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Activating transcription factor 3 promotes intestinal epithelial cell apoptosis in Crohn’s disease
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Activating transcription factor 3 promotes intestinal epithelial cell apoptosis in Crohn’s disease ⁎
Liugen Gua, , Zhenming Gea, Yamin Wanga, Meiqin Shena, Ping Zhaob a b
Department of Gastroenterology, The Second Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China Department of Clinical Laboratory, The Second Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Activating transcription factor 3 Crohn’s disease Intestinal epithelial cell Apoptosis p53 Bax
Intestinal epithelial cell (IEC) apoptosis plays a vital role in the pathogenesis of Crohn’s disease (CD), which is an inflammatory bowel disease (IBD). Activating transcription factor 3 (ATF3) modulates apoptosis under stress via regulating the p53 pathway. However, the expression and function of ATF3 in CD are unclear. In the present study, ATF3, p53, and p53 target gene Bax expression increased in CD patients; a mouse 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced CD model; and a TNF-α-treated HT29 cell colitis model. ATF3 knockdown effectively decreased TNF-α-induced p53 and Bax expression, as well as inhibited the apoptosis of HT29 cells. Additionally, ATF3 enhanced the stability and transcription activity of p53 via interacting with p53. In summary, these data indicated that ATF3 might promote IEC apoptosis in CD via up-regulating the stability and transcription activity of p53, implying a novel molecular target for CD therapy.
1. Introduction Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is one of the most common inflammatory diseases in China [1,2]. Apoptosis causes intestinal epithelial cell (IEC) shedding, which in turn leads to barrier loss [3]. Abnormal apoptosis detected in the intestinal epithelium of IBD patients [4] is considered one of the major courses that accelerates IBD. Several animal studies further confirm the central role of IEC apoptosis in the pathogenesis of CD. For instance, the conditional signal transducer and activator of transcription 3 (STAT3) knockout of intestinal epithelial cell (IECs) mice are highly susceptible to experimental colitis, with important defects in epithelial restitution and enhanced apoptosis [5]. Aberrant intestinal epithelial cell (IEC) apoptosis impairs the mucosal barrier, which leads to intestinal hyper-permeability, invasion of luminal antigens and commensal microflora, and triggers the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). TNF-α further induces IEC apoptosis, and this vicious feedback eventually results in the clinical signs and symptoms of IBD [6,7]. Activating transcription factor 3 (ATF3), a member of the ATF/ CREB family of transcription factors [8], is maintained at a low level in normal and quiescent cells. As a highly versatile stress sensor, ATF3 is induced by a variety of stresses including DNA damage, oxidative stress and endoplasmic reticulum stress [9]. Therefore, ATF3 acts as an adaptive response gene participating in a variety of cellular processes
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including immune response [10], atherogenesis [11], cell cycle [12], glucose homeostasis [13], and apoptosis [14]. With regard to apoptosis, ATF3 has double pro-apoptotic or anti-apoptotic roles depending on cell type and physiologic circumstances. ATF3 plays tumor suppressing roles in different cancer types, including colon cancer [15] and esophageal squamous cell carcinomas (ESCC) [16]. In contrast, ATF3 functions as a tumor promoter in hepatocytes [17] and breast cancer [18]. However, the relationship between ATF3 and IEC apoptosis in CD is unknown. ATF3 contains a central leucine zipper domain (Zip) that is well characterized as a mediator of protein–protein interaction [19]. ATF3 binds to p53 via this domain, and as a consequence, p53 ubiquitination catalyzed by E3 ubiquitin ligase murine double minute 2 (MDM2) [20,21] is blocked, leading to up-regulation of p53 tumor suppressor activity, independent of ATF3 transcriptional activity [22]. Exposure to cellular stress triggers transcription factor p53 to induce cell growth arrest [23] or apoptosis [24]. The intrinsic apoptotic pathway is dominated by the B cell lymphoma-2 (Bcl-2) family of proteins, which govern the release of cytochrome c from the mitochondria [25]. BCL2associated X (Bax) is the first member of this group shown to be induced by p53 [26]. It is revealed that p53 expression is up-regulated in noncancerous tissues of CD patients [27]. Bax expression also dramatically increases in rat dextran sulfate sodium (DSS)-induced colitis model [28]. Here, we show that ATF3 might promote IEC apoptosis in CD via up-
Corresponding author. E-mail address: [email protected] (L. Gu).
https://doi.org/10.1016/j.prp.2018.04.013 Received 28 January 2018; Received in revised form 6 April 2018; Accepted 17 April 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Gu, L., Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.04.013
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Fig. 1. ATF3 is up-regulated in the inflamed region of intestinal tissues from CD patients. (A) IHC analysis of ATF3 and p53 in mucosal biopsies from inflamed (n = 10) and non-inflamed tissues (n = 10). Scale bar = 100 μm. The bar graph indicates the positive cell ratio of AFT3 and p53. *,#P < 0.05 vs non-inflamed tissues. (B) ATF3, p53 and Bax expression in non-inflamed and inflamed colon tissues from healthy control and CD patients, respectively, were detected by western blot. GAPDH was used as a loading control. The bar chart shows quantification of ATF3, p53 and Bax protein level relative to GAPDH. n = 3, *,#,&P < 0.05 vs noninflamed tissues.
regulating the stability and transcription activity of p53 using CD patient colon tissues; a mouse 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis model; and an HT29 cell inflammatory model. Our results present a novel mechanism of IEC apoptosis in CD.
until use for immunohistochemistry analysis. Written informed consent was obtained before specimen collection.
2. Materials and methods
All animal care and surgical procedures were performed according to Guide for the Care and Use of Laboratory Animals promulgated by National Research Council in 1996 and supported by the Chinese National Committee for Use of Experimental Animals for Medical Purposes, Jiangsu Branch. BALB/c mice (8–10 weeks, weight 18–20 g) were obtained from the Department of Experimental Animal Center, Nantong University. Chemical colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS; Sigma Chemical Co., St. Louis, MO, USA). As previously described [30], mice were fasted for 24 h. Then, they were anesthetized through intraperitoneal injection of sodium pentobarbital (0.3% solution). A 3.5 F catheter was carefully inserted through the anus into the colon, and the tip was 4 cm proximal to the anal verge. To induce colitis, 0.1 ml of 2.5% (w/v) TNBS solution in 50% ethanol was injected slowly into the lumen of the colon via a catheter fitted to a 1 ml syringe. In the EtOH (ethanol) group, mice
2.2. Mouse colitis model
2.1. Human intestinal tissues The intestinal tissue specimens from the terminal ileum and rectum were obtained from patients with newly diagnosed CD (n = 10) and healthy subjects with non-inflammatory conditions of the gastrointestinal tract (n = 10) under endoscopy at the Second Affiliated Hospital of Nantong University from 2014 to 2017. The CD patients had diagnoses based on standard criteria, including clinical presentation as well as endoscopic, radiologic, and/or pathologic confirmation [29]. CD patients who had infectious colitis or colorectal cancer or who had received anti-inflammation drug therapy within 6 months were excluded. Some biopsy specimens were used for western blot, and other biopsies were immediately fixed in formalin and embedded in paraffin 2
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2.5. Western blot After determining the protein concentration of colon tissue samples with Bradford assay (Bio-Rad, Hercules, CA, USA), the samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was then blocked with 5% skim milk and incubated overnight at 4 °C with the primary antibodies, including anti-ATF3 (mouse; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p53 (rabbit; 1:1000; Santa Cruz); anti-Bax (rabbit; 1:500; Santa Cruz), and anti-GAPDH (rabbit; 1:1000, Santa Cruz). Finally, the membrane was incubated with HRP-conjugated secondary antibodies (Dako Corporation, Santa Barbara, CA, USA) for 2 h and visualized using an ECL system (Pierce Company, Rockford, IL, USA). Data were analyzed with ImageJ software (NIH, Bethesda, MD, USA). Each group was first standardized with GAPDH. 2.6. Immunohistochemistry (IHC) The IHC analysis of colon tissues with ATF3 (mouse, 1:200, Santa Cruz) and p53 (rabbit, 1:200, Santa Cruz) antibodies was performed as previously described [32]. The IHC images were examined by researchers who were unaware of the clinical findings or animal grouping. These slices were visualized with a Leica light microscope (Leica, DM 5000B; Germany). We examined the sections and counted the cells with strong or moderate brown staining; the cells with weak or no staining were counted as positive or negative ATF3 or p53 cells, respectively, from each group at higher magnification. The images were analyzed with ImageJ software (1.41 v, US National Institutes of Health, USA). 2.7. siRNAs and transfection
received 0.1 ml of 50% ethanol. To confirm the distribution of TNBS throughout the entire colon, including the cecum and appendix, mice were kept vertical for 1 min and then returned to their cages.
Human ATF3 (NM_001674.3) siRNAs were purchased from Thermo Fisher Scientific (catalog number AM16708), while a scrambled siRNA with a sequence of 5′-UUCUCCGAACGUGUCACGU-3′ (GenePharma, Shanghai, China) was used as negative control. The siRNAs were transfected into HT29 cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, the cells were treated with TNF-α (50 ng/ml) for 24 h and then subjected to subsequent experiments.
2.3. Assessment of TNBS-induced colitis
2.8. Annexin V/PI staining
To evaluate the severity of colitis, the animals were monitored daily for weight, piloerection, water/food consumption, stool consistency, and the presence of blood in the feces and at the anus. Animals were sacrificed by cervical dislocation at 3 d (n = 5). Normal mice were also sacrificed (n = 5). The colon was removed quickly and gently cleared of stool as soon as mice were sacrificed. After that, colonic tissues were embedded in paraffin and stained with hematoxylin and eosin. The different degree of inflammation on microscopic colon sections graded from 0 to 4 (0, no signs of inflammation; 1, very low level; 2, low level of leukocytic infiltration; 3, high level of leukocytic infiltration, high vascular density, and thickening of the colon wall; 4, transmural infiltrations, loss of goblet cells, high vascular density, and thickening of the colon wall) [31].
The flow cytometry assay was performed to measure the degrees of both apoptosis and necrosis using an ApoScreen Annexin V kit-FITC according to the manufacturer’s instructions (catalog number 1001002; Southern Biotechnology, Birmingham, AL, USA). Briefly, HT29 cells were digested by 0.1% trypsin and resuspended in cold binding buffer at concentrations between 105 and 106 cells/ml. Ten microliters of labeled annexin V-FITC was added to 100 μl of the cell suspension. After 15 min incubation on ice, 380 μl binding buffer and 10 μl propidium iodide (PI) solution were added to the cell suspension. Subsequently, the number of stained cells was assessed via flow cytometer (BD FACSAriaII; BD Bioscience, San Jose, CA, USA).
2.4. Cell culture and stimulation
After treatment with TNF-α, HT29 cells were collected and lysed using lysis buffer supplemented with PMSF. Then, an equal amount of protein was subjected to anti-ATF3 antibody or anti-p53 antibody (Santa Cruz) following overnight incubation at 4 °C. Then, protein-antibody immunoprecipitates were collected by protein A/G plus-agarose at 4 °C (Santa Cruz). After 2 h of incubation, pellets were washed 5 times with lysis buffer and resuspended in sample buffer. Finally, the ATF3 or p53 protein was analyzed by western blot [33].
Fig. 2. TNBS-induced colitis model was performed successfully. (A) The changes of body weight n = 3, *P < 0.05 vs the EtOH group. (B) Colonic tissue structure was detected by HE stain with TNBS or EtOH treatment. (C) Histological scores in mice. n = 3, *P < 0.05 vs the EtOH group.
2.9. Co-immunoprecipitation (Co-IP)
The human colon epithelial cell line HT29 cells (Cell library, China Academy of Science, Shanghai, China) were cultured in RPMI 1640 medium, with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C, in a 95% O2/5% CO2 atmosphere. For further analysis, the cells were treated by TNF-α (Human origin, Sigma Chemical Co., St. Louis, MO, USA). 3
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Fig. 3. ATF3 is up-regulated and associated with IEC apoptosis in TNBS-induced colitis. (A) IHC analysis of ATF3 and p53 in colon tissues from EtOH (n = 3) and TNBS (n = 3) groups. Scale bar = 100 μm. The bar graph indicates the positive cell ratio for ATF3 and p53. n = 3, *P < 0.05 and #P < 0.01 vs the EtOH group. (B) Immunofluorescence assay of ATF3 and p53 in colon tissues from EtOH and TNBS groups. The bar chart shows the positive cell ratio for ATF3 and p53. n = 3, * P < 0.05 vs the EtOH group. (C) ATF3, p53 and Bax protein level was measured by western blot for EtOH and TNBS groups. The bar chart shows the data analysis of the relative protein level. n = 3, *P < 0.05 vs the EtOH group. (D) The correlation between ATF3 and p53 expression in EtOH and TNBS groups was analyzed. n = 9, *P = 0.0002, R2 = 0.7713. (E) The correlation between ATF3 and Bax expression levels was analyzed. n = 9, *P = 0.0028, R2 = 0.6615. 4
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Fig. 4. ATF3 promotes HT29 cell apoptosis. (A) ATF3, p53, and Bax expression was measured by western blot for different concentrations of TNF-α treatment. The bar chart shows the relative level of ATF3, p53 and Bax compared to GAPDH. n = 3, *,#,&P < 0.05 vs 0 ng/ml group. (B) ATF3, p53, and Bax protein level was measured by western blot for different time points of TNF-α (50 ng/ml) treatment. The bar chart shows relative level of ATF3, p53 and Bax compared to GAPDH. n = 3, *,#,&P < 0.05 vs 0 h group. (C) ATF3 expression was measured by western blot after scrambled siRNA or ATF3 siRNA was transfected in HT29 cells. The bar chart shows the relative level of ATF3 compared to GAPDH. n = 3, *P < 0.05 vs the control group. (D) ATF3, p53 and Bax protein level in HT29 cells treated with or without TNF-α (50 ng/ml) for 24 h after scrambled siRNA or ATF3 siRNA transfection was measured by western blot. The bar chart shows relative level of ATF3, p53 and Bax compared to GAPDH. *P < 0.01 vs the scrambled siRNA group. (E) Flow cytometry assay was performed to measure HT29 cell apoptosis after TNF-α (50 ng/ ml) treatment for 24 h. The bar graph shows the statistical quantification of apoptotic cells in control, TNF-α, TNF-α plus scrambled siRNA, and TNF-α plus ATF3 siRNA groups. *P < 0.05 vs the TNF-α plus scrambled siRNA group.
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Fig. 5. ATF3 enhances the stability and transcription activity of p53 via interacting with p53. (A, B) The interaction between ATF3 and p53 is shown by Co-IP assays of HT29 cell lysates. (C) CHX chase assay of p53 in HT29 cells with scrambled siRNA or ATF3 siRNA transfection. Relative p53 levels were quantified by densitometry. *P < 0.05 vs scrambled siRNA group. (D) Dual luciferase reporter assay of p53 target gene Bax in HT29 cells with or without TNF-α treatment (50 ng/ml, 24 h) plus scrambled siRNA or ATF3 siRNA transfection.
Assay System (Promega, Madison, Wis, USA), and normalized against Renilla luciferase activity.
2.10. Cycloheximide chase assay HT29 cells were transfected with scrambled siRNA or ATF3 siRNA and treated with 50 mg/ml cycloheximide (CHX) 30 h after transfection. Then, the cells were harvested at 0 h, 0.5 h, 1 h, 2 h, and 4 h and subjected to lysis and western blot with anti-p53, anti-ATF3, and antiGAPDH antibodies (Santa Cruz).
2.12. Statistical analysis All results were expressed as the mean ± SEM. Data were compared by Student'st test. The level of significance was set at a P value of 0.05. Each experiment consisted of at least three replicates per condition.
2.11. Dual luciferase reporter assay HT29 cells were seeded at 3 × 104 cells/well in 24-well plates and allowed to settle for 24 h. Subsequently, the cells were transfected with pmiR-REPORT-Bax 3′-untranslated region (3′-UTR) reporter plasmid and a Renilla luciferase vector. Twenty-four hours later, the cells were stimulated with TNF-α (50 ng/ml) and transfected with scrambled siRNA or ATF3 siRNA. Twenty-four hours after transfection, relative luciferase activity was measured using the Dual-Luciferase Reporter
3. Results 3.1. ATF3 is up-regulated and associated with IEC apoptosis of CD patients Increased IEC apoptosis greatly destroys the intestinal mucosal integrity and contributes to colitis development [34]. First, IHC and western blot were used to determine the expression and localization of 6
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interacted with ATF3 (Fig. 5A and B). CHX chase assay showed ATF3 knockdown shortened the half-life of p53 with TNF-α treatment, indicating that ATF3 enhanced the stability of p53 (Fig. 5B). Additionally, ATF3 knockdown inhibited the transcription activity of p53 induced by TNF-α treatment, which was detected by double luciferase reporter assay (Fig. 5B). The data suggested that ATF3 enhanced the stability and transcription activity of p53 via interacting with p53.
ATF3 and p53 in inflamed and non-inflamed CD samples. ATF3 and p53 staining was stronger in the inflamed samples than that in the noninflamed samples (Fig. 1A). Additionally, ATF3 and p53 were primarily localized in the nuclei of IECs. Western blot revealed that ATF3 expression levels were higher in the inflamed samples than that in the non-inflamed samples from CD patients (Fig. 1B). Consistent with previous studies [35,36], the expression of p53 and its target gene Bax was up-regulated in the inflamed tissues (Fig. 1B). These data showed that ATF3 and p53 expression was up-regulated in the inflamed CD samples, suggesting that ATF3 might play a role in IEC apoptosis of CD.
4. Discussion Under physiological conditions, the mRNA level of ATF3 is relatively low in most cell types. Induction of ATF3 mRNA by stress signals, such as mechanical injury, chemicals, and ischemia/reperfusion, is an early event and returns to a low level after several hours [40]. In our study, ATF3 protein level increased in CD patients, mouse TNBS-induced CD model and TNF-α-treated HT29 cell colitis model. ATF3 is known to be responsive to stress such as reactive oxygen species (ROS) [41]. ROS-induced ATF3 crucially increases susceptibility to secondary infections during sepsis-associated immunosuppression (SAIS) [42]. Moreover, the ROS/ATF3/C/EBP-homologous protein (CHOP) pathway plays a negative role in mechanisms, by which echinacoside (ECH) protects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in Parkinson’s disease (PD) [43]. Oxidative stress has been proposed as a mechanism underlying the pathophysiology of CD [44]. The immune cells that reach the mucosa in Crohn’s disease release a number of ROS that are potentially detrimental [45]. Whether ROS produced during CD induces the expression of ATF3 needs further investigation. The ATF3 isoforms can heterodimerize with each other and with other transcription factors, such as p53 [46], c-Jun [47], ATF2 [48], and Smad3 [49]. Depending on its partner, target promoter, or cellular context, ATF3 functions either as a transcriptional activator or repressor [50]. We found ATF3 enhanced the stability of p53 via interacting with p53 in HT29 cells, in line with previous research [51]. Additionally, ATF3 also up-regulated the transcription activity of p53 in HT29 cells following TNF-α exposure, indicating that ATF3 might act as a transcription activator in the development of CD. The sporadic form of colorectal cancer (CRC) and IBD-related CRC have very few clinical differences; however, a two-fold higher rate of mortality is reported in the latter form [52,53]. In the four step pathway to CRC tumorigenesis, p53 inactivation usually occurs at a later stage leading to the transition into a carcinoma and represents a major driver event in CRC [54]. Consistent with previous studies [55,56], increased p53 expression in the inflamed colon tissues of CD patients was observed in our study. Researchers found that p53 overexpression in CD patients is associated with dysplasia, which may progress to a higher grade of neoplasia over time [57]. Is ATF3 associated with p53 mutation and tumorigenesis in CD patients? This question will be answered in future research. ATF3 binds to the promoters of pro-inflammatory cytokine genes, resulting in the inhibition of cytokine production, therefore protecting the host cell from lipopolysaccharide (LPS)-induced endotoxic shock [9]. Furthermore, LPS-induced ATF3 competes with nuclear factor kappa B (NF-κB) for binding to the promoter region of cytokine genes [9], and once induced, ATF3 negative feedback regulates production of TLR-mediated cytokines [58]. ATF3 up-regulation also represses interleukin 6 (IL-6) expression during Neisseria gonorrhoeae infection [59]. Collectively, these findings indicate that ATF3 acts as a negative regulator of cytokine production during gram-negative bacterial infection. However, ATF3 positively regulates innate immunity upon pneumococcus (a gram-positive bacterium) infection by enhancing TNF-α, interleukin 1β (IL-1β), and interferon γ (IFN-γ) expression and modulating bacterial clearance [60]. The functions of ATF3 in the inflammatory response of CD call for exploration. In summary, the study suggests that ATF3 might promote IEC apoptosis by interacting with p53 to enhance the stability and
3.2. Mouse TNBS-induced colitis To further study the functions of ATF3, we used a mouse TNBSinduced CD model, a well-established model of colonic inflammation resembling certain prominent clinical and morphological features of human CD [37]. Body weight started to decrease at 2 d and touched the bottom at 3 d (Fig. 2A). An HE stain showed the CD-like pathological changes in TNBS-induced mice colons, including inflammatory cell infiltration within propria lamina, formation of ulceration, depletion of epithelial cells, edema, and thickened colon wall (Fig. 2B). The histological scores reflecting inflammation level peaked at 3 d and then declined (Fig. 2C). The results indicated that the mouse TNBS-induced colitis model was performed successfully. 3.3. ATF3 is up-regulated and associated with IEC apoptosis in TNBSinduced colitis TNBS-induced colitis peaked at 3 d, which was selected for further experiments. Immunohistochemistry found the stronger staining of ATF3 and p53 predominantly in IECs in the TNBS group than that in the EtOH group (Fig. 3A). The up-regulated expression and co-localization of ATF3 and p53 with IEC nuclei was observed in the colon tissues of the TNBS group (Fig. 3B). Furthermore, western blot showed ATF3, p53 and Bax expression increased in TNBS-induced colitis at 3 d (Fig. 3C). Additionally, ATF3 expression was positively correlated with p53 and Bax expression in the colon (Fig. 3D and E), revealing a positive link between ATF3 and IEC apoptosis. 3.4. ATF3 promotes apoptosis in colonic epithelial cells To identify the role of ATF3 in IEC apoptosis, HT29 cells were incubated with TNF-α, a key cytokine that triggers IEC apoptosis and participates in CD pathogenesis [38]. To determine the appropriate concentration of TNF-α to induce apoptosis, we incubated HT29 cells with different concentrations of TNF-α for 24 h [39]. ATF3, p53 and Bax expression increased in a dose-dependent manner (Fig. 4A). Thus, we treated HT29 cells with 50 ng/ml TNF-α for different times, finding a time-dependent up-regulation of ATF3, p53, and Bax expression, with the peak at 48–72 h (Fig. 4B). Then, HT29 cells were transfected with ATF3 siRNA, and the knockdown efficiency was confirmed by western blot (Fig. 4C), showing that ATF siRNA3 obviously inhibited ATF3 expression. Thus, ATF3 siRNA3 was used for consequent experiments. ATF3 knockdown dramatically inhibited TNF-α-induced p53 and Bax expression (Fig. 4D). Moreover, flow cytometry-based annexin V/PI staining showed that compared to the control group, TNF-α stimulation induced HT29 cell apoptosis (Fig. 4E). Interestingly, ATF3 knockdown clearly alleviated TNF-α-induced cell apoptosis. Taken together, our results suggested that ATF3 might facilitate IEC apoptosis during CD. 3.5. ATF3 enhances the stability and transcription activity of p53 via interacting with p53 The interaction between endogenous ATF3 and p53 in HT29 cells after TNF-α treatment was determined by Co-IP, showing that p53 7
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transcription activity of p53 in CD. Further studies about the detailed mechanism of ATF3 in IECs may provide novel insights into the pathophysiology of CD and lead to new therapeutic approaches for the treatment of CD.
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Activating transcription factor 3 promotes intestinal epithelial cell apoptosis in Crohn’s disease ⁎
Liugen Gua, , Zhenming Gea, Yamin Wanga, Meiqin Shena, Ping Zhaob a b
Department of Gastroenterology, The Second Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China Department of Clinical Laboratory, The Second Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Activating transcription factor 3 Crohn’s disease Intestinal epithelial cell Apoptosis p53 Bax
Intestinal epithelial cell (IEC) apoptosis plays a vital role in the pathogenesis of Crohn’s disease (CD), which is an inflammatory bowel disease (IBD). Activating transcription factor 3 (ATF3) modulates apoptosis under stress via regulating the p53 pathway. However, the expression and function of ATF3 in CD are unclear. In the present study, ATF3, p53, and p53 target gene Bax expression increased in CD patients; a mouse 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced CD model; and a TNF-α-treated HT29 cell colitis model. ATF3 knockdown effectively decreased TNF-α-induced p53 and Bax expression, as well as inhibited the apoptosis of HT29 cells. Additionally, ATF3 enhanced the stability and transcription activity of p53 via interacting with p53. In summary, these data indicated that ATF3 might promote IEC apoptosis in CD via up-regulating the stability and transcription activity of p53, implying a novel molecular target for CD therapy.
1. Introduction Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is one of the most common inflammatory diseases in China [1,2]. Apoptosis causes intestinal epithelial cell (IEC) shedding, which in turn leads to barrier loss [3]. Abnormal apoptosis detected in the intestinal epithelium of IBD patients [4] is considered one of the major courses that accelerates IBD. Several animal studies further confirm the central role of IEC apoptosis in the pathogenesis of CD. For instance, the conditional signal transducer and activator of transcription 3 (STAT3) knockout of intestinal epithelial cell (IECs) mice are highly susceptible to experimental colitis, with important defects in epithelial restitution and enhanced apoptosis [5]. Aberrant intestinal epithelial cell (IEC) apoptosis impairs the mucosal barrier, which leads to intestinal hyper-permeability, invasion of luminal antigens and commensal microflora, and triggers the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α). TNF-α further induces IEC apoptosis, and this vicious feedback eventually results in the clinical signs and symptoms of IBD [6,7]. Activating transcription factor 3 (ATF3), a member of the ATF/ CREB family of transcription factors [8], is maintained at a low level in normal and quiescent cells. As a highly versatile stress sensor, ATF3 is induced by a variety of stresses including DNA damage, oxidative stress and endoplasmic reticulum stress [9]. Therefore, ATF3 acts as an adaptive response gene participating in a variety of cellular processes
⁎
including immune response [10], atherogenesis [11], cell cycle [12], glucose homeostasis [13], and apoptosis [14]. With regard to apoptosis, ATF3 has double pro-apoptotic or anti-apoptotic roles depending on cell type and physiologic circumstances. ATF3 plays tumor suppressing roles in different cancer types, including colon cancer [15] and esophageal squamous cell carcinomas (ESCC) [16]. In contrast, ATF3 functions as a tumor promoter in hepatocytes [17] and breast cancer [18]. However, the relationship between ATF3 and IEC apoptosis in CD is unknown. ATF3 contains a central leucine zipper domain (Zip) that is well characterized as a mediator of protein–protein interaction [19]. ATF3 binds to p53 via this domain, and as a consequence, p53 ubiquitination catalyzed by E3 ubiquitin ligase murine double minute 2 (MDM2) [20,21] is blocked, leading to up-regulation of p53 tumor suppressor activity, independent of ATF3 transcriptional activity [22]. Exposure to cellular stress triggers transcription factor p53 to induce cell growth arrest [23] or apoptosis [24]. The intrinsic apoptotic pathway is dominated by the B cell lymphoma-2 (Bcl-2) family of proteins, which govern the release of cytochrome c from the mitochondria [25]. BCL2associated X (Bax) is the first member of this group shown to be induced by p53 [26]. It is revealed that p53 expression is up-regulated in noncancerous tissues of CD patients [27]. Bax expression also dramatically increases in rat dextran sulfate sodium (DSS)-induced colitis model [28]. Here, we show that ATF3 might promote IEC apoptosis in CD via up-
Corresponding author. E-mail address: [email protected] (L. Gu).
https://doi.org/10.1016/j.prp.2018.04.013 Received 28 January 2018; Received in revised form 6 April 2018; Accepted 17 April 2018 0344-0338/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Gu, L., Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.04.013
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Fig. 1. ATF3 is up-regulated in the inflamed region of intestinal tissues from CD patients. (A) IHC analysis of ATF3 and p53 in mucosal biopsies from inflamed (n = 10) and non-inflamed tissues (n = 10). Scale bar = 100 μm. The bar graph indicates the positive cell ratio of AFT3 and p53. *,#P < 0.05 vs non-inflamed tissues. (B) ATF3, p53 and Bax expression in non-inflamed and inflamed colon tissues from healthy control and CD patients, respectively, were detected by western blot. GAPDH was used as a loading control. The bar chart shows quantification of ATF3, p53 and Bax protein level relative to GAPDH. n = 3, *,#,&P < 0.05 vs noninflamed tissues.
regulating the stability and transcription activity of p53 using CD patient colon tissues; a mouse 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis model; and an HT29 cell inflammatory model. Our results present a novel mechanism of IEC apoptosis in CD.
until use for immunohistochemistry analysis. Written informed consent was obtained before specimen collection.
2. Materials and methods
All animal care and surgical procedures were performed according to Guide for the Care and Use of Laboratory Animals promulgated by National Research Council in 1996 and supported by the Chinese National Committee for Use of Experimental Animals for Medical Purposes, Jiangsu Branch. BALB/c mice (8–10 weeks, weight 18–20 g) were obtained from the Department of Experimental Animal Center, Nantong University. Chemical colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS; Sigma Chemical Co., St. Louis, MO, USA). As previously described [30], mice were fasted for 24 h. Then, they were anesthetized through intraperitoneal injection of sodium pentobarbital (0.3% solution). A 3.5 F catheter was carefully inserted through the anus into the colon, and the tip was 4 cm proximal to the anal verge. To induce colitis, 0.1 ml of 2.5% (w/v) TNBS solution in 50% ethanol was injected slowly into the lumen of the colon via a catheter fitted to a 1 ml syringe. In the EtOH (ethanol) group, mice
2.2. Mouse colitis model
2.1. Human intestinal tissues The intestinal tissue specimens from the terminal ileum and rectum were obtained from patients with newly diagnosed CD (n = 10) and healthy subjects with non-inflammatory conditions of the gastrointestinal tract (n = 10) under endoscopy at the Second Affiliated Hospital of Nantong University from 2014 to 2017. The CD patients had diagnoses based on standard criteria, including clinical presentation as well as endoscopic, radiologic, and/or pathologic confirmation [29]. CD patients who had infectious colitis or colorectal cancer or who had received anti-inflammation drug therapy within 6 months were excluded. Some biopsy specimens were used for western blot, and other biopsies were immediately fixed in formalin and embedded in paraffin 2
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2.5. Western blot After determining the protein concentration of colon tissue samples with Bradford assay (Bio-Rad, Hercules, CA, USA), the samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was then blocked with 5% skim milk and incubated overnight at 4 °C with the primary antibodies, including anti-ATF3 (mouse; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p53 (rabbit; 1:1000; Santa Cruz); anti-Bax (rabbit; 1:500; Santa Cruz), and anti-GAPDH (rabbit; 1:1000, Santa Cruz). Finally, the membrane was incubated with HRP-conjugated secondary antibodies (Dako Corporation, Santa Barbara, CA, USA) for 2 h and visualized using an ECL system (Pierce Company, Rockford, IL, USA). Data were analyzed with ImageJ software (NIH, Bethesda, MD, USA). Each group was first standardized with GAPDH. 2.6. Immunohistochemistry (IHC) The IHC analysis of colon tissues with ATF3 (mouse, 1:200, Santa Cruz) and p53 (rabbit, 1:200, Santa Cruz) antibodies was performed as previously described [32]. The IHC images were examined by researchers who were unaware of the clinical findings or animal grouping. These slices were visualized with a Leica light microscope (Leica, DM 5000B; Germany). We examined the sections and counted the cells with strong or moderate brown staining; the cells with weak or no staining were counted as positive or negative ATF3 or p53 cells, respectively, from each group at higher magnification. The images were analyzed with ImageJ software (1.41 v, US National Institutes of Health, USA). 2.7. siRNAs and transfection
received 0.1 ml of 50% ethanol. To confirm the distribution of TNBS throughout the entire colon, including the cecum and appendix, mice were kept vertical for 1 min and then returned to their cages.
Human ATF3 (NM_001674.3) siRNAs were purchased from Thermo Fisher Scientific (catalog number AM16708), while a scrambled siRNA with a sequence of 5′-UUCUCCGAACGUGUCACGU-3′ (GenePharma, Shanghai, China) was used as negative control. The siRNAs were transfected into HT29 cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, the cells were treated with TNF-α (50 ng/ml) for 24 h and then subjected to subsequent experiments.
2.3. Assessment of TNBS-induced colitis
2.8. Annexin V/PI staining
To evaluate the severity of colitis, the animals were monitored daily for weight, piloerection, water/food consumption, stool consistency, and the presence of blood in the feces and at the anus. Animals were sacrificed by cervical dislocation at 3 d (n = 5). Normal mice were also sacrificed (n = 5). The colon was removed quickly and gently cleared of stool as soon as mice were sacrificed. After that, colonic tissues were embedded in paraffin and stained with hematoxylin and eosin. The different degree of inflammation on microscopic colon sections graded from 0 to 4 (0, no signs of inflammation; 1, very low level; 2, low level of leukocytic infiltration; 3, high level of leukocytic infiltration, high vascular density, and thickening of the colon wall; 4, transmural infiltrations, loss of goblet cells, high vascular density, and thickening of the colon wall) [31].
The flow cytometry assay was performed to measure the degrees of both apoptosis and necrosis using an ApoScreen Annexin V kit-FITC according to the manufacturer’s instructions (catalog number 1001002; Southern Biotechnology, Birmingham, AL, USA). Briefly, HT29 cells were digested by 0.1% trypsin and resuspended in cold binding buffer at concentrations between 105 and 106 cells/ml. Ten microliters of labeled annexin V-FITC was added to 100 μl of the cell suspension. After 15 min incubation on ice, 380 μl binding buffer and 10 μl propidium iodide (PI) solution were added to the cell suspension. Subsequently, the number of stained cells was assessed via flow cytometer (BD FACSAriaII; BD Bioscience, San Jose, CA, USA).
2.4. Cell culture and stimulation
After treatment with TNF-α, HT29 cells were collected and lysed using lysis buffer supplemented with PMSF. Then, an equal amount of protein was subjected to anti-ATF3 antibody or anti-p53 antibody (Santa Cruz) following overnight incubation at 4 °C. Then, protein-antibody immunoprecipitates were collected by protein A/G plus-agarose at 4 °C (Santa Cruz). After 2 h of incubation, pellets were washed 5 times with lysis buffer and resuspended in sample buffer. Finally, the ATF3 or p53 protein was analyzed by western blot [33].
Fig. 2. TNBS-induced colitis model was performed successfully. (A) The changes of body weight n = 3, *P < 0.05 vs the EtOH group. (B) Colonic tissue structure was detected by HE stain with TNBS or EtOH treatment. (C) Histological scores in mice. n = 3, *P < 0.05 vs the EtOH group.
2.9. Co-immunoprecipitation (Co-IP)
The human colon epithelial cell line HT29 cells (Cell library, China Academy of Science, Shanghai, China) were cultured in RPMI 1640 medium, with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C, in a 95% O2/5% CO2 atmosphere. For further analysis, the cells were treated by TNF-α (Human origin, Sigma Chemical Co., St. Louis, MO, USA). 3
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Fig. 3. ATF3 is up-regulated and associated with IEC apoptosis in TNBS-induced colitis. (A) IHC analysis of ATF3 and p53 in colon tissues from EtOH (n = 3) and TNBS (n = 3) groups. Scale bar = 100 μm. The bar graph indicates the positive cell ratio for ATF3 and p53. n = 3, *P < 0.05 and #P < 0.01 vs the EtOH group. (B) Immunofluorescence assay of ATF3 and p53 in colon tissues from EtOH and TNBS groups. The bar chart shows the positive cell ratio for ATF3 and p53. n = 3, * P < 0.05 vs the EtOH group. (C) ATF3, p53 and Bax protein level was measured by western blot for EtOH and TNBS groups. The bar chart shows the data analysis of the relative protein level. n = 3, *P < 0.05 vs the EtOH group. (D) The correlation between ATF3 and p53 expression in EtOH and TNBS groups was analyzed. n = 9, *P = 0.0002, R2 = 0.7713. (E) The correlation between ATF3 and Bax expression levels was analyzed. n = 9, *P = 0.0028, R2 = 0.6615. 4
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Fig. 4. ATF3 promotes HT29 cell apoptosis. (A) ATF3, p53, and Bax expression was measured by western blot for different concentrations of TNF-α treatment. The bar chart shows the relative level of ATF3, p53 and Bax compared to GAPDH. n = 3, *,#,&P < 0.05 vs 0 ng/ml group. (B) ATF3, p53, and Bax protein level was measured by western blot for different time points of TNF-α (50 ng/ml) treatment. The bar chart shows relative level of ATF3, p53 and Bax compared to GAPDH. n = 3, *,#,&P < 0.05 vs 0 h group. (C) ATF3 expression was measured by western blot after scrambled siRNA or ATF3 siRNA was transfected in HT29 cells. The bar chart shows the relative level of ATF3 compared to GAPDH. n = 3, *P < 0.05 vs the control group. (D) ATF3, p53 and Bax protein level in HT29 cells treated with or without TNF-α (50 ng/ml) for 24 h after scrambled siRNA or ATF3 siRNA transfection was measured by western blot. The bar chart shows relative level of ATF3, p53 and Bax compared to GAPDH. *P < 0.01 vs the scrambled siRNA group. (E) Flow cytometry assay was performed to measure HT29 cell apoptosis after TNF-α (50 ng/ ml) treatment for 24 h. The bar graph shows the statistical quantification of apoptotic cells in control, TNF-α, TNF-α plus scrambled siRNA, and TNF-α plus ATF3 siRNA groups. *P < 0.05 vs the TNF-α plus scrambled siRNA group.
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Fig. 5. ATF3 enhances the stability and transcription activity of p53 via interacting with p53. (A, B) The interaction between ATF3 and p53 is shown by Co-IP assays of HT29 cell lysates. (C) CHX chase assay of p53 in HT29 cells with scrambled siRNA or ATF3 siRNA transfection. Relative p53 levels were quantified by densitometry. *P < 0.05 vs scrambled siRNA group. (D) Dual luciferase reporter assay of p53 target gene Bax in HT29 cells with or without TNF-α treatment (50 ng/ml, 24 h) plus scrambled siRNA or ATF3 siRNA transfection.
Assay System (Promega, Madison, Wis, USA), and normalized against Renilla luciferase activity.
2.10. Cycloheximide chase assay HT29 cells were transfected with scrambled siRNA or ATF3 siRNA and treated with 50 mg/ml cycloheximide (CHX) 30 h after transfection. Then, the cells were harvested at 0 h, 0.5 h, 1 h, 2 h, and 4 h and subjected to lysis and western blot with anti-p53, anti-ATF3, and antiGAPDH antibodies (Santa Cruz).
2.12. Statistical analysis All results were expressed as the mean ± SEM. Data were compared by Student'st test. The level of significance was set at a P value of 0.05. Each experiment consisted of at least three replicates per condition.
2.11. Dual luciferase reporter assay HT29 cells were seeded at 3 × 104 cells/well in 24-well plates and allowed to settle for 24 h. Subsequently, the cells were transfected with pmiR-REPORT-Bax 3′-untranslated region (3′-UTR) reporter plasmid and a Renilla luciferase vector. Twenty-four hours later, the cells were stimulated with TNF-α (50 ng/ml) and transfected with scrambled siRNA or ATF3 siRNA. Twenty-four hours after transfection, relative luciferase activity was measured using the Dual-Luciferase Reporter
3. Results 3.1. ATF3 is up-regulated and associated with IEC apoptosis of CD patients Increased IEC apoptosis greatly destroys the intestinal mucosal integrity and contributes to colitis development [34]. First, IHC and western blot were used to determine the expression and localization of 6
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interacted with ATF3 (Fig. 5A and B). CHX chase assay showed ATF3 knockdown shortened the half-life of p53 with TNF-α treatment, indicating that ATF3 enhanced the stability of p53 (Fig. 5B). Additionally, ATF3 knockdown inhibited the transcription activity of p53 induced by TNF-α treatment, which was detected by double luciferase reporter assay (Fig. 5B). The data suggested that ATF3 enhanced the stability and transcription activity of p53 via interacting with p53.
ATF3 and p53 in inflamed and non-inflamed CD samples. ATF3 and p53 staining was stronger in the inflamed samples than that in the noninflamed samples (Fig. 1A). Additionally, ATF3 and p53 were primarily localized in the nuclei of IECs. Western blot revealed that ATF3 expression levels were higher in the inflamed samples than that in the non-inflamed samples from CD patients (Fig. 1B). Consistent with previous studies [35,36], the expression of p53 and its target gene Bax was up-regulated in the inflamed tissues (Fig. 1B). These data showed that ATF3 and p53 expression was up-regulated in the inflamed CD samples, suggesting that ATF3 might play a role in IEC apoptosis of CD.
4. Discussion Under physiological conditions, the mRNA level of ATF3 is relatively low in most cell types. Induction of ATF3 mRNA by stress signals, such as mechanical injury, chemicals, and ischemia/reperfusion, is an early event and returns to a low level after several hours [40]. In our study, ATF3 protein level increased in CD patients, mouse TNBS-induced CD model and TNF-α-treated HT29 cell colitis model. ATF3 is known to be responsive to stress such as reactive oxygen species (ROS) [41]. ROS-induced ATF3 crucially increases susceptibility to secondary infections during sepsis-associated immunosuppression (SAIS) [42]. Moreover, the ROS/ATF3/C/EBP-homologous protein (CHOP) pathway plays a negative role in mechanisms, by which echinacoside (ECH) protects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in Parkinson’s disease (PD) [43]. Oxidative stress has been proposed as a mechanism underlying the pathophysiology of CD [44]. The immune cells that reach the mucosa in Crohn’s disease release a number of ROS that are potentially detrimental [45]. Whether ROS produced during CD induces the expression of ATF3 needs further investigation. The ATF3 isoforms can heterodimerize with each other and with other transcription factors, such as p53 [46], c-Jun [47], ATF2 [48], and Smad3 [49]. Depending on its partner, target promoter, or cellular context, ATF3 functions either as a transcriptional activator or repressor [50]. We found ATF3 enhanced the stability of p53 via interacting with p53 in HT29 cells, in line with previous research [51]. Additionally, ATF3 also up-regulated the transcription activity of p53 in HT29 cells following TNF-α exposure, indicating that ATF3 might act as a transcription activator in the development of CD. The sporadic form of colorectal cancer (CRC) and IBD-related CRC have very few clinical differences; however, a two-fold higher rate of mortality is reported in the latter form [52,53]. In the four step pathway to CRC tumorigenesis, p53 inactivation usually occurs at a later stage leading to the transition into a carcinoma and represents a major driver event in CRC [54]. Consistent with previous studies [55,56], increased p53 expression in the inflamed colon tissues of CD patients was observed in our study. Researchers found that p53 overexpression in CD patients is associated with dysplasia, which may progress to a higher grade of neoplasia over time [57]. Is ATF3 associated with p53 mutation and tumorigenesis in CD patients? This question will be answered in future research. ATF3 binds to the promoters of pro-inflammatory cytokine genes, resulting in the inhibition of cytokine production, therefore protecting the host cell from lipopolysaccharide (LPS)-induced endotoxic shock [9]. Furthermore, LPS-induced ATF3 competes with nuclear factor kappa B (NF-κB) for binding to the promoter region of cytokine genes [9], and once induced, ATF3 negative feedback regulates production of TLR-mediated cytokines [58]. ATF3 up-regulation also represses interleukin 6 (IL-6) expression during Neisseria gonorrhoeae infection [59]. Collectively, these findings indicate that ATF3 acts as a negative regulator of cytokine production during gram-negative bacterial infection. However, ATF3 positively regulates innate immunity upon pneumococcus (a gram-positive bacterium) infection by enhancing TNF-α, interleukin 1β (IL-1β), and interferon γ (IFN-γ) expression and modulating bacterial clearance [60]. The functions of ATF3 in the inflammatory response of CD call for exploration. In summary, the study suggests that ATF3 might promote IEC apoptosis by interacting with p53 to enhance the stability and
3.2. Mouse TNBS-induced colitis To further study the functions of ATF3, we used a mouse TNBSinduced CD model, a well-established model of colonic inflammation resembling certain prominent clinical and morphological features of human CD [37]. Body weight started to decrease at 2 d and touched the bottom at 3 d (Fig. 2A). An HE stain showed the CD-like pathological changes in TNBS-induced mice colons, including inflammatory cell infiltration within propria lamina, formation of ulceration, depletion of epithelial cells, edema, and thickened colon wall (Fig. 2B). The histological scores reflecting inflammation level peaked at 3 d and then declined (Fig. 2C). The results indicated that the mouse TNBS-induced colitis model was performed successfully. 3.3. ATF3 is up-regulated and associated with IEC apoptosis in TNBSinduced colitis TNBS-induced colitis peaked at 3 d, which was selected for further experiments. Immunohistochemistry found the stronger staining of ATF3 and p53 predominantly in IECs in the TNBS group than that in the EtOH group (Fig. 3A). The up-regulated expression and co-localization of ATF3 and p53 with IEC nuclei was observed in the colon tissues of the TNBS group (Fig. 3B). Furthermore, western blot showed ATF3, p53 and Bax expression increased in TNBS-induced colitis at 3 d (Fig. 3C). Additionally, ATF3 expression was positively correlated with p53 and Bax expression in the colon (Fig. 3D and E), revealing a positive link between ATF3 and IEC apoptosis. 3.4. ATF3 promotes apoptosis in colonic epithelial cells To identify the role of ATF3 in IEC apoptosis, HT29 cells were incubated with TNF-α, a key cytokine that triggers IEC apoptosis and participates in CD pathogenesis [38]. To determine the appropriate concentration of TNF-α to induce apoptosis, we incubated HT29 cells with different concentrations of TNF-α for 24 h [39]. ATF3, p53 and Bax expression increased in a dose-dependent manner (Fig. 4A). Thus, we treated HT29 cells with 50 ng/ml TNF-α for different times, finding a time-dependent up-regulation of ATF3, p53, and Bax expression, with the peak at 48–72 h (Fig. 4B). Then, HT29 cells were transfected with ATF3 siRNA, and the knockdown efficiency was confirmed by western blot (Fig. 4C), showing that ATF siRNA3 obviously inhibited ATF3 expression. Thus, ATF3 siRNA3 was used for consequent experiments. ATF3 knockdown dramatically inhibited TNF-α-induced p53 and Bax expression (Fig. 4D). Moreover, flow cytometry-based annexin V/PI staining showed that compared to the control group, TNF-α stimulation induced HT29 cell apoptosis (Fig. 4E). Interestingly, ATF3 knockdown clearly alleviated TNF-α-induced cell apoptosis. Taken together, our results suggested that ATF3 might facilitate IEC apoptosis during CD. 3.5. ATF3 enhances the stability and transcription activity of p53 via interacting with p53 The interaction between endogenous ATF3 and p53 in HT29 cells after TNF-α treatment was determined by Co-IP, showing that p53 7
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transcription activity of p53 in CD. Further studies about the detailed mechanism of ATF3 in IECs may provide novel insights into the pathophysiology of CD and lead to new therapeutic approaches for the treatment of CD.
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