Objective Raf kinase inhibitor protein (RKIP) appears to control cancer cell metastasis and its expression in colonic tissue is related to colonic cancer development. We sought to identify the roles of RKIP in maintaining homeostasis of GI tract.
Design The expression of RKIP was determined by immunohistochemistry and western blot analysis. RKIP knockout and wild-type mice were administered dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce experimental colitis, and the mice were assessed based on colitis symptoms and biochemical approaches. The mechanism was analysed using immunoprecipitation and pull-down experiments.
Results The RKIP expression is positively correlated with the severity of IBD. RKIP deficiency protects mice from DSS-induced or TNBS-induced colitis and accelerated recovery from colitis. RKIP deficiency inhibits DSS-induced infiltration of acute-phase immune cells and reduces production of proinflammatory cytokines and chemokines in colon. RKIP deficiency inhibits DSS-induced or TNBS-induced colonic epithelial barrier damage and intestinal epithelial cell (IEC) apoptosis. RKIP deficiency also inhibits tumour necrosis factor-alpha-induced IEC apoptosis and colitis. Mechanistically, RKIP enhances the induction of P53-upregulated modulator of apoptosis by interacting with TGF-β-activated kinase 1 (TAK1) and promoting TAK1-mediated NF-κB activation. This is supported by the observation that TAK1 activation is positively correlated with the expression of RKIP in human clinical samples and the development of IBD.
Conclusions RKIP contributes to colitis development by promoting inflammation and mediating IEC apoptosis and might represent a therapeutic target of IBD.
- COLONIC DISEASES
- IBD BASIC RESEARCH
Statistics from Altmetric.com
Significance of this study
What is already known on this subject?
Abnormal intestinal epithelial cell (IEC) apoptosis accelerates IBD.
Raf kinase inhibitor protein (RKIP) expression in colonic tissue is related to colon cancer.
RKIP is a critical regulator of physiological processes, including cancer cell metastasis, invasion and apoptosis.
What are the new findings?
Colonic RKIP expression is positively correlated with the severity of inflammatory colitis in humans and mice.
RKIP deficiency relieves experimental colitis and contributes to the recovery from dextran sulfate sodium-induced colitis.
RKIP deficiency inhibits IEC apoptosis and preserves the integrity of the intestinal epithelial barrier.
RKIP promotes IEC apoptosis via the TGF-β-activated kinase 1 (TAK1)/NF-κB/P53-upregulated modulator of apoptosis axis. TAK1 activation is positively correlated with the expression of RKIP in human clinical samples and the development of IBD.
How might it impact on clinical practice in the foreseeable future?
Targeting RKIP expression might be a new strategy for the treatment of IBD.
IBD consists of UC and Crohn's disease (CD), which are major chronic disorders that affect the GI tract in humans. Apoptosis causes intestinal epithelial cell (IEC) shedding, which in turn leads to barrier loss.1 Abnormal apoptosis is considered as one of the major courses that accelerates IBD. Increased apoptosis of IEC results in disruption of intestinal mucosal integrity and barrier functions in murine IBD models.2–4 Increased IEC apoptosis has also been detected in acute inflammatory colonic tissue from the biopsies of patients with UC and CD.3 ,5 Aberrant IEC apoptosis impairs the mucosal barrier, which leads to intestinal hyperpermeability, invasion of luminal antigens and commensal microflora, and triggers the production of proinflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) and interferons. TNF-α and interferon gamma (IFN-γ) further induce IEC apoptosis,6 ,7 and this vicious feedback eventually results in the clinical signs and symptoms of IBD. P53-upregulated modulator of apoptosis (PUMA) has been shown to be an important regulator of IEC apoptosis.3 However, the molecular mechanisms that regulate IEC apoptosis during the development of IBD remain elusive.
Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding protein family, and it is widely expressed in both prokaryotic and eukaryotic organisms. RKIP modulates and controls crucial intracellular signalling networks, including the signalling cascades of Raf/MEK/ERK, NF-κB, glycogen synthase kinase-3B and G-protein-coupled receptors;8–11 therefore, it is involved in the regulation of a variety of physiological processes such as cell differentiation, cell cycle, apoptosis and controlling cardiac contractile force.12–15 RKIP has been shown to promote chemotherapy-induced apoptosis in a number of cancers.16 Recently, Moen, E. L. reported that Helicobacter pylori induce gastric cancer cell apoptosis via the STAT3-Snail-RKIP loop.17
RKIP expression in colonic tissue has been shown to be related to colonic cancer development;18 however, its functions in other GI tract diseases remain unclear. In this study, we found that RKIP expression is significantly increased in patients with UC and CD as well as in mouse experimental colitis models, and its expression is correlated with the severity of colitis. RKIP deficiency protects mice from dextran sulfate sodium (DSS)/2,4,6-trinitrobenzenesulfonic acid (DSS/TNBS) or TNF-α-induced IEC apoptosis and colitis. Our studies have further shown that RKIP regulates IEC apoptosis in colitis via the TGF-β-activated kinase 1(TAK1)/NF-κB/PUMA axis. Collectively, our findings demonstrate that RKIP is involved in the pathogenesis of colitis by mediating IEC apoptosis induced by intestinal inflammation and injury. Thus, RKIP may serve as a new diagnostic marker and therapeutic target for IBD.
RKIP expression is increased in IBD
We first recruited two cohorts (cohort1 and cohort2) of study subjects from Xinhua Hospital and the Second Affiliated Hospital of Zhejiang University, School of Medicine (SAHZU), and used the anti-RKIP antibody to trace the RKIP expression in the colonic biopsies. As shown in figure 1A, the RKIP protein level in IECs was markedly enhanced in both patients with CD and patients with UC compared with normal control subjects. In the samples from cohort1 (Xinhua Hospital), all of the UC patient biopsies (34/34) exhibited strong RKIP staining, whereas only 10.3% of the normal control subjects (3/29) showed strong RKIP staining (p<0.001; table 1).
In the SAHZU cohorts, strong RKIP expression in IECs was observed in 75.0% (15/20) of the UC patient biopsies and 70.0% (14/20) of the CD patient biopsies, whereas only 18.2% (4/22) of the normal control subjects showed strong RKIP staining (p<0.01; table 2).
Importantly, RKIP expression was higher in the patients with moderate or severe colitis than in those with mild colitis (figure 1B). Moreover, RKIP expression was downregulated in the colonic mucosa collected from patients with IBD during remission compared with the colonic tissue from the same patients with IBD with active disease (figure 1C, see online supplementary figure S1A), indicating that RKIP expression was positively correlated with the severity of colitis. Similar to previous observations in human IBD, RKIP expression in IECs was dramatically elevated in mouse after DSS or TNBS treatment (figure 1D). Consistently, RKIP induction was increased with the development of DSS-induced colitis in mice (figure 1E).
To further explore the kinetic of RKIP induction, we sacrificed DSS-treated mice daily to determine the RKIP expression in IECs and monitored the disease symptoms in the mice. RKIP expression was significantly increased on the second day after DSS treatment (see online supplementary figure S1B, C). However, inflammatory cells could hardly be observed in the colonic tissue until the fourth day after DSS treatment (see online supplementary figure S1D). The obvious disease symptoms including marked inflammatory cell infiltration, the significant increase of disease activity index (DAI) scores and colon length shortening were observed on the fourth day after DSS treatment (see online supplementary figure S1D, E). These results suggest that RKIP expression precedes the appearance of disease symptoms and contributes to the colitis development.
RKIP-deficient mice are resistant to DSS-induced or TNBS-induced experimental colitis
To explore the function of RKIP in colitis development, RKIP knockout (RKIP-KO) mice and control wild-type (WT) littermates were initially challenged in an acute experimental colitis model using 3% DSS. The death rate of RKIP-KO mice was significantly decreased compared with that of the control littermates (figure 2A). Simultaneously, RKIP-KO mice and WT mice were challenged with 2.5% DSS for 5 days, and the mice were monitored daily for clinical disease. Although all of the DSS-treated mice developed colitis from day 4, body weight loss and DAI score were significantly lower in the KO mice than in WT mice (figure 2B, C). WT mice displayed more severe diarrhoea, rectal bleeding and colon shortening than the KO mice (figure 2C–E). Haematological examination showed that WT and KO mice did not exhibit obvious pathological signs in the intestine under basal conditions. However, DSS treatment induced substantially less epithelial damage and crypt architecture disruption in RKIP-KO mice compared with the WT littermates (figure 2F, G).
We next determined whether RKIP deficiency might protect mice in another colitis model induced by TNBS. Both WT and KO mice began to lose body weight on the first day after TNBS rectal injections, and maximum weight loss was observed on days 3–4 after TNBS injections. However, TNBS induced less body weight loss, colon shortening and disease activity in the RKIP-KO mice (see online supplementary figure S2A–D). Histological examination demonstrated greater loss of crypts, increased severity of focal ulceration and increased inflammation in the colons of WT mice compared with that of RKIP-KO mice (see online supplementary figure S2E, F). These data indicate that RKIP contributes to DSS-induced and TNBS-induced colonic damage and colitis.
The DSS-induced IBD consisted of two stages: acute (day 5) stage and recovery (days 9–20) stage.19 ,20 The most severe clinical colitis was observed on day 9 in both the WT and RKIP-KO mice. After the ninth day, the mice started to recover, and RKIP-KO mice gained up to 90% of their original body weight, whereas WT mice only gained up to 70%–80% (see online supplementary figure S3A). In addition, WT mice suffered from much more severe diarrhoea and rectal bleeding than the RKIP-KO mice (see online supplementary figure S3B). Histopathological changes in the different pathological stages of colitis were monitored by H&E staining. Similar to the body weight changes, the architecture destruction and epithelium damage in RKIP-KO mice were less severe compared with their control littermates (see online supplementary figure S3C). These results indicate that RKIP deficiency facilitates recovery from DSS-induced colitis in mice.
RKIP deficiency restricts inflammatory responses in colon tissue
IBD is an inflammation-driven chronic disease with high levels of inflammatory cell infiltration into local tissue.20 As shown in figure 3A and online supplementary figure S4A, the colons of RKIP-deficient mice contained fewer infiltrating F4/80+ macrophages and GR-1+ neutrophil inflammatory cells during the recovery phase of DSS-induced colitis (at days 9 and 20) but not at early stages (day 5), indicating that RKIP contributes to DSS-induced colitis after the initiation of the inflammatory response. Moreover, DSS treatment dramatically induced mucosal proinflammatory cytokine and chemokine (TNF-α, interleukin (IL) 1β, IL-6, KC, CXCL2 and CCL20) expression in WT mice, and the induction of these cytokines and chemokines was substantially attenuated in RKIP-KO mice (figure 3B, C and see online supplementary figure S4B), indicating that the RKIP-KO colons are less inflamed than the WT colons.
Absence of RKIP in non-hematopoietic cells relieves the DSS-induced colitis
To determine the relevant cell compartment responsible for the reduced GI inflammation in the RKIP-KO mice, bone marrow chimaera experiments were performed. Lethally irradiated control littermates and RKIP-KO mice were reconstituted with bone marrow cells from WT mice. The reconstituted mice with RKIP deficiency in non-hematopoietic cells exhibited reduced loss of body weight and lower DAI compared with the WT chimaeras after DSS treatment (figure 4A, B). The colon phenotype, colon shortening and histological score from RKIP deficiency in mice with non-hematopoietic cells (WT→KO) were also decreased compared with that of WT mice chimaeras (WT→WT) (figure 4C–F). Collectively, these data implicate that the lack of RKIP in the epithelial or stromal cells protect the mice against DSS-induced colitis.
RKIP deficiency maintains intestinal barrier integrity by suppressing DSS/TNBS-induced IEC apoptosis
It has been reported that IBD in patients and experimental colitis in mice involve an abnormal intestinal barrier.21 To investigate if RKIP affects intestinal barrier function in the DSS-induced colitis model, we determined FITC-dextran in the serum of WT and KO mice 7 days after DSS treatment. The WT and KO mice displayed similar epithelial permeability before DSS treatment (figure 5A). However, the WT mice displayed higher serum FITC-dextran concentrations compared with the RKIP-KO mice after DSS treatment (figure 5A), indicating that RKIP is important in the control of mucosal permeability upon DSS treatment. The tight junction protein ZO-1 has been used to indicate the colonic epithelial integrity in the experimental colitis.22 ,23 As shown in figure 5B, obvious decreases in ZO-1 in focal regions were observed in the colons of WT mice 4 days after the TNBS treatment or 7 days after the DSS treatment. In contrast, the colons of the RKIP-KO mice maintained a relatively normal ZO-1 pattern in the epithelium (figure 5B).
IEC apoptosis is thought to be one of the major accelerators of the disruption of intestinal epithelial integrity. Compared with WT littermates, the level of TUNEL-positive epithelial cells in RKIP-KO mice was markedly reduced in the DSS/TNBS-induced colitis models (figure 5C). Consistent with these observations, activated caspase3-positive staining cells in RKIP-KO mice epithelium were also significantly less than that in WT mice after exposure to DSS (see online supplementary figure S5A). PUMA, an important mediator of IEC apoptosis in colitis,3 ,24 was upregulated in DSS-treated or TNBS-treated WT mice. However, DSS-induced or TNBS-induced PUMA expression was dramatically suppressed in RKIP-KO mice (figure 5D). Simultaneously, we examined the mRNA level of PUMA and other apoptosis-related proteins including Bax, Bid, Bik, Bim, P53, Bcl-2, Bcl-XL, Mcl-1 and Bag1 by quantitative real-time PCR (RT-PCR) and only the induction of PUMA by DSS was reduced in RKIP deficiency mice (see online supplementary figure S5B). Western blotting also showed that PUMA induction and caspase3 activation in colonic tissue were attenuated in the TNBS-treated RKIP-KO mice (figure 5E). PUMA is transcriptionally induced by NF-κB in colon tissue.3 Consistently, NF-κB activation induced by DSS or TNBS treatment was impaired in the RKIP-KO mice (figure 5E, see online supplementary figure S5C). Notably, the expression of RKIP in IEC was dramatically induced by the DSS or TNBS treatment (figure 5E, see online supplementary figure S5C). These data suggest that RKIP impairs the intestinal barrier via the NF-κB/PUMA/caspase3-dependent apoptosis pathway.
RKIP promotes TNF-α-induced apoptosis and the activation of TAK1/NF-κB/PUMA axis in HCT116 colonic cancer cells
Critical to IBD pathogenesis, TNF-α and IFN-γ can induce IEC apoptosis, and TNF-α-blocking agents have shown clinical treatment efficacy in both patients and mice.25 We overexpressed RKIP or silenced RKIP in the HCT116 colonic cancer cells, and they were then stimulated with human TNF-α for 48 h to induce cell apoptosis.26 The efficiency of RKIP overexpression or silencing was confirmed by western blot (see online supplementary figure S6A). As shown in figure 6A, B and online supplementary figure S6B,C, RKIP overexpression promoted TNF-α-induced cell apoptosis, whereas RKIP silencing inhibited it in HCT116 cells. In addition, RKIP promoted TNF-α-induced PUMA expression and caspase3, NF-κB activation but had no effect on the expression of other apoptosis-related proteins such as P53, Bcl-2 and Bcl-XL (figure 6B, see online supplementary figure S6C, D). Consistently, TNF-α-induced activation of NF-κB and caspase3 were significantly decreased in RKIP-deficient MEFs (see online supplementary figure S6D). TNF-α-induced expression of PUMA was also significantly decreased in RKIP-deficient MEFs (see online supplementary figure S6D). We next knocked down the gene of PUMA in the HCT116 cell to detect if PUMA is required for the effect of RKIP on promoting TNF-α-induced apoptosis. As shown in figure 6C, D, the expression of PUMA in HCT 116 cell silenced with PUMA-specific small interfering RNA could hardly be detected. RKIP overexpression promoted TNF-α-induced cell apoptosis and caspase3 activation in HCT116 cells, and this promotion was vanished in the PUMA-silencing HCT116 cells, indicating that PUMA is required for RKIP promoting TNF-α-induced cell apoptosis.
The activation of TAK1, which is critical for NF-κB activation,27 ,28 was highly increased by RKIP in the TNF-α-treated HCT116 cells (figure 6B, see online supplementary figure S6C). Immunofluorescence staining assay and western blot showed that the colonic tissue of RKIP-KO mice displayed a lower P-TAK1 level in vivo both in DSS-induced and TNBS-induced colitis model (figure 6E, see online supplementary figure S6E). Furthermore, phosphorylation of TAK1 was dramatically increased in colonic tissue from patients with IBD (figure 6F, G). The expression of RKIP and P-TAK1 in 22 paired IBD samples was analysed. As shown in figure 6H, there was a pronounced positive correlation between RKIP expression and P-TAK1 level in the samples.
RKIP deficiency ameliorates TNF-α-induced IEC apoptosis
We further evaluated the effect of RKIP deficiency in mice on TNF-induced pathology. Compared with WT mice, which displayed severe symptoms associated with TNF-α toxicity, including hypothermia and diarrhoea, RKIP-KO mice displayed less severe symptoms, and their body temperatures began to decrease at 4 h after injection. Approximately 2 h later, their body temperature began to recover. At sacrifice, the KO mice body temperature had recovered to the original temperature, whereas the WT mice body temperature was still low at 30°C (figure 7A). We assessed inflammation by quantifying proinflammatory cytokine and chemokine production after TNF-α injection. KO mice exhibited lower levels of IL-6 and MCP-1 in the circulation after TNF-α challenge than WT littermates (figure 7B). Treatment with TNF-α resulted in damage to the WT mice's colonic architecture, with extensive epithelial destruction and a loss of crypt-villus structure. However, RKIP-KO littermates maintained the tissue integrity following treatment with TNF-α (figure 7C). TUNEL assay showed that TNF-α-challenged WT mice suffered from much higher epithelial cell apoptosis than RKIP-KO mice (figure 7D, E). Accordingly, RKIP-KO mice displayed lower intensity of PUMA and P-TAK1 staining in the colonic section (figure 7F, see online supplementary figure S7A), and more ZO-1-positive staining in epithelial cells than WT mice after TNF-α injection (see online supplementary figure S7B). It is worth noting that RKIP expression was strongly induced by TNF-α treatment both in vitro (figure 7G, see online supplementary figures S6C, D and S7E) and in vivo (see online supplementary figure S7C). These results demonstrated that RKIP deficiency can relieve TNF-α-induced intestinal cell apoptosis and protect mice from TNF-α-related colitis.
It was reported that polycomb protein enhancer of zeste homolog 2 (EZH2) suppressed RKIP expression in malignant cancer cell.29 Microarray analysis of mouse colonic tissue showed that EZH1 and EZH2 were significantly downregulated upon DSS treatment (see online supplementary figure S7D), which was confirmed by RT-PCR. TNF-α treatment also greatly inhibited the mRNA expression and protein level of EZH1 and EZH2 while enhancing the expression of RKIP (figure 7G, see online supplementary figure S7E). Overexpression of EZH1 in HCT116 cells completely reversed TNF-α-induced expression of RKIP (figure 7H). Furthermore, EZH1 expression was significantly inhibited in IEC upon TNF-α treatment or DSS/TNBS treatment (figure 7I, see online supplementary figure S7F), whereas EZH2 expression was slightly reduced (figure 7I). These results suggest that EZH1 might be involved in RKIP upregulation in TNF-α-induced intestinal inflammation.
RKIP interacts with TAK1 and promotes TAK1 dimerisation and autophosphorylation
To investigate the underlying mechanisms involved in the positive regulation of TNF-α-activated TAK1/NF-κB/PUMA axis pathway by RKIP, we immunoprecipitated RKIP from TNF-α-treated HCT116 cell lysates. RKIP physically associated with TAK1 in untreated cells but not TRAF2. TNF-α treatment promoted the interaction between TAK1 and RKIP (figure 8A, B and data not shown). Moreover, RKIP bound to TAK1 in colon tissue, and this binding was enhanced by DSS treatment (figure 8C). When transiently overexpressed, RKIP also interacted with TAK1 but not TRAF2 (data not show). RKIP directly interacted with TAK1, as MPB-TAK1 was able to pull down GST-RKIP and vice versa (figure 8D, E), both of which were expressed and purified from the Escherichia coli BL21 strain. Domain-mapping experiments showed that RKIP bound to the C-terminal of TAK1 (aa 303–579) (figure 8F), which is essential for TAK1 intermolecular interaction and autophosphorylation.30 Therefore, we transfected the kinase mutant,30 Flag-TAK1 (K63W) and WT EGFP-TAK1 with or without Myc-RKIP into 293 T cells and examined the phosphorylation status of Flag-TAK1 (K63W). As shown in figure 8G, RKIP overexpression enhanced WT EGFP-TAK1 and Flag-TAK1 (K63W) phosphorylation at Thr187, which is the autophosphorylation site of TAK1.31 Furthermore, RKIP promotes TAK1 phosphorylation in a dose-dependent manner (figure 8H). Transient transfection and coimmunoprecipitation experiments demonstrated that RKIP promoted TAK1 self-association (figure 8I). Collectively, these data indicate that RKIP binds with TAK1 and enhances TAK1 dimerisation and autophosphorylation.
RKIP has been demonstrated as a metastasis suppressor, and reduced RKIP expression is related to a number of metastatic cancers such as colorectal cancer, prostate cancer and breast cancer.18 ,29 ,32 In this study, we demonstrated that RKIP contributes to the development of IBD in humans and mice. RKIP expression in the colon is greatly induced in patients with CD and UC and in mouse with experimental colitis. RKIP deficiency in mice restricts DSS-induced and TNBS-induced IEC apoptosis and maintains the integrity of the epithelial barrier, leading to experimental colitis resistance.
Numerous reports have implicated RKIP in disease-associated conditions. RKIP loss is tightly associated with a number of aggressive cancers, and RKIP may serve as a positive predictive marker of cancer progression and metastasis.32–34 In this study, we found that RKIP expression was positively correlated with the severity of inflammatory colitis in humans and mice. RKIP deficiency protects mice from DSS/TNBS-induced acute colitis and accelerates intestinal recovery after DSS challenge, demonstrating that RKIP promotes intestinal epithelium injury. Furthermore, RKIP deficiency in the non-hematopoietic systems significantly relieves DSS-induced colitis, indicating that RKIP expressed in the epithelium is responsible for the regulation of IBD.
Apoptotic IEC death has been implicated as a major homeostatic and pathogenic mechanism of IBD,2 ,3 and RKIP deficiency inhibits DSS/TNBS-induced IEC apoptosis. The FITC-dextran permeability experiment suggests that RKIP deficiency is helpful in maintaining the integrity of colonic epithelial barrier, which leads to the resistance of RKIP-KO mice to experimental colitis. Furthermore, we found that the induction of PUMA, a critical regulator controlling IEC apoptosis in a P53-independent manner, was significantly suppressed in RKIP-KO mice after TNBS or DSS treatment. The activation of NF-κB, which mediates inflammation-induced PUMA upregulation in IEC, is impaired in RKIP-KO mice, indicating that RKIP promotes apoptosis by inducing PUMA through the activation of NF-κB. RKIP deficiency also suppresses DSS-induced colonic inflammation as monitored 9 days after DSS treatment. However, on day 5 after DSS treatment, there were no significant differences in inflammatory cell infiltration between the WT and RKIP-KO mice, suggesting that RKIP deficiency might suppress DSS-induced colonic inflammation by maintaining the integrity of colonic epithelial barrier.
TNF-α is a major cytokine that is responsible for PUMA induction and IEC apoptosis in colitis.3 ,26 ,35 Consistent with the findings from the DSS and TNBS model, RKIP deficiency restricted TNF-α-induced PUMA expression and IEC apoptosis and protected mice from TNF-α-induced colitis in mice. In HCT116 colonic tumour cells, RKIP promoted the activation of TAK1/NF-κB/PUMA axis and increased TNF-α-induced apoptosis. It is reported that tumour progression locus 2 (TPl2) governs the MAP3K-mediated MEK1/ERK1/2 activation in (TNF receptor) signalling.36 RKIP has no effect on TNF-α-induced ERK1/2 activation, suggesting that RKIP functions in TNF-α signalling in a Tpl2-independent manner. We further demonstrated that RKIP interacts with TAK1 in both HCT116 and colon tissue and that TNF-α or DSS treatment increases the interaction between RKIP and TAK1. RKIP binds to C-terminal of TAK1 and promotes TAK1 dimerisation and autophosphorylation. The absence of RKIP in IECs substantially inhibited DSS, TNBS and TNF-α-induced TAK1 activation and also inhibited NF-κB, caspase3 activation and PUMA induction. Collectively, RKIP contributes to IEC apoptosis and colitis by promoting the TAK1/NF-κB /PUMA pathway (see online supplementary figure S8). These data extend the pathological roles of RKIP and add a new dimension to the multiple actions of RKIP, directly modulating the catalytic activities of kinase instead of interrupting the interactions of kinases with their substrates.11
RKIP was considered as an inhibitor of the NF-κB pathway, as reported by Yeung that RKIP overexpression inhibits the TNF-α-induced NF-κB activation by luciferase reporter assay in 293 T cells.10 However, Yeung et al recently reported that RKIP silencing inhibits IL-1β-induced TAK1/NF-κB activation in COS-1 cells.37 These results indicate that RKIP might play different roles in the NF-κB pathway in different cells. TAK1 is a proinflammatory effector that contributes to activation of the NF-κB pathway38 and is a key regulator of inflammation.39 The role of TAK1 in the development of IBD is still controversial. TAK1-KO in IECs results in intestinal inflammation,40 indicating that TAK1 protects the mice against colitis. Recently, Takahashi reported that the mucin 1 oncoprotein promotes the intestinal inflammation via activating TAK1 signalling,41 indicating that TAK1 signalling promotes colitis. In our study, we have demonstrated that the P-TAK1 level in IECs was robustly enhanced in human IBD and experimental colitis. Furthermore, the TAK1 activation was significantly associated with RKIP upregulation.
In summary, our data demonstrated that RKIP contributes to colitis by promoting IEC apoptosis and mucosal epithelial barrier damage. Because of the positive relationship between RKIP expression and colitis severity in patients with IBD, RKIP may be a predictive marker of IBD and therapeutic target for the treatment of IBD.
Materials and methods
The experimental license to use human paraffin-embedded colon sections was approved by the Medical Research Ethics Committee of Zhejiang University. In addition, informed consent was obtained from all of the subjects involved, and the experiments were conducted according to the principles expressed in the Declaration of Helsinki.
Induction of colitis
Acute colitis was induced by oral administration of 2.5%–3% DSS or rectal injection of TNBS (200 mg/kg) as previously described.42 Briefly, RKIP-KO mice or WT littermates were fed with 2.5% DSS for 5 days or rectal injection of TNBS on day 0 and followed by regular drinking water until the end of the study, and colonic tissue was collected on the indicated day. Except for survival analysis, the mice were fed with 3% DSS for 6 days and followed by regular drinking water until the end of the study.
All data are expressed as the mean ±SEM. Statistical significance between two experimental groups was assessed using Student’s t test. Three or more groups were compared based on an analysis of variance and Pearson's χ2 tests with a 95% CI to evaluate the clinical specimens. A value of p <0.05 was considered statistically significant.
We thank Dr Huazhang An for critical reading of the manuscript.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
- Data supplement 1 - Online supplement
Contributors XW and JL designed research. WL, CM, FS, RL, YJ and ZC performed research. TZ, LF, KS and ZL collected the clinical sample. HH contributed the reagents. XW, JL and WL analysed data and wrote the paper.
Funding This work was supported by grants from the National Basic Research Program of China (973) (2014CB542101), the National Natural Science Foundation of China (81230014, 31400740, 81272353,) and the Natural Science Foundation of Zhejiang Province (LR13C080001, LY15H160012).
Competing interests None declared.
Patient consent Obtained.
Ethics approval The Medical Research Ethics Committee of Zhejiang University.
Provenance and peer review Not commissioned; externally peer reviewed.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.