Mechanism of Regulation of Na-H Exchanger in Inflammatory Bowel Disease: Role of TLR-4 Signaling Mechanism
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- Siddique, I. & Khan, I. Dig Dis Sci (2011) 56: 1656. doi:10.1007/s10620-010-1524-7
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We investigated the role of toll-like receptor-4 (TLR-4) signal transduction in the regulation of Na-H exchanger-1 isoform (NHE-1) in ulcerative colitis (UC).
Colonic biopsies from control and UC patients were selected from four groups: controls (group 1), untreated UC patients (group 2), UC patients treated with 5′-aminosalicylic acid (5′-ASA) plus steroid (group 3), and UC patients treated with 5′-ASA plus azathioprine (AZA) (group 4). Patients presenting with abdominal pain (n = 13) and a normal colon on endoscopy served as controls. NHE-1, TLR-4, MyD88, NFkB and actin protein levels were estimated using Western blot analysis and sodium pump activity (PNPase) by a spectrophotometeric method. Myeloperoxidase (MPO) activity and histologic evaluation confirmed inflammation.
PNPase activity decreased significantly (P < 0.05) in the untreated UC patients as compared to the controls or treated UC groups 3 and 4. There was a significant decrease of NHE-1 and a significant increase (P < 0.05) of TLR-4, MyD88 and NFkB protein levels in the untreated UC or 5′-ASA plus steroid treated UC patients as compared to the controls. NHE-1, TLR-4, MyD88 and NFkB protein levels were not significantly different in 5′-ASA plus AZA treated biopsies as compared to controls. The level of actin remained unaltered. Inflammatory cells’ infiltration and MPO activity increased significantly in the untreated UC, but was significantly lower in the treated UC groups 3 and 4 (P < 0.05).
These findings suggest that NHE-1 in UC is regulated by NFkB induced through TLR-4 and MyD88 signaling mechanism. These findings identify TLR-4 as a putative therapeutic target for IBD.
KeywordsIBDUlcerative colitisCrohn’s colitisNa+/H+ exchangerSodium pumpMyeloperoxidase
Inflammatory bowel disease (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) are associated with defect in homeostasis of cations as revealed by altered expression of several cation transporters [1–5]. These changes are responsible for motility dysfunction, diarrhea and pain commonly seen in IBD [5–7]. The Na-H exchanger plays an important role in cation homeostasis [8, 9]. It performs electroneutral uptake of Na+ at the expense of H+ secretion across the plasma membrane in the GI tract and kidney [8, 9]. Several NHE isoforms have been reported which show tissue-specific expression and activity [8, 9]. Among these, the NHE-1 isoform plays a predominant role in the regulation of intracellular pH (pHi) homeostasis, while NHE-3 mainly contributes to NaCl uptake . These isoforms thus regulate cell volume and size, cell differentiation and apoptosis. Earlier reports indicate a decrease in intracellular and luminal pH in CD and active UC, suggestive of a pathogenic role of NHE-1 . The sodium pump fuels NHE, and hence changes in its activity may aggravate electrolyte homeostasis. It is becoming clear that NHE-1, NHE-3 and the sodium pump contribute to the pathogenesis of IBD [1–4]. These changes in NHE-1 expression in an animal model and NHE-3 in human CD and UC are not reversed by anti-inflammatory treatment [2, 12, 13]. Therefore, in the present study we examined the combined effects of two anti inflammatory substances which target two different sites: (1) 5′-aminosalicylic acid [5′-ASA] plus steroids [5′-ASA + steroid], and (2) 5′-ASA plus azathioprine [5′-ASA + AZA]. 5′-ASA blocks production of prostaglandins and leukotrienes and is used to induce remission in active UC and is also useful in maintaining remission . Systemic corticosteroids are useful for acute flare-ups of most forms of IBD . Azathioprine is used as an immunomodulatory agent in IBD. Genomic studies have shown down-regulation of various immune and inflammation related genes including tumor necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor receptor super family member 7, and α 4-integrin . The exact etiology of IBD is not known, but it is well established that these conditions result from immune dysregulation . NFkB, a transcription factor, is the hallmark of immune responses . A large number of pathways including toll-like receptor-4 (TLR-4) induce NFkB. TLR-4 plays an important role in gut innate immunity, protection and is implicated in human IBD [19–21]. TLR-4 acts through a down stream regulator, MyD88, which initiates a signal transduction cascade leading to induction of NFkB. Therefore, to investigate the role of TLR-4 in the regulation of NHE in UC, expression of TLR-4, MyD88, and NFkB was examined in UC biopsies.
Materials and Methods
Collection of Colonic Biopsies
The method of biopsy collection was the same as described earlier [2, 3]. Briefly, patients with chronic diarrhea, who had blood in the stools, and suspected to have UC were invited to participate in this study. Twenty three cases were confirmed to have UC based on their clinical presentation, laboratory investigations, radiological findings as well as typical colonoscopic findings and histopathology as described earlier [2, 3]. The patients who were diagnosed with conditions other than UC were excluded from the study. Thirteen patients presenting with abdominal pain or discomfort, cramps or bloating without diarrhea and diagnosed as irritable bowel syndrome (IBS) served as normal controls. Colonoscopy was performed under conscious sedation after bowel preparation with 4 l of polyethylene glycol (PEG) oral lavage.
Group 1: Normal controls (n = 13)
Group 2: UC (Untreated UC, new cases, n = 7)
Group 3: UC (Treated with 5′-ASA + steroid, n = 8)
Group 4: UC (Treated with AZA + 5′-ASA, n = 8)
The dose of 5′-ASA in patients in groups 3 and 4 was 2.4–3.2 g of mesalamine (5′-ASAcol®) per day. The dose of steroids in patients in group 3 ranged from 20 to 40 mg of prednisolone per day. The patients in group 4 received azathioprine at a dose of 2–2.5 mg/kg daily.
The study was conducted in accordance with the Declaration of Helsinki, and the protocol and the statement of informed consent were approved by the ethical committee of the Medical Research Council, Faculty of Medicine, Kuwait University. All patients gave an informed consent prior to inclusion in the study.
Biopsy samples were homogenized to prepare crude microsomes in 2 ml of ice-cold 3-(morpholino)-propanesulphonic (MPOS) buffer, pH 7.4 as described earlier [2, 3]. Mitochondrial and nuclear debris were removed from the lysates by centrifugation at 500g (Beckman) for 10 min at 4°C. Crude microsomal pellets were obtained by centrifugation of the supernatants at 120,000g for 45 min at 4°C (Beckman). The pellets were suspended in 100 μl MOPS buffer and total protein contents were measured using a dye binding assay kit (Biorad). Proteins in the crude microsomes were separated on an 8% polyacrylamide gel [12, 22], and then transferred to a nitrocellulose membrane (Biorad) electrophoretically [2, 3, 12]. Membranes were washed with phosphate buffered saline (PBS), blocked with 5% nonfat milk solution and were incubated with anti-NHE-1  and actin antibodies (Sigma) for 1 h at room temperature [2, 3]. After washing with PBS, the membranes were incubated with anti-rabbit 2° antibody-HRP conjugate (Sigma) for 1 h at room temperature. Then the filters were washed with PBS thoroughly and the signals were developed using the ECL kit components 1 and 2 (Amersham). Band densities were obtained by scanning the film using a densitometer. All steps were carried out at 4°C unless specified otherwise.
MPO activity was estimated following the method as described earlier [13, 19]. Briefly, biopsy samples were homogenized using 14 mM hexadecyltrimethylammonium bromide (HTAB) containing 50 mM potassium phosphate buffer, pH 6.0, with a polytron (Janke and Kunkle, Germany). Supernatants were obtained by centrifugation and were used to measure MPO activity in the presence of O-dianisidine solution containing H2O2 [13, 19]. Enzyme activity was expressed as units/mg tissue. Enzyme unit is defined as number of μmoles of H2O2 converted to product per minute per mg tissue at room temperature.
Sodium Pump Activity
Ouabain-sensitive K-stimulated p-nitrophenylphosphatase (PNPase) activity represents ATPase activity of the sodium pump and was measured as described earlier [2, 3, 24] by recording optical density of the reaction mixture at 420 nm spectrophotometrically (Beckman 5000). Enzyme unit was calculated using the molar extinction coefficient of p-nitrophenol , and is defined as μmoles of the product released per min per mg protein at 37°C.
ECL Western Blot Analysis
Expression of NHE-1, TLR-4, MyD88, NFkB and actin was examined in colonic biopsies using ECL Western blot analysis with specific antibodies following the procedure used earlier [2, 3, 19]. Specific polyclonal antibodies against NHE-1 were the same as used earlier , whereas polyclonal antibodies against TLR-4, NFkB and MyD88 were obtained from RDI, USA. Monoclonal anti actin antibodies were obtained from Sigma Co. Crude microsomes were separated on an 8% polyacrylamide gel and transferred onto nitrocellulose membrane (BioRad) electrophoretically overnight . NC membranes were blocked with non fat milk solution and treated with specific primary antibodies for NHE-1, TLR-4, MyD88, NFкB, and actin. Primary antibodies were used in 1:3,000 (anti TLR-4), 1:1,000 (anti MyD88), 1:1,000 (anti NFкB) and 1:2,000 (monoclonal anti-actin) dilutions for 3 h with gentle shaking at room temperature . The membranes were then washed with PBS and incubated with appropriate dilution (1:2,000) of anti-rabbit secondary antibody-horse radish peroxidase conjugate (Sigma) or mouse anti-actin secondary antibody (Jackson ImmunoResearch, USA). Specific bands were developed using Amersham chemiluminescence reagent kit (Amersham Bioscience) and the membrane was then exposed to an X-ray film (Kodak). Band densities were obtained using a densitometer (SYnGene, Chemi Genuis Bio Imaging System).
Data are presented as mean ± standard deviation (SD). Significance was calculated using non parametric unpaired, two tailed t test, and P < 0.05 was considered statistically significant.
Age of the subjects recruited for this study
Mean age (years ± SD)
Control (n = 13)
39 ± 6
38 ± 12
UC (n = 23)a
32 ± 9
35 ± 12
Crude Microsomes’ Yield
Crude microsomes yield (mg/gm tissue)
Mean crude microsomes
5.7 ± 0.20
2. New active UC (active)
4.9 ± 0.50
3. ASA + steroid treated UC
5.1 ± 0.80
4. ASA + AZA treated UC
4.8 ± 0.15
Sodium Pump Activity
TLR-4, MyD88 and NFkB Expression
The levels of these proteins were increased significantly (P < 0.05) in the biopsies from the untreated UC or 5′-ASA + steroid treated UC patients as compared to the controls (Figs. 4, 5b–d). However, the level of these proteins was significantly lower in 5′-ASA + AZA treated patients as compared to the untreated UC or 5′-ASA + steroid treated UC patients (Figs. 4, 5b–d).
The basis of this study came from our recent findings in which we have reported suppression of NHE-1 and -3 isoforms in both Crohn’s disease and ulcerative colitis [2, 3]. Although anti-inflammatory treatments are able to reverse inflammatory processes, both in humans and experimental colitis, changes in the expression of NHE remains irreversible [2, 3]. For example, we have recently reported that suppression of NHE in humans was not reversed by 5′-ASA, a treatment used in IBD . 5′-ASA inhibits prostaglandins therefore suggesting that these mediators are not involved in the regulation of NHE in IBD. In this study we further investigated the combined effects of multi target treatments on the reversibility of suppression of NHE-1 using mucosal biopsies from the colons of UC patients. When 5′-ASA was combined with steroid, there was suppression of inflammatory mediators which was not associated with reversal in the IBD-induced suppression of NHE-1, suggesting that NHE-1 is not regulated by inflammatory mediators. Reason for this discrepancy is not known but deserves some speculation. Among the possibilities, degree of inflammation and or the region involved may be an important factor contributing to this discrepancy. Our findings demonstrate reversal in the inflammatory markers such as MPO and histology by 5′-ASA + steroid, or 5′-ASA + AZA treatments, but the combined treatment of 5′-ASA together with AZA was able to reverse the suppression of NHE-1 significantly. NHE-1 isoform is present in the basolateral domain in epithelial cells and its suppression together with that of sodium pump activity should decrease intracellular pH and increase intracellular Na+. Our findings of PNPase are consistent with others and our previous findings [1–3]. These changes will lead to decreased NaCl and water uptake from colonic lumen and contribute to diarrhea. Since corticosteroid inhibits inflammatory mediators, but did not reverse IBD-induced changes in the expression of NHE, we believe that reversibility of NHE-1 suppression is mainly due to AZA. It is worth mentioning that a study has reported that UC and CD-induction of nitric oxide synthase remained unaffected by corticosteroids . Our findings therefore may be interpreted that persistent changes in certain parameters such as NHE even during the treatment might make these conditions relapse on discontinuation of the treatment. With regard to the underlying mechanism of NHE-1 regulation in IBD it is important to note that AZA is an immunomodulatory agent which affects the immune regulatory genes . Since NFkB is the hallmark of immune responses we investigated its role and the involvement of TLR-4 in the regulation of signal transduction of NHE-1 in IBD. There are multiple pathways including TLR-4 that can influence NFkB. An association of TLR-4 with IBD is well known. TLR-4 activation leads to recruitment of an important down-stream regulator called MyD88 which subsequently causes activation of the cascade leading ultimately to induction of NFkB. Our findings showed induction of TLR-4, MyD88 and NFkB expression in untreated active UC patients and 5′-ASA + steroid treated patients. Interestingly, IBD induced expression of these proteins was significantly suppressed by 5′-ASA + AZA. Azathioprine has been shown to inhibit TNF-α which is induced through induction of NFkB. However, since steroids inhibit TNF-α and other inflammatory mediators, it is less likely that they play a role in the regulation of NHE-1 expression in IBD cases used in this study. Cell wall contents of certain pathogens/bacteria are known to induce TLR-4 and hence it may be a possible mechanism of induction of NFkB. Whether azathioprine influence the population of microflora in the GI tract, however, remains to be explored. Nevertheless, these findings suggest that NHE-1 is regulated in human UC via NFkB involving through induction of TLR-4 and MyD88 upstream regulator.
In conclusion, we demonstrate that suppression of the NHE-1 protein in the UC is associated with an induction of TLR-4, MyD88 and NFkB expression. These changes are resistant to 5′-ASA and steroid treatment, but are reversed by azathioprine. Persistent changes in the expression of NHE-1 during certain treatments may be an important mechanism of remission upon discontinuation of the treatments. These findings signify the TLR-4 signaling mechanism as a putative target for intervention in IBD. Furthermore, examination of level of NHE-1 in IBD may be used as an indicator to predict outcome of the treatment.
The Kuwait University Research Administration is gratefully acknowledged for financial support through research grant # MB05/04, as well as Mr Abdul Kadir for his technical assistance.