disease is thought to be due to abnormal activation of the intestinal mucosal immune system and environmental factors [18]. Recently, the morbidity of IBD has increased markedly. Due to the high incidence rate of IBD, the pathogenesis of IBD has attracted great attention. In this study, we found that GRHPR played an important role in IECs apoptosis. We found that the level of GRHPR protein was up-regulated in TNBS-induced colitis as compared with controls using Western blot. Furthermore, immunohistochemical analysis showed that GRHPR were mainly located in IECs. When knocking down GRHPR expression in HT-29 cells, both Western blot and flow cytometry assay showed that cell apoptosis was significantly inhibited. To investigate the clinical significance of GRHPR up-regulation in CD, we used inflamed and normal colonic biopsy samples obtained respectively from CD patients and normal controls. It was verified that GRHPR showed higher expression in CD patients, and was mainly located in intestinal epithelial cells. The data were in accordance with those of murine experimental colitis. GRHPR, also called GlxR, was a cAMP-responsive regulator, and was confirmed as the transcriptional repressor of the aceB gene, encoding malate synthase of the glyoxylate cycle [6,14]. It not only activated gapA expression [28,29] but also participated in various physiological functions, including carbon and nitrogen metabolism, respiration, SOS and stress responses, and cell division [2,16,17,29]. Only little is known about GRHPR except for
the fact that its dysfunction could result in the formation of primary hyperoxaluria type 2 (PH2) [7,15], which was regulated by peroxisome proliferator-activated receptor ␣ (PPAR ␣) [9]. However, one recent study demonstrated that GRHPR was negatively correlated with HCC proliferation [25]. In our study, increased GRHPR expression was also observed in murine experimental colitis and CD patients. Since enterocyte apoptosis is a typical characteristic of CD, we speculated that GRHPR might participate in IECs apoptosis. Then we gave 100 ng/ml TNF-␣ to stimulate HT-29 cells to establish an IEC in vitro apoptosis model, which demonstrated a time-dependent increase in GRHPR expression with accumulation of active caspase-3 and cleaved PARP. Furthermore, Western blot and flow cytometry assay showed that knockdown of GRHPR by siRNA caused decreased apoptosis in IECs. Taken together, these results revealed that GRHPR might be involved in the intestinal epithelium aberrant apoptosis. Because GRHPR is a metabolism enzyme, it is still unknown whether the enzyme itself or its metabolites lead to activation of apoptotic pathway. In summary, we investigated GRHPR expression in colonic tissues with or without inflammation. We found that the expression of GRHPR was up-regulated in human CD and experimental colitis, and the molecule was mainly located in IECs. Furthermore, knockdown of GRHPR expression could inhibit IECs apoptosis, indicating that GRHPR might play an important role in promoting overactive apoptosis in murine experimental colitis and CD patients. However, further studies are warranted to explore the molecular mechanism of GRHPR-regulating apoptosis in intestinal homeostasis. Acknowledgments This study was supported by the Natural Science Foundation of China (No. 81201252), the Jiangsu Province’s Key Provincial Talents
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