Inhibition of CREB‐mediated ZO‐1 and activation of NF‐κB‐induced IL‐6 by colonic epithelial MCT4 destroys intestinal barrier function

Abstract Objective Inflammatory bowel disease (IBD) is a disorder intestinal inflammation and impaired barrier function, associated with increased epithelial expression of monocarboxylate transporter 4 (MCT4). However, the specific non‐metabolic function and clinical relevance of MCT4 in IBD remain to be fully elucidated. Methods Lentivirus‐mediated overexpression of MCT4 was used to assess the role of MCT4 in transcriptionally regulating ZO‐1 and IL‐6 expression by luciferase assays, WB and ChIP. IP was used to analyse the effect of MCT4 on the interaction NF‐κB‐CBP or CREB‐CBP, and these MCT4‐mediated effects were confirmed in vivo assay. Results We showed that ectopic expression of MCT4 inhibited ZO‐1 expression, while increased pro‐inflammatory factors expression, leading to destroy intestinal epithelial barrier function in vitro and in vivo. Mechanistically, MCT4 contributed NF‐κB p65 nuclear translocation and increased the binding of NF‐κB p65 to the promoter of IL‐6, which is attributed to MCT4 enhanced NF‐κB‐CBP interaction and dissolved CREB‐CBP complex, resulting in reduction of CREB activity and CREB‐mediated ZO‐1 expression. In addition, treatment of experimental colitis with MCT4 inhibitor α‐cyano‐4‐hydroxycinnamate (CHC) ameliorated mucosal intestinal barrier function, which was due to attenuation of pro‐inflammation factors expression and enhancement of ZO‐1 expression. Conclusion These findings suggested a novel role of MCT4 in controlling development of IBD and provided evidence for potential targets of IBD.


| INTRODUC TI ON
Despite tremendous efforts have been made to improve the effectiveness of treatment, inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), remains the leading cause of cancer-related healthy people in the world. 1,2 Growing evidences showed that IBD is a disorder of dysregulation by inflammation accompanied by impaired intestinal barrier function, [3][4][5] which is an vital event in treatment of IBD. For instance, pro-inflammatory cytokine IL-6 and TNF-α increased intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. 6 In addition, disruption of tight junction is regarded as one of the earliest hallmarks of epithelial injury, leading to the loss of cell polarity and tissue disorganization. 7 Thus, deciphering the key molecules that enable anti-inflammatory and protection of intestinal barrier function are urgently needed to improve therapeutic outcomes.
A key intracellular signalling pathway that governs intestinal barrier function is MAPK signalling. The major downstream transcription factors of the MAPK pathway are the cAMP-response element binding protein (CREB) and nuclear factor-κB (NF-κB). 8,9 Phosphorylation of CREB at ser133, but not Ser142, directly or indirectly activated by ERK1/2, p38MAPK and other stimuli, [10][11][12] increases its transcriptional activity and target genes expression, such as muc2 13 and tight junction protein 1 (ZO-1). 14,15 Phosphorylation at this residue promotes CREB binding to transcriptional co-activator CBP, which lead to displacement of NF-κB from the same interaction domain on CBP. 16,17 Formation of CREB-CBP complex promotes the expression of anti-inflammatory cytokines(eg, IL-4, IL-10 and IL-13), 18 thereby suppressing expression of pro-inflammatory cytokines(eg, IL-6 and TNF-α) activated by the NF-κB-CBP complex, 12,19 suggesting the biological action is maintained in part by the activity of CREB and NF-κB. Nevertheless, the upstream and role of both in intestinal epithelial barrier function have not yet been fully illustrated.
Recent study have showed that proliferative cells at the base of the intestinal crypt are characterized by a glycolytic metabolic phenotype, 20 whereas differentiated cells have an oxidative phosphorylation phenotype, which is in line with the findings showed by sun et.al 21

and
Wang et.al, 22 implying that nutritional states are closely associated with alteration of intestinal barrier function. Interesting, our previous study has revealed monocarboxylate transporter 4 (MCT4) expression is significantly increased in intestinal mucosal tissue of IBD patients, which is correlated with intestinal mucosal inflammation, 23 and MCT4 is involved in establishment and maintenance of epithelial polarity and lactate transporters. 24,25 However, the effect of MCT4 that has a global influence on IBD has not yet been illustrated. In this study, we further demonstrated that MCT4 destroyed intestinal barrier function by reduction of ZO-1 expression and promotion of pro-inflammatory IL-6 expression in vivo and in vitro. Mechanically, ectopic expression of MCT4 significantly inhibited phosphorylation of CREB(Ser133), leading to reduce ZO-1 expression, while promoted phosphorylation of Ser536 on p65, resulting in enhancement of IL-6 expression. Most importantly, endogenous CBP interacted with CREB, and this interaction was dramatically disrupted by ectopic expression of MCT4, which in turn promotes CBP-NF-κB complex. What's more, α-cyano-4-hydroxycinnamate (CHC), an inhibitor of MCT4, alleviated dextran sulphate sodium (DSS)-induced colitis in vivo. Collectively, these findings provide the mechanism by which MCT4 regulated IBD and support MCT4 inhibitor used as an effective therapeutic approach to improve IBD.

| Reagents and antibodies
CaCO 2 and HT-29 cells were obtained from American Type Cell Collection (Manassas, VA). Cell culture medium and foetal bovine serum were purchased from Life Technologies, and α-cyano-4-hydroxycinnamate (C2020) was supplied by Sigma. Antibody against MCT4 (sc-50329) and CBP (sc-7300 X) were from Santa Cruz Biotechnology. were purchased from Abclonal. Phospho-p65 (Ser536) (No.3033) and p65 (No.8242) were purchased from Cell Signaling Technology. Other reagents used in this study were purchased from Sigma.

| Plasmid and siRNA
Plasmid and siRNA-targeted NF-κB p65 were purchased from GenePharma. which was due to attenuation of pro-inflammation factors expression and enhancement of ZO-1 expression.

Conclusion:
These findings suggested a novel role of MCT4 in controlling development of IBD and provided evidence for potential targets of IBD.

| Cell culture and transfection
CaCO 2 and HT-29 cell line was cultured in DMEM supplemented with 10% foetal bovine serum and maintained in a humidified incubator at 37°C and 5% CO 2 . For transfection, plasmids or siRNA was transfected into cells with hilymax (H357) and lipofectamine3000 (L-3000), respectively, following the manufacturer's instructions.

| Lentivirus-mediated overexpression of MCT4
The lentivirus vectors Lv-CTL and Lv-MCT4 were purchased from Genepharma, and puromycin was purchased from Sigma and used to select for stably transfected cells. MCT4 expression at protein level was verified by Western blotting.

| RNA Extraction and quantitative realtime PCR
Total RNA was extracted using Trizol (life technologies), converted to cDNA using the All-in-One™ First-Strand cDNA Synthesis Kit (Genecopoeia™, FulenGen) and amplified by PCR using the All-in-One™ qPCR Mix (Genecopoeia™, FulenGen) according to the manufacturer's instructions. Primer sequences for Claudins were from Sangon Biotech, and other primers for indicated genes used in this study were listed in Table 1.

| Immunoprecipitation and immunoblotting
As described in our previous study, cells were lysed in ice-cold

| Luciferase reporter assay
Cells were co-transfected with the indicated promoter reporter plasmid (ZO-1-luc or IL-6-Luc) and internal control plasmid

| DSS-induced colitis and Intestinal Permeability Assays
Colitis mice were induced by 2% (w/v) dextran sulphate sodium (DSS, Millipore Corporation) in drinking water for 5 days, followed by CHC or vehicle treatment for a period of 14 days. The body weight changes and overall survival were recorded daily. On day 20, fluorescent conjugated dextran (10 kDa with Cy7) was gavaged into mice, and then, 1 hour later animals were studied using multispectral fluorescent capture followed by digital X-ray imaging.

| In vitro barrier function assessment
Twelve-well plate Millicells (0.4 μm, Millipore Corporation) were used for transepithelial electrical resistance (TEER) assays as described previously. Briefly, 0.5 mL CaCO 2 cells at the density of 4 × 10 5 cells/ mL were seeded in the apical chamber that bathed in the basal chamber with 1.0 mL DMEM complete medium for 21 days. Voltage was measured daily using EVOM (WPI), which was multiplied by the area of filter (1.12 cm 2 ) to obtain the TEER in Ohm cm 2 . DMEM complete medium in apical and basal chamber was refreshed every day. The permeability of FITC-dextran (Sigma) across the CaCO 2 cell monolayer was measured as previously described with modifications.
At 21 days, 1.0 mg/mL FITC-dextran was added on the apical side of monolayers after washed twice with PBS. One millilitre cells in the basal chamber were taken at indicated point, and 1.0 mL prewarmed fresh medium was added after each sampling to replenish basal medium. The fluorescence emission at 520 nm was measured with excitation at 490 nm using Synergy H1 microplate reader.

| Immunohistochemistry (IHC) and immunofluorescence(IF)
Immunohistochemistry and immunofluorescence were performed as described in our previous work. 26,27 The sections were depar-

| Statistical analysis
All analysis was conducted using GraphPad Prism V software. A P value < .05 was considered statistical significant. Statistical differences among groups were determined by Student's t test, one sample t tests, one-way ANOVA or two-way ANOVA were used to determine the significance.

| MCT4 is significantly increased in the colonic epithelium of IBD patients and DSS-induced colitis
In previous study, we demonstrated that a higher levels of MCT4 expression in intestinal mucosa of IBD patients were compared with healthy controls. 23 Consistently, as shown in Figure 1A

| MCT4 destroyed intestinal barrier function through suppression of ZO-1 expression
It has been reported that dysfunction of intestinal mucosal barrier caused the invasion of luminal pathogens into the lamina propria, which further triggered the dysregulation of immune responses, leading to chronic colonic inflammation. 28 To clarify whether up- Although our data suggested that MCT4 was essential for CREB-dependent transcription of ZO-1, the exact mechanisms remained unclear. Activation of CREB promoted its nuclear translocation to activate CREB-dependent genes transcription, 31 and we utilized immunofluorescence to analyse the influence of MCT4 on nuclear distribution of CREB. As shown in Figure 3E-F, CREB nuclear translocation was decreased to 25% of that in the control cells by introduction of MCT4. Meanwhile, the level of nuclear CREB in undetectable in IBD mice mucosa was compared with that in healthy control ( Figure 3G). In line with this, a subcellular fractionation analysis showed that MCT4 led to a significant increase in the amount of cytosolic CREB( Figure 3H), indicating MCT4 has an important role in regulation of CREB translocation.
Phosphorylation of CREB (Ser133) is critical for CREB translocation in response to various stimuli, 32-34 next, we sought to further elucidate the possible mechanism underlying CREB nuclear translocation. As shown in Figure 3I;

| MCT4 enhanced NF-κB binding to IL-6 promoter by promotion of phosphorylation and nuclear translocation of NF-κB p65
It is well known that activation of NF-κB p65 and its targets involved in the pathogenesis of autoimmune diseases, including IBD. 37,38 IL-6, a cytokines critical to the pathogenesis of IBD, was found to dramatically increase in IECs transduced with Lv-MCT4 compared with controls. To explore whether MCT4-induced IL-6 expression could be attributed to upregulation of NF-κB activity, as shown in Figure 5A-B, IL-6 transactivation was significantly increased in HT-29 cells with MCT4 overexpression, which was markedly reduced in response to NF-κB depletion, while inhibition of MCT4 by CHC dramatically attenuated IL-6 transactivation. These phenomena were attributed to the binding of NF-κB p65 to IL-6 promoter was drastically increased in response to MCT4 overexpression ( Figure 5C). Moreover, silencing of the NF-κB p65 gene using siRNA resulted in abrogation of IL-6 expression caused by MCT4 ( Figure 5D), indicating that MCT4 has a significant role in NF-κB-mediated transactivation of IL-6.
Our data suggested that MCT4 was essential for NF-κB-dependent transcription of IL-6; the exact mechanism remained unclear.
To explore whether MCT4 contributed to IL-6 expression by promotion of NF-κB activity, we found that the relative abundance of NF-κB p65 in nucleus was decreased to 25% of control group by  Figure 5E-F), and NF-κB p65 in nucleus was evaluated in colonic epithelial of patient with IBD compared with healthy donors ( Figure 5G). In addition, CHC treatment strongly reduced NF-κB nuclear translocation in CaCO 2 cells ( Figure 5H), whereas there was a significant activation of NF-κB p65 phosphorylation at Ser536 in HT-29 and CaCO 2 cells with MCT4 overexpression ( Figure 5I).
Conversely, stimulation by CHC in IECs led to a dramatic inhibition of NF-κB p65 (Ser536) ( Figure 5J). Taken together, these results demonstrated that MCT4 mediated IL-6 expression via activation of NF-κB pathway.

| MCT4 regulated the interaction between CBP and NF-κB or CREB
The above results showed that MCT4 contributed to NF-κB-mediated inflammatory reaction and decreased CREB-induced ZO-1 expression that led to destroy barrier function, a critical step in the transcriptional regulation mediated by NF-κB or CREB is the interaction of each of these transcription factors with the co-activator CBP. 39,40 In order to better understand the driving forces controlling IBD by MCT4, we investigated the potential role of MCT4 in the complex of NF-κB-CBP and CREB-CBP complex in IECs. As shown in Figure 6A, immunofluorescence showed that endogenous NF-κB was co-localized with CBP, which was disrupted by CHC stimulation for 1h. Consistent with this result, IP of CBP showed a markedly reduction of NF-κB-CBP interaction and a strong increase in CREB-CBP complex in CaCO 2 cells with CHC treatment for 1 hour ( Figure 6B); in contrary, overexpression of MCT4 significantly disrupted CREB-CBP interaction and contributed to the formation of NF-κB-CBP complex ( Figure 6C). These results suggested that the expression level of MCT4 was critical for the interaction of NF-κB/CREB and CBP.

| CHC improved intestinal barrier function and alleviated DSS-induced colitis in vivo
Our study indicated that MCT4 has an important role in the pathogenesis of IBD, and we further assessed the effect of MCT4 inhibitor CHC on intestinal inflammation and intestinal epithelial integrity in vivo. Chronic colitis in C57BL/6 mice induced by 2% DSS in drinking water for 7 days, followed by intraperitoneal administration of CHC or vehicle controls for 14 days. CHC-treated mice were found to be protected from experimental colitis as predicted (mean bodyweight) and improved overall survival ( Figure 7A-B). Imaging live mice after oral administration of label dextran revealed that CHC treatment resulted in improvement of intestinal barrier integrity: fluorescence in the intestinal lumen was slightly increased in DSS-induced colitis followed by CHC treatment compared with DSS group ( Figure 7C left panel). In a confirmatory assay, circulating concentrations of Cy7-label dextran after gavage in CHC treatment were significantly lower than in DSS group, showing a better barrier function ( Figure 7C right panel), and a longer colon length compared with the DSS group ( Figure 7D). Most importantly, a significant reduction of IL-6 was observed in DSS + CHC mice compared with that of DSS

| D ISCUSS I ON
Up to now, no available reports on the function of MCT4 in inflammatory bowel disease. In this study, we further rigorously demonstrated a novel signalling pathway driven by MCT4 in regulating IBD by modulation of shift balance between NF-κB-CBP and CREB-CBP complex. As shown in Figure 8,  Most importantly, MCT4 contributes to interaction of NF-κB p65 with CBP, which in turn to disrupt CREB-CBP complex, depicting the alteration of MCT4 is critical to modulate shift balance between NF-κB-CBP and CREB-CBP complex, and these results implied proper expression of MCT4 is critical to improve IBD cytokines activated by CREB-CBP complex. 19 In line with this, we found that overexpression of MCT4 inhibited CREB activity and nuclear translocation, resulting in ablation of CREB-DNA binding activity and formation of CREB-CBP complex, and decreased ZO-1 expression.
The function of MCT4 in IBD majorly depends on its expression level, which indirectly modulates the shift balance between the CREB-CBP interaction and the NF-κB-CBP complex. Interestingly, we found the contribution of MCT4 to NF-KB replaced with CREB, leading to form NF-κB-CBP complex, which further not only activated phosphorylation of NF-κB p65(Ser536) and nucleus translocation, resulting in increase of pro-inflammatory cytokines expression, such as IL-6 and TNF-α, but also attenuated level of CREB(Ser133) activity and nucleus translocation, led to decrease ZO-1 and antiinflammatory cytokines expression. However, the limitation of this study is that changes of MCT4 expression and activity in IBD remain to be addressed, and further work is required to demonstrate how MCT4 regulated CREB/NF-κB activity, whether in MAPK-dependent way, although lacking strong evidences to confirm it at present.
In conclusion, normal expression of MCT4 is essential to coordi-

ACK N OWLED G EM ENTS
We would like to thank prof. Deng and prof. Chen in department of cell biology of Southern Medical University for kindly supporting.
We also thank Dr. Yuanwen Xie and Dr. Songyu Li for supporting in data analysis and funding.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no conflict of interests. and use committee. Written informed consent was given by the caregiver of the child for his clinical records used, which are not publicly available since the database is currently not anonymous and contains all patient's name; however, it could be available upon request.

AUTH O R CO NTR I B UTI
Animal study: All animal experiments were approved by Southern Medical University animal committee and performed at Southern Medical University.

DATA ACCE SS I B I LIT Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.