Immunol Res DOI 10.1007/s12026-015-8651-3

Epithelial-specific ETS-1 (ESE1/ELF3) regulates apoptosis of intestinal epithelial cells in ulcerative colitis via accelerating NF-jB activation Liren Li1 • Xianjing Miao1 • Runzhou Ni1 • Xiaobing Miao5 • Liang Wang1 Xiaodan Gu1 • Lijun Yan4 • Qiyun Tang4 • Dongmei Zhang2,3

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Ó Springer Science+Business Media New York 2015

Abstract Epithelial-specific ETS-1 (ESE1), also named as ELF3, ERT and ESX, belonging to the ETS family of transcription factors, exerts multiple activities in inflammation, epithelial differentiation and cancer development. Previous data demonstrated that ESE1 synergizes with NF-jB to induce inflammation and drive tumor progress, and the nuclear translocation of ESE1 promotes colon cells apoptosis. However, the expression and biological functions of ESE1 in ulcerative colitis (UC) remain unclear. In this study, we reported for the first time that ESE1/ELF3 was over-expressed in intestinal epithelial cells (IECs) of patients with UC. In DSS-induced colitis mouse models, we Liren Li and Xianjing Miao have contributed equally to this study. & Qiyun Tang [email protected] & Dongmei Zhang [email protected] 1

Department of Gastroenterology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province, People’s Republic of China

2

Department of Pathogen Biology, Medical College, Nantong University, Nantong, People’s Republic of China

3

Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province, People’s Republic of China

4

Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210000, Jiangsu Province, People’s Republic of China

5

Department of Pathology, Affiliated Cancer Hospital of Nantong University, 30 North Tongyang Road, Pingchao, Nantong 226361, Jiangsu Province, People’s Republic of China

observed the up-regulation of ESE1/ELF3 accompanied with the elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and NF-jB activation indicators [phosphorylated NF-jB p65 subunit (p-p65) and p-IjB] in colitis IECs. Increased co-localization of ESE1/ ELF3 with active caspase-3 (and p-p65) in IECs of the DSSinduced colitis group further indicated the possible involvement of ESE1/ELF3 in NF-jB-mediated IEC apoptosis in UC. Employing the TNF-a-treated HT-29 cells as an IEC apoptosis model, we confirmed the positive correlation of ESE1/ELF3 with NF-jB activation and caspase-dependent IEC apoptosis in vitro. Immunoprecipitation and immunofluorescence assay revealed the physical interaction and increased nuclear translocation of ESE1/ELF3 and the NF-jB p65 subunit in TNF-a-treated HT-29 cells. Knocking ESE1/ELF3 down by siRNA significantly alleviated TNF-ainduced NF-jB activation and cellular apoptosis in HT-29 cells. Taken together, our data suggested that ESE1/ELF3 may promote the UC progression via accelerating NF-jB activation and thus facilitating IEC apoptosis. Keywords Ulcerative colitis  Epithelial-specific ETS-1  Intestinal epithelial cell  Apoptosis

Introduction Ulcerative colitis (UC), a broad phenotypic variant of inflammatory bowel diseases (IBD), is characterized by diffuse, continuous, superficial and ulcerating inflammation confined to the colon [1]. Patients with UC manifest relapsing clinical symptoms such as bloody diarrhea, abdominal pain, weight loss and developing colon cancer with a fivefold overall relative risk compared with the control population [2, 3]. As no curative therapy currently

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exists [4], so the safety and efficacy of treatment in patients with UC has raised some concerns. The pathogenesis of UC is still poorly understood, some researches have shown that genetic susceptibility factors [5], environmental factors [6] and immunological factors [7] all contribute to the pathogenesis of the disease, and the frontline among these factors lies in the intestinal epithelial homeostasis [8]. Epithelial homeostasis acts as a first line of the defense against varieties of harmful substances including luminal antigens and bacteria in the intestines. In UC, persistently elevated mucosal cytokine expression and leukocyte infiltration lead to the homeostasis destruction, including IEC apoptosis and subsequent crypt hyper-proliferation, which contribute to widespread tissue ulceration and epithelial hyperplasia [9]. This is the evidence that increased IEC apoptosis induced by inflammation participates in the damage of intestinal epithelial homeostasis. However, underlying molecular mechanisms that regulate IEC apoptosis during UC still remain unclear. Epithelial-specific ETS-1 (ESE1), also named ELF3, ERT, ESX and EPR-1, is the prototypic member of a novel subset of the ETS transcription factor family including ESE-2 and ESE-3 [10]. In view of being highly restricted cells of epithelial origin [11], ESE1/ELF3 is expressed in several tissues including kidney, prostate, small intestine, colon, ovary, pancreas, liver and placenta, with particularly high expression in the gastrointestinal tract [12, 13]. ESE1/ELF3 has been reported to be involved in a variety of pathophysiologic processes, including cancer and inflammatory disorders [12, 14, 15]. Ets factors are defined by a highly conserved DNAbinding domain called the Ets domain which binds to a purine-rich GGAA/T core motif in enhancer and promoter regions of target genes [16]. Therefore, ESE1 also contains a pointed domain, which is involved in protein–protein interactions [17]. For instance, ESE1 interacts with Sp1 and AP-1 proteins to induce squamous differentiation marker expression in bronchial epithelial cells [18]. In addition, ESE1 interacts with NF-jB to induce nitric oxide synthase during terminal differentiation of epidermal and primary keratinocytes [19, 20]. Moreover, interactions between ESE1/ ELF3 and NF-jB result in sustained activation of NF-jB to drive prostate cancer progression [21]. Recently, clinical studies demonstrated that the expression and nuclear translocation of ESE1 can be induced by inflammatory cytokines (IL-1b and TNF-a) [22], and cytoplasmic accumulation of ESE1 can be involved in the development of colon cancer, while nuclear translocation of ESE1 enhances colon cells apoptosis [23]. Based on all these findings, this paper puts forward our presumption that ESE1/ELF3 constitutively activates NF-jB to promote IEC apoptosis. Due to the established pro-apoptosis effect of ESE1/ ELF3, it is tempting to speculate that ESE1/ELF3 might be responsible for the pathophysiology of UC by regulating

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IEC apoptosis. The aim of this study was to investigate the intestinal expression of ESE1/ELF3 in UC patients and experimental models for UC and then to discuss its involvement in IECs apoptosis.

Materials and methods Mucosal biopsy specimens The study was performed at the Department of Gastroenterology of the Affiliated Hospital of Nantong University from September 2012 to June 2014 under a protocol approved by its ethical committee. Mucosal biopsy specimens were prospectively collected from inflamed and non-inflamed areas of patients with clinically and macroscopically active UC (n = 20), while control samples were obtained from the normal areas of healthy subjects (n = 20). Biopsy specimens were immediately fixed in formalin and embedded in paraffin until use for immunohistochemistry analysis. Written informed consent was obtained before specimen collection. Animals and experimental colitis Experiments were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (National Research Council, 1996, USA) and were approved by the Chinese National Committee to Use of Experimental Animals for Medical Purposes, Jiangsu Branch. We obtained seven- to eight-weekold female C57Bl/6 mice (n = 21) weighing 18–20 g from the Experimental Animal Center of Nantong University. Induction of acute and chronic dextran sulfate sodium (DSS) colitis was performed as described previously and is depicted in Fig. 2a [24–26]. In brief, 2.5 or 4 % DSS wt/ vol (molecular weight, 36,000–50,000; MP Biomedicals) was dissolved in tap water and given ad libitum. Animals were monitored for changes in body weight, stool consistency and presence of blood in the stool. Mice were killed after 7 days (acute colitis), 10 days (recovery phase) or 4 weeks (chronic colitis). Assessment of colitis activity To examine the severity of colitis, we evaluated the body weight and disease activity index (DAI). The DAI was determined by scoring changes in animal weight, occult blood, gross bleeding and stool consistency. We used five grades of weight loss (0, no loss or weight gain; 1, 1–5 % loss; 2, 5–10 % loss; 3, 10–20 % loss; 4, 20 % loss), three grades of stool consistency (0, normal; 2, loose; and 4, diarrhea) and three grades of occult blood (0, negative; 2,

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occult blood positive; and 4, gross bleeding). After determination of the DAI, mice were euthanized, the entire colon was removed from the cecum to the anus. Subsequently, colonic tissue was fixed in 4 % buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. According to a well-established scoring system described previously, histological score on microscopic cross sections was assigned as follows: (0), no signs of inflammation; (1), very low level; (2), low level of leukocytic infiltration; (3), high level of leukocytic infiltration, high vascular density, thickening of the colon wall; and (4), transmural infiltrations, loss of goblet cells, high vascular density, thickening of the colon wall [27]. Cell culture and stimulation The human colon epithelial cell line HT-29 was purchased from Cell library, China Academy of Science. HT-29 cells were grown in 1640 (GibCo BRL, Grand Island, NY, USA) medium containing 10 % fetal bovine serum (FBS), 100 U/ ml penicillin and 100 lg/ml streptomycin (GibCo BRL, Grand Island, NY, USA). Cultures were incubated at 37 °C in a 95 % air/5 % CO2 atmosphere. Cells were treated with different concentrations (0.1, 1, 10, 100 ng/ml) of TNF-a (Sigma-Aldrich, USA). Cultured HT-29 cells were collected, washed with phosphate buffer solution (PBS) and suspended in hypotonic buffer to achieve nuclear extracts. The cultured cells were homogenized, meanwhile nuclei were pelleted. Then, we removed the cytoplasmic extracts and resuspended nuclei in a low-salt buffer. A high-salt buffer was added to release soluble proteins from the nuclei, and the nuclei were removed by centrifugation. The nuclear extracts were dialyzed into a moderate salt solution. Western blot analysis For Western blot analysis, colon tissues were dissected and flash-frozen at -80 °C. To prepare the lysates, frozen samples were weighed and minced on ice. The samples were homogenized in lysis buffer (1 % NP-40, 50 mmol/L Tris, Ph = 7.5, 5 mmol/L EDTA, 1 % SDS, 1 % sodium deoxycholate, 1 % Triton-X100, 1 mmol/L PMSF, 10 lg/ mL aprotinin and 1 lg/mL leupeptin) and centrifuged at 12,000 rpm and 4 °C for 20 min to collect the supernatant. Cell cultures for immunoblot were lysed with sodium lauryl sulfate loading buffer and stored at -80 °C until use. After determining the protein concentration with the Bradford assay (Bio-Rad, Hercules, CA, USA), protein samples were subjected to SDS–polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5 % fat-free milk in TBST

(20 mM Tris, 150 mM NaCl, 0.05 % Tween-20) for 2 h at room temperature, and then, the filters were washed with TBST for three times and incubated overnight with polyclonal antibody against using the primary antibodies against anti-ESE1 antibody (anti-mouse, 1:500; Santa Cruz), Bcl-xl (anti-mouse, 1:1000; Santa Cruz), cleaved caspase-3 (antirabbit, 1:1000; Cell Signaling), cleaved PARP (anti-rabbit, 1:1000; Cell Signaling),b-actin (anti-mouse, 1:500; Santa Cruz), p65 (anti-rabbit, 1:500; Santa Cruz), p-p65 (antirabbit, 1:1000; Cell Signaling), p-IjB (anti-mouse, 1:1000; Cell Signaling), LaminA/C (anti-mouse, 1:500; Santa Cruz) and a-tublin (anti-rabbit, 1:1000; Santa Cruz) at 4 °C overnight. At last, the membrane was incubated with second antibody goat anti-mouse or goat anti-rabbit conjugated horseradish peroxidase (1:2000, Southern-Biotech) for 2 h and visualized using an enhanced chemiluminescence system (ECL; Pierce Company, USA). Immunohistochemical studies Mice colon samples were freshly isolated and frozen in OCT (Sakura Finetek, USA) or fixed in 10 % neutral buffered formalin and embedded in paraffin wax. Immunohistochemical studies were performed using paraffinembedded sections (4 lm). All sections were deparaffinized and rehydrated, and thereafter, the sections were processed in 10 mM citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 20 min to retrieve the antigen. After rinsing in PBS, sections were first treated with 3 % hydrogen peroxide to block endogenous peroxidase activity and then blocked by 1.5 % normal goat serum for 20 min. For analysis of ESE1, sections were incubated 2 h at room temperature with primary antibody against ESE1 (diluted 1:500). All slides were processed using the peroxidase– anti-peroxidase method (DAKO, Hamburg, Germany). After rinsing in PBS, the peroxidase reaction was visualized by incubating the sections with the liquid mixture DAB (0.1 % phosphate buffer solution, 0.02 % diaminobenzidine tetrahydrochloride and 3 % H2O2). After rinsing in water, the sections were counterstained with hematoxylin, dehydrated and cover-slipped. Immunofluorescent studies Additional sets of sections from mice were used for double immunofluorescence staining. Frozen sections were first blocked with 10 % normal serum-blocking solution species the same as the secondary antibody, containing 3 % (w/v) BSA and 0.1 % Triton X-100 and 0.05 % Tween-20 2 h at RT in order to avoid unspecific staining. Then, the sections were double-stained with primary antibodies against ESE1 (anti-mouse, 1:500) and anti-active caspase-3 (anti-rabbit, 1:1000, Cell Signaling). Briefly, sections were incubated

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with both primary antibodies overnight at 4 °C, subsequently with secondary antibodies (donkey anti-rabbit Alexa FluorÒ 488, 1:500; donkey anti-mouse Alexa FluorÒ 568, 1:1,000) (both from Life Technologies Corporation, Paisley, UK) for 2 h at room temperature. To show the nucleus of cells, sections were counterstained with 4, 6diamidino-2-phenylindole (DAPI; 0.1 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) for 40 min at 30 °C. The stained sections were examined under a Leica fluorescence microscope (Leica, DM 5000B; Leica CTR 5000; Germany). To perform cell immunofluorescent staining, cells were fixed in formaldehyde for 1 h at room temperature. After washing with 1 9 PBS, cells were permeabilized with 0.05 % Triton X-100 in 1 9 PBS for 15 min and incubated with pre-block buffer (3 % BSA, 0.02 % Triton X-100 in 1 9 PBS) for 15 min before being probed with primary antibodies [28]. And then, cells were stained with anti-ESE1 antibody (anti-mouse, 1:50) and anti-p65 antibody (anti-rabbit, 1:50) at 4 °C overnight, followed by secondary antibody for 2 h at room temperature. Nuclei were stained with DAPI (1:1000, Santa Cruz).

A1, 50 -TCTTCTGATGAGCTCAGTT-30 of siRNA2, 50 -A CTACTTCAGTGCGATGTA-30 of siRNA3 and 50 - CCC TAATTTATGTGCTATA-30 of siRNA4, while the control target was 50 -UUCUCCGAACGUGUCACGU-30 . Transfected cells were cultured for 48 h before using.

Coimmunoprecipitaton and immunoblotting

Cell counting kit (CCK)-8 cell viability assays

HT-29 cells were grown to confluence in 10-cm dishes and stimulate with 100 ng/ml TNF-a. At 2 h after stimulating, the growth medium was aspirated, and cells were washed three times with cold phosphate-buffered saline (PBS). Cells were further lysed in plates by using 1 ml of radioimmunoprecipitation assay buffer (150 mM NaCl, 0.5 % Nonidet P-40, 50 mM Tris (pH 8.0), 2 mM PMSF, Complete protease inhibitor mixture) and then collected in microcentrifuge tubes, where cell lysates were purged 10 times through the 27-gauge syringe, followed by a 30-min incubation with gentle rotation at 4 °C. After centrifugation at 4 °C for 30 min at 14,0009g, the supernatant was used for coimmunoprecipitation with 1 lg of specific antibodies or control IgG (Santa Cruz Biotechnology), shaken for 2 h(4 °C), mixed with 30 ll of protein A/G (Santa Cruz Biotechnology), incubated for another 2 h(4 °C) and washed three times with washing buffer. Proteins bound to the beads were boiled in 60 ll of loading buffer followed by Western blotting.

Cell viability was measured using the commercial CCK-8 assays in accordance with the manufacturer’s protocol. Briefly, ESE1 siRNA-treated cells or control cells were seeded at a density of 1 9 105 per well in volumes of 100 lL into a 96-well plate. After adhering, cells were incubated in the presence of 100 ng/ml TNF-a with RPMI 1640 medium containing 10 % FBS. Following treatments, the CCK-8 reagents (Dojindo, Kumamoto, Japan) were added to each well at due time and incubated for additional 1.5 h at 37 °C. The absorbency was measured at a test wavelength of 490 nm and a reference wavelength of 650 nm with a microplate reader (Bio-Rad). Untreated (control) cells served as the indicator of 100 % cell viability.

siRNA-mediated reduction in ESE1 expression HT-29 cells were seeded in 6-well plates at a density of 400,000 cells per well in 1640 medium with Glutamax supplemented with 1 % fetal bovine serum. After 4 h, medium was replaced by serum-free medium. For transient transfection, the ESE1 siRNA vector or the non-specific vector was transfected using SuperFectinTM II and plus reagent in OptiMEM (Invitrogen). The target of ESE1 siRNA was 50 -CATGAGGTACTACTACAAA-30 of siRN

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Flow cytometry-based annexin V/PI staining The flow cytometry assay was performed to measure the degrees of both apoptosis and necrosis using an ApoScreen Annexin V kit (Southern Biotechnology, Birmingham, AL, USA) according to the manufacturer’s protocol. Briefly, HT-29 cells were digested by 0.1 % trypsin and resuspended in cold binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 Mm CaCl2, 0.1 % BSA) at concentrations between 105 and 106 cells/ml. Ten microliters of labeled annexin V was added to 100 ll of the cell suspension. After 15-min incubation on ice, 380 ll binding buffer and 10 ll PI solution were added to the cell suspension. Subsequently, the number of stained cells was assessed via flow cytometer (BD FACSAriaII).

Statistical analysis All data were expressed as mean ± SEM. Data were compared using the Student’s t test. P\0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition.

Results The expression of ESE1/ELF3 was increased in IECs of patients with UC To assess the intestinal expression and localization of ESE1/ELF3 in UC, we performed immunohistochemistry

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analysis on endoscopic mucosal biopsy tissues from noninflamed and inflamed areas of patients with UC and normal individuals. Consistent with the previous findings [12], only weak staining of ESE1/ELF3 was observed in normal mucosal biopsies. However, ESE1/ELF3 staining was stronger in tissues from inflamed regions compared with non-inflamed regions mainly detected in nucleus of IECs, and both were more detectable than in tissues from normal controls (Fig. 1a). Immunoblotting analysis revealed that ESE1 expression was significantly increased in tissue from inflamed areas of patients with UC (Fig. 1b). Since overexpression of ESE1/ELF3 was detected in IECs of patients with UC compared with normal controls, further experiment was required to certify the specific contribution of ESE1/ELF3 to the pathophysiology of the disease and related molecular mechanisms. DSS-induced experimental colitis was established The above observations prompted us to use mouse models to further investigate the role of ESE1/ELF3 in colitis. We established the DSS-induced colitis mouse model of acute colitis, recovery from acute intestinal inflammation and chronic colitis (Fig. 2a), which was a well-established model of acute and chronic colonic inflammation resembling several prominent clinical and morphological features of human UC [29]. Compared with the healthy group, in acute colitis and recovery from acute intestinal inflammation, the body weight of mice began to decline at day 3 and reached the minimum at day 7. While in chronic colitis, the mice body weight gradually decreased from the beginning of the experiment and exhibited significant loss ultimately (Fig. 2b). The clinical scores for weight loss, bleeding and diarrhea composed the disease activity index (DAI). Following giving DSS, the DAI of mice was gradually increased in three groups. After stopping DSS administration, DAI gradually decreased in the recovery groups (Fig. 2c). H&E-stained colon sections of three groups of murine models showed that DSS induced inflammatory cell infiltration within the lamina propria, focal loss of crypts, depletion of epithelial cells and disseminated fibrosis, which was parallel with previous reports (Fig. 2d) [30], while no histological alteration was observed in the intestinal segments of control mice (Fig. 2d). Collectively, the above data indicated that three murine models of colitis induced by DSS were well established in our system. ESE1/ELF3 was up-regulated and translocated to the nucleus during DSS-induced colitis To characterize the expression of ESE1/ELF3 during the course of DSS-induced colitis, the levels of ESE1/ELF3 at different group of murine colitis models were evaluated by

immunoblot analysis. Compared with the healthy group, the expression of ESE1/ELF3 was increased in all the three DSS-treated groups and demonstrated the most apparent over-expression in chronic DSS-induced colitis (Fig. 3a). Previous reports implied that the activity of ESE1/ELF3 can be regulated by its subcellular localization [31], and strong nuclear localization of ESE1/ELF3 has been demonstrated in colorectal cancers [23, 32]. To identify the subcellular distribution of ESE1/ELF3 during DSS-induced colitis, we applied immunohistochemistry analysis. The three DSS-treated groups, especially the chronic DSS-induced colitis, exhibited strong staining of ESE1/ELF3 in nucleus of IECs; on the contrary, only weak staining signal in IEC cytoplasm was detected in the healthy group, which was in line with the immunohistochemistry results of the patient biopsies (Fig. 3b). Due to chronic model complying with ulcerative colitis, immunofluorescent staining was performed with DAPI to further identify the intracellular location of ESE1/ELF3 in this group. In the healthy group, a weak staining of ESE1/ELF3 was located in cytoplasm of IECs. In contrast, we observed a considerable increase in ESE1/ELF3 nuclear signal intensity in chronic DSS-induced colitis, which was in accordance with the result from immunohistochemistry (Fig. 3c). These findings demonstrated that ESE1/ELF3 protein was elevated and translocated to the nucleus in IECs during DSS-induced colitis. ESE1/ELF3 was relevant to IEC apoptosis and NF-jB activation in DSS-induced colitis Multiple studies displayed the increase in IEC apoptosis in DSS-induced colitis [33]. Apoptosis induction in our experimental system was confirmed by immunoblot analysis of active caspase-3 and cleaved PARP, the biochemical markers of apoptosis, along with the anti-apoptotic Bcl-2 family member Bcl-xl. As predicted, the level of active caspase-3 and cleaved PARP was significantly increased in DSS-induced colitis, which was similar with the expression pattern of ESE1/ELF3, while the expression of Bcl-xl was strikingly decreased (Fig. 4a). Moreover, with the aid of double immunofluorescent staining, we observed the enhanced co-localization of ESE1/ELF3 with active caspase-3 in chronic DSS-induced group compared with healthy group (Fig. 4b). Based on the previous observation that activation of ESE1/ ELF3 via inflammation enhanced the nuclear translocation and activation of NF-jB in prostate cancers [21], we hypothesized that NF-jB might also be an important downstream effector of ESE1/ELF3 in regulating apoptosis of IECs in UC. We found that there was no obvious expression change of p65, a subunit of NF-jB. However, compared with the healthy group, the expression of phosphorylated p65 (p-p65) along with p-IjB, the indicators of NF-jB activation as well as IjB degradation, was increased in the DSS groups,

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which was in accordance with the expression pattern of ESE1/ELF3 and apoptotic biochemical markers (Fig. 4c). Moreover, enhanced co-localization of ESE1/ELF3 with phosphorylated p65 was observed in chronic DSS-induced group compared with healthy group (Fig. 4d), indicating that ESE1/ELF3 might play a pivotal part in IEC apoptosis by constitutively activating NF-kB.

Fig. 1 The expression of ESE1 was increased in IECs of patients with UC. a Immunohistochemistry analysis of ESE1 in mucosal biopsies tissues derived from inflamed as well as non-inflamed areas of patients with UC and tissues from normal controls. ESE1 staining was stronger in tissues from inflamed regions compared with non-inflamed regions mainly detected in the nuclei of IECs. The UC group (n = 20) contained 10 males and 10 females; the mean age was 44.5 years. The normal control group (n = 20) contained four males and six females; the mean age was 45.2 years. Scale bar column 100 lm. b Immunoblotting analysis of ESE1 expression. ESE1 expression was significantly increased in tissues from inflamed areas of patients with UC compared with noninflamed regions of UC and normal controls. The bar graph indicated the density of ESE1 versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05)

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Fig. 2 Multiple indicators were used to assess the success of DSS- c induced colitis model. a Acute recovery and chronic colitis were induced as depicted in the schematic. The changes of b body weight, c DAI and d colonic tissue structure by H&E-staining during the development of DSS-induced colitis in mice. Each value indicates the mean ± SEM for six mice. *Significant difference at P \ 0.05 compared with control mice receiving water alone. Scale bar column 100 lm

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Fig. 3 ESE1/ELF3 was up-regulated and translocated to the nucleus during DSS-induced colitis. a Western blot showed ESE1 overexpression in DSS-induced colitis compared with healthy group. The bar graph indicated the density of ESE1 versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). b Immunohistochemistry analysis of ESE1 expression in colonic mucosa of DSS-colitis mice. Scale bar column 100 lm. c Immunofluorescent staining for ESE1 in colonic mucosa of the DSS-colitis mice. Scale bars 100 lm

ESE1/ELF3 was associated with TNF-a-induced apoptosis and NF-jB activation in human HT-29 cells Based on the in vivo studies covering UC patients and animal models, we speculated that ESE1/ELF3 might be involved in IEC apoptosis induced by inflammation via constitutively activating NF-jB pathway. To confirm this postulation, we performed in vitro experiments using the human IEC line HT-29 cells incubated with tumor necrosis factor-a (TNF-a), a well-defined pro-inflammatory cytokine which closely related with the pathogenesis of IBD [34–38]. Western blot demonstrated that TNF-a stimulated ESE1/ELF3 expression in a dose-dependent pattern in HT29 cells; 100 ng/ml TNF-a significantly up-regulated ESE1/ELF3 level and greatly induced the expression of cellular apoptotic markers (active caspase-1 and cleaved PARP) (Fig. 5a). Therefore, this concentration was used for all the subsequent experiments. We next treated HT-29

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cells for increasing time with 100 ng/ml TNF-a and found significantly enhanced ESE1 level beginning at 0.5 h and reached the peak at 2 h (Fig. 5b). The similar concomitant up-regulation of active caspase-3 and cleaved PARP was also observed in a time-dependent manner (Fig. 5b). Given the activity of ESE1/ELF3 was regulated by its subcellular localization [31], effects of TNF-a on nuclear translocation of ESE1 were investigated. HT-29 cells were treated by TNF-a for 0, 0.5, 2 and 4 h, separated into cytoplasmic and nuclear fractions, and analyzed by immunoblot studies. While ESE1/ELF3 level in the cytoplasm was at lower level, a slight increase compared with control cells after TNF-a treatment, the amount of ESE1/ELF3 in the nuclei was clearly elevated, beginning at 0.5 h and reaching the peak at 2 h (Fig. 5c). Then, immunofluorescence revealed that ESE1/ELF3 began appearing in nuclei at 0.5 h and reached peak at 2 h, in accordance with immunoblot studies (Fig. 5d). These findings demonstrated that TNF-a treatment up-regulated the expression and increased the nuclear accumulation of ESE1/ELF3 in HT-29 cells. Various pro-inflammatory stimuli (TNF-a and IL-1a) induced the transcription of pro-apoptosis-related target genes by activating NF-jB in different cell types [39, 40]. Based on the previous observation that activation of ESE1/ ELF3 via inflammation enhanced nuclear translocation constitutively activates NF-jB and drives prostate cancer progression [21], we hypothesized that ESE1/ELF3 might promote TNF-a-induced apoptosis in a NF-jB-dependent way. To verify this postulation, we treated HT-29 cells for increasing time with 100 ng/ml TNF-a and found significantly enhanced p-p65 levels with beginning at 0.5 h and peaking at 2 h. The similar concomitant up-regulation of p-IjB was also observed in a time-dependent manner; however, there was no obvious variation of p65 post-TNFa stimulation (Fig. 6a). Subcellular protein fractionation and Western blot analysis detected a clear increase in p-p65 amount in the nuclei 2 h after TNF-a administration (Fig. 6b), which is consistent with the intracellular transportation of ESE1/ELF3 during TNF-a treatment (Fig. 5c). To further define the contribution of ESE1/ELF3 to NF-jB activation, we examined the expression and intracellular localization of p-p65 in HT-29 cells. Immunofluorescence revealed that both the level and nuclear co-localization of p-p65 and ESE1/ELF3 were elevated in HT-29 cells 2 h after TNF-a treatment (Fig. 6c). Immunoprecipitation with an antibody directed to ESE1/ELF3 coimmunoprecipitated p65 in stimulated HT-29 cells. In addition, ESE1/ELF3 was immunoprecipitated, along with p65, by an anti-p65 antibody, confirming the physical interaction between ESE1/ELF3 and the NF-jB subunits (Fig. 6d). In a word, ESE1/ELF3 might be involved in TNF-a-induced apoptosis by constitutively activating NF-jB in human HT-29 cells.

Immunol Res Fig. 4 Association of ESE1 with IEC apoptosis and NF-jB activation in DSS-induced colitis. a Western blot showed expression of active caspase-3, cleaved PARP and Bcl-xl in DSS-induced colitis. The bar graph indicated the density of active caspase-3, cleaved PARP and Bcl-xl versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). b Double immunofluorescent staining for ESE1 and active caspase-3 in colonic mucosa of the mice treated with DSS. Scale bars 100 lm. c Western blot showed expression of p65, p-p65 and pIjB in DSS-induced colitis. The bar graph indicated the density of p65, p-p65 and p-IjB versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). d Double immunofluorescent staining for ESE1 and p-p65 in colonic mucosa of the mice treated with DSS. Scale bars 100 lm

Inhibition of ESE1/ELF3 restrained TNF-a-induced NF-jB activation and apoptosis in human HT-29 cells To get a deeper understanding of the function of ESE1 in IEC apoptosis, we knocked down the expression of endogenous ESE1 by siRNA in TNF-a-treated HT-29 cells. Western blot analysis showed that siRNA–ESE1, especially ESE1 siRNA3, had a significant effect in reducing ESE1/ELF3 expression in HT-29 cells compared with control siRNA (Fig. 7a). Knockdown of ESE1/ELF3 resulted in down-regulation of p-p65 and p-IjB levels in TNF-a-treated HT-29 cells (Fig. 7b), indicating that NFjB might be an important downstream effector of ESE1/ ELF3 in regulating IEC apoptosis. In addition, inhibiting ESE1/ELF3 expression significantly reduced TNF-a-induced up-regulation of active caspase-3 and cleaved PARP (Fig. 7b). Furthermore, the flow cytometry-based annexin

V/PI staining revealed that ESE1/ELF3 knockdown notably alleviated TNF-a-induced apoptosis in HT-29 cells (Fig. 7c), and the alleviation of the TNF-a-induced reduction in HT-29 cell viability by ESE1 siRNA3 was analyzed by the CCK-8 assay (Fig. 7d). These observations implied that ESE1/ELF3 might facilitate the caspase-dependent IECs apoptosis at least partly mediated by promoting NF-jB activation.

Discussion Intestinal epithelial cells (IECs) organizing as a single cell layer which covers the intestine provide essential roles for host defense and homeostasis, including participating in mucosal immune responses and maintaining barrier function [41–43]. Adjustment of IECs apoptosis is one way that the intestinal epithelium maintains or returns to

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Immunol Res Fig. 5 TNF-a-enhanced expression and nuclear translocation of ESE1 in human HT-29 cells. a Western blot showed expression of ESE1, active caspase-3 and cleaved PARP in HT-29 cells treated by different concentrations of TNF-a. The bar graph indicated the density of ESE1, active caspase-3 and cleaved PARP versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). b Western blot showed expression of ESE1, Bcl-xl, active caspase-3 and cleaved PARP in TNF-a (100 ng/ml)-treated HT-29 cells at different time points. The bar graph indicated the density of ESE1, active caspase-3 and cleaved PARP versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). c The detection of ESE1 was performed in cytoplasmic and nuclear extracts from control and TNFa (100 ng/ml)-treated groups at 0, 0.5, 2 and 4 h. d Immunofluorescent staining for ESE1 in TNF-a-treated cells was performed (original magnification 9200)

homeostasis [44]. During UC, IECs apoptosis is observed in active inflammatory sites [44, 45]. Clinical therapy with TNF-neutralizing antibody effectively decreases IEC apoptosis and promotes mucosal repair, but the mechanisms for TNF-mediated tissue injury in UC remain unclear [46, 47]. In the current study, we took new insight into the molecular mechanisms for IEC apoptosis regulated by ESE1/ELF3 in UC. Epithelial-specific ETS-1 (ESE1) protein is part of the ETS family of transcription factors and is also named as ELF3 [13], ERT [48] and ESX [49]. ESE1 proteins contain a highly conserved DNA-binding domain that recognizes the purine-rich GGA (A/T). ESE1 proteins are

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constitutively expressed in many types of epithelia, including liver, lung and intestine [12], and regulate terminal differentiation of the epidermis [12, 50]. ESE1 has various functions in the transcriptional regulation of genes involved in inflammation progress, epithelial differentiation and development of cancer [51], relying on exact molecular mechanisms and cell context. The present study supports the crucial role of ESE1 in UC by inducing IECs apoptosis. Using a murine model of DSS-induced colitis that mimics UC, we discovered that ESE1/ELF3 was significantly up-regulated, companied by a concomitant increasing of caspase-3 enzyme activity and PARP cleavage, key markers of cell apoptosis [52, 53]. Furthermore, high

Immunol Res Fig. 6 ESE1 was involved in TNF-a-induced NF-jB activation in human HT-29 cells. a Western blot showed expression of p65, p-p65 and pIjB in TNF-a (100 ng/ml)treated HT-29 cells at different time points. The bar graph indicated the density of p65, pp65 and p-IjB versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). b The detection of p-p65 was performed in cytoplasmic and nuclear extracts from control and TNFa (100 ng/ml)-treated groups at 0, 0.5, 2 and 4 h. c Double immunofluorescent staining for ESE1 and p-p65 in TNF-atreated HT-29 cells at 2 h (original magnification 9200). d Lysates of HT-29 cells treated with TNF-a were immunoprecipitated with antibodies against ESE1/ELF3 and analyzed by immunoblotting with the indicated antibodies

level of ESE1/ELF3 was detected in IECs and appeared to co-localize with active caspase-3, indicating the association of ESE1/ELF3 with IEC apoptosis. Then, the in vitro experiment in human HT-29 cells revealed that ESE1/ELF3 expression was increased in TNF-a-induced cellular apoptosis model. Furthermore, knocking ESE1/ ELF3 down by RNA interference (RNAi) in HT-29 cells could alleviate TNF-a-induced apoptosis assessed by active caspase-3, cleaved PARP, flow cytometry and CCK-8 cell viability assay, indicating that ESE1/ELF3 might exert its pro-apoptotic function in IECs. Taken together, our present study suggested that under the inflammatory

microenvironments, over-expressed ESE1/ELF3 might promote the caspase-mediated IEC apoptosis in UC. The molecular mechanism that ESE1/ELF3 regulates IEC apoptosis in UC remains to be explored. As an important transcription factor, ESE1/ELF3 may function as a novel mediator of the inflammatory response, which is supported by the findings that ESE1 can activate the expression of several genes in response to pro-inflammatory mediators. For example, iNOS is activated by ESE1 in endothelial cells, and COX-2 is another target for ESE1 in monocyte/macrophages. Matrix metalloproteinases (MMPs) 1 and 13, which are involved in cartilage and bone destruction in RA and OA, are

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Fig. 7 Knockdown of ESE1 attenuated TNF-a-induced NF-jB activation and cellular apoptosis in human HT-29 cells. a Western blot assay determined the knockdown efficiency of ESE1 by siRNA. We detected ESE1 expression by Western blot after transfected with ESE1–siRNA in HT-29 cells, then stimulated with TNF-a, while ESE1–siRNA3 achieved the best down-regulation effect. The bar graph indicated the density of ESE1 versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). b ESE1–siRNA3 declined the expression of p-p65, p-IjB, active caspase-3 and cleaved PARP in TNF-a stimulated HT-29 cells. The bar graph indicated the

density of p-p65, p-IjB, active caspase-3 and cleaved PARP versus b-actin. Data were presented as mean ± SEM (n = 3, *P \ 0.05). c Flow cytometry assay showed that ESE1–siRNA3 reduced significant number of annexin V?/PI- cells after TNF-a stimulating. d Cell viability was measured by Cell Counting Kit-8 (CCK-8) cell viability assay. HT-29 cells were untreated (control) or treated with 100 ng/ml TNF-a for different time points. Untreated (control) cells served as the indicator of 100 % cell viability. The data were normalized to that of control and presented as mean ± SEM (n = 3, *P \ 0.05)

regulated by ESE1 as well. These genes are involved in the initiation and perpetuation of inflammation and tissue destruction [54]. During angiotensin II-mediated vascular inflammation and remodeling, ESE1 synergizes with NF-jB to induce the expression of NOS2 [32]. Moreover, activation of ESE1/ELF3 via inflammation enhanced the nuclear translocation and activation of NF-jB and drove prostate cancer

progression [21]. Based on the above reports, we hypothesized that NF-jB might also be an important downstream effector of ESE1/ELF3 in regulating apoptosis of IECs in UC. Here, we found that the level of p-p65 and p-IjB was increased during DSS-induced colitis in vivo and TNF-induced cellular apoptosis in vitro. Furthermore, we observed the enhanced co-localization of ESE1/ELF3 with p-p65 both

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in intestinal epithelium of experimental colitis and in TNF-astimulated HT-29 cells. Coimmunoprecipitation assay further confirmed the physical interaction between ESE1/ELF3 and the NF-jB subunits in TNF-a-treated HT-29 cells. Inhibiting the ESE1/ELF3 expression by siRNA greatly attenuated TNF-a-stimulated phosphorylation of p65 and IjB in HT-29 cells. All of our data suggested that in UC, ESE1/ELF3 might play a crucial role in IEC apoptosis at least partly through facilitating NF-jB activation. The activity of ESE1/ELF3 can be regulated by its subcellular localization [31]. In the present study, we found that TNF-a resulted in the translocation of ESE1 protein to the nucleus in HT-29 cells. Previous findings suggested that this temporal pattern of nuclear ESE1 translocation and binding of ESE1 in the NF-jB promoter [19, 21] nicely corresponded to TNF-induced activation of p65 gene expression at the transcriptional level. Under our experimental conditions, ESE1 nuclear translocation was almost exclusively observed in cells 0.5 h after administration of TNF-a, reaching the peak at 2 h, which is consistent with the intracellular transportation of p-p65. Immunofluorescent double staining further confirmed the up-regulated accumulation and co-localization of p65 and ESE1 in the nuclear following TNF-a exposure. After knocking ESE1/ELF3 down by RNAi in TNF-a-treated HT-29 cells, p-p65 level in the nuclear was at lower level comparing with control RNAi. In a word, the inflammatory factors, such as TNF-a, may not only stimulate ESE1 expression but also modulate its subcellular distribution and thus regulate IEC apoptosis in UC through constitutively activating NF-jB. The recent study reported that ESE1 could inhibit proliferation and induce apoptosis in human colorectal cancer cells through an EGR-independent pathway [23], indicating the potential beneficial effect of ESE1 in development of cancer. Here, we discovered the phenomenon that ESE1 was up-regulated and promoted IEC apoptosis in UC, which should damage the intestinal epithelial homeostasis and drive UC progression. Considering a relatively higher risk of developing colorectal cancer in individuals with UC than the general population [55], we speculated that ESE1-induced IEC apoptosis might play different effects on different stages of the disease. Moreover, the exact functions of ESE1 might greatly depend on its specific molecular partners within IECs under certain pathological conditions. Taken together, our study demonstrated for the first time that intestinal expression of ESE1/ELF3 was significantly up-regulated in UC. Moreover, the expression of ESE1 in acute phase is similar with remission phase. So, the grade of activity of UC does not influence the expression of ESE1. Both in vivo and in vitro experiments are compatible with the hypothesis that increased levels of ESE1/ELF3 might promote IECs apoptosis at least partly through activating NF-jB pathway. Further research is needed to demonstrate

the exact activities and detailed molecular mechanisms of ESE1/ELF3 in the intestinal epithelial homeostasis and UC development. Thus, ESE1/ELF3 could represent a novel target for future therapies in human UC. Acknowledgments This work was supported by National Basic Research Program of China (973 Program, No. 2012CB822104); National Natural Science Foundation of China (81201252, 81470806, 81171140, 81472272); Nantong City Social Development Projects funds (HS2012032); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); the Natural Science Foundation of Jiangsu Province Grant (BK20141496).

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