Searching for the Answer to Irritable Bowel Syndrome in the Colonic Mucosa: SERTainty and UnSERTainty

Abstract

See “Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome” by Camilleri M, Andrews CN, Bharucha AE, Carlson PJ, Ferber I, Stephens D, Smyrk TC, Urrutia R, Aerssens J, Thielemans L, Göhlmann H, Van Den Wyngaert I, and Coulie B, on page 17. Abdominal pain and discomfort with disturbed bowel habit but normal standard investigations account for nearly half of all gastrointestinal (GI) consultations. Even using the more exact Rome criteria,1Longstreth G.F. Thompson W.G. Chey W.D. Houghton L.A. Mearin F. Spiller R.C. Functional bowel disorders.Gastroenterology. 2006; 130: 1480-1491Abstract Full Text Full Text PDF PubMed Scopus (3858) Google Scholar this group remains heterogeneous with respect to both symptoms and response to treatment. One effect of this is that one of the most effective treatments, the 5HT3 receptor antagonists,2Cremonini F. Delgado-Aros S. Camilleri M. Efficacy of alosetron in irritable bowel syndrome: a meta-analysis of randomized controlled trials.Neurogastroenterol Motil. 2003; 15: 79-86Crossref PubMed Scopus (190) Google Scholar benefit only 1 in 7 of irritable bowel syndrome (IBS) patients with diarrhea (IBS-D). The search for biomarkers, objective reproducible measures that, by assessing some disease process, reliably predict response to treatment remains elusive. As gastroenterologists we have ready access to endoscopic mucosal biopsies, which have therefore seemed an attractive area to look. Numerous recent studies have indicated that, although normal by routine hematoxylin and eosin staining, endoscopic biopsies of IBS-D patients show increases in T lymphocytes,3Gwee K.A. Leong Y.L. Graham C. McKendrick M.W. Collins S.M. Walters S.J. et al.The role of psychological and biological factors in postinfective gut dysfunction.Gut. 1999; 44: 400-406Crossref PubMed Scopus (705) Google Scholar, 4Dunlop S.P. Jenkins D. Neal K.R. Spiller R.C. Relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS.Gastroenterology. 2003; 125: 1651-1659Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 5Chadwick V.S. Chen W. Shu D. Paulus B. Bethwaite P. Tie A. Wilson I. Activation of the mucosal immune system in irritable bowel syndrome.Gastroenterology. 2002; 122: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar mast cells,6Barbara G. Stanghellini V. De Giorgio R. Cremon C. Cottrell G.S. Santini D. Pasquinelli G. Morselli-Labate A.M. Grady E.F. Bunnett N.W. Collins S.M. Corinaldesi R. Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome.Gastroenterology. 2004; 126: 693-702Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar and 5HT-containing enteroendocrine cells.4Dunlop S.P. Jenkins D. Neal K.R. Spiller R.C. Relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS.Gastroenterology. 2003; 125: 1651-1659Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar However, cell counting is tedious and somewhat subjective; moreover, cell density is easily altered by tissue edema or in the case of enterochromaffin (EC) cells by poor section orientation. Many workers have therefore moved to more quantitative, hopefully more reliable assays, although these are somewhat limited by the very small amount of tissue available (typically 10–15 mg per endoscopic biopsy). Assessing mRNA is an attractive option; even such small biopsies in our hands yield 40–500 ng of RNA, adequate for scores of assays. The study in this issue of Gastroenterology7Camilleri M. Andrews C.N. Bharucha A.E. Carlson P.J. Ferber I. Stephens D. Smyrk T.C. Urrutia R. Aerssens J. Thielemans L. Göhlmann H. Van Den Wyngaert I. Coulie B. Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome.Gastroenterology. 2007; 132: 17-25Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar assessed mRNA for both the serotonin transporter (SERT) and p11. The study followed on from an earlier study published in Gastroenterology8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar that indicated that mRNA for both SERT and tryptophan hydroxylase (the rate-limiting enzyme for 5HT synthesis) were markedly depressed in IBS patients with both constipation (IBS-C) and diarrhea as well as in patients with ulcerative colitis. Current interest in SERT relates to the important role 5HT plays in gut physiology9Gershon M.D. Review article: serotonin receptors and transporters—roles in normal and abnormal gastrointestinal motility.Aliment Pharmacol Ther. 2004; 20: 3-14Crossref PubMed Scopus (439) Google Scholar and the recent recognition of the benefits of 5HT3 receptor antagonists in some IBS-D patients. 5HT is released from the mucosa in response to a variety of stimuli, both from the lumen (pressure, bacterial toxins, nutrients) and from intrinsic and extrinsic nerves of the gut.10Racke K. Reimann A. Schworer H. Kilbinger H. Regulation of 5-HT release from enterochromaffin cells.Behav Brain Res. 1996; 73: 83-87Crossref PubMed Scopus (200) Google Scholar The 5HT released acts on both enterocytes and perhaps more importantly on mucosal nerve endings, mediating secretomotor responses, as well as moderating afferent signals and hence gut sensations. The concentration of 5HT found in the vicinity of the afferent nerves depends not only on the rate of release of 5HT, but also on its rate of reuptake. 5HT, being a charged amine at physiologic pH, requires sodium-linked active transport to pass the cell membrane and it is this serotonin transporter (SERT), which has been the focus of much recent interest. Fourteen IBS-C and 26 IBS-D were compared with 25 healthy controls. All underwent endoscopic biopsy of the sigmoid and rectal mucosa 1 hour after 2 hypertonic (1384 mmol/L) Fleet sodium phosphate enemas. Bowel symptom questionnaires were used at the time of the study to confirm IBS symptoms, the IBS subtype (diarrhea or constipation) as well as to exclude IBS in the controls. The biopsies were immediately placed in RNA Later solution, which considerably improves RNA quality and were then stored prior to assay in a batch. mRNA was quantified by quantitative real-time polymerase chain reaction (PCR). Assays were done for SERT (SLC6A4) and p11 (SA100A10 exons 1-2 and 2-3). These were compared with SART-1 (squamous cell carcinoma antigen recognized by T cells), ACTB (β-actin), and BM2 (β-2 microglobulin). A batch correction of the data was performed to control for day-to-day variability in the assays. Groups were well matched for gender, age, and body mass index. Six out of 25 controls, 8 out of 16 IBS-C, and 12 out of 25 IBS-D were taking either antidepressants or serotonin-modulating agents. The study found no difference in the mRNA for SERT compared with reference genes between any of the groups in the sigmoid or the rectal mucosa. This finding was true whether SERT mRNA was expressed as a ratio to SART 1 or ACTB as was done in the previous study.8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar The authors also looked at p11 (see below). The authors expressed p11 (S100A10) mRNA compared with B2M, which is a serum protein found in association with the major histocompatibility complex class I heavy chain on the surface of nearly all nucleated cells. The expression of this gene is very similar to the p11, making it a suitable comparator. In contrast to the negative findings with SERT, they found a significant increase of p11 in the sigmoid but not the rectal mucosa. The changes in the sigmoid were most obvious in IBS-C patients for both exon 1 and 2 of the gene. The surprising failure to reproduce earlier findings of alterations in SERT mRNA8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar led to extensive further analyses for which the authors are to be commended. Importantly, because this control has not been reported before, they examined reproducibility of their assays by assessing repeated biopsies a median of 82 days apart in 10 subjects. This showed reasonable stability in the sigmoid colon, but very poor reproducibility in the rectum with almost no correlation between the biopsies taken at different times, which is of some concern and requires some explanation. One key issue is sampling error if the organ undergoing biopsy is heterogeneous. One source of heterogeneity is the presence or absence of lymphoid follicles so that colonic biopsies from the same individual can have a very different proportion of lymphocytes and hence very different mRNA for genes expressed differentially in lymphocytes versus enterocytes. There may also be collection artifacts because the rectum, more so than the sigmoid, is subject to uncontrolled and hence variable low-level trauma during endoscopy. It also is maximally affected by the undiluted hypertonic phosphate enema, which is known to injure the mucosa11Leriche M. Devroede G. Sanchez G. Rossano J. Changes in the rectal mucosa induced by hypertonic enemas.Dis Colon Rectum. 1978; 21: 227-236Crossref PubMed Scopus (29) Google Scholar and alter both histology and enzyme levels.12Love R.R. Verma A.K. Surawicz T.S. Morrissey J.F. Colon ornithine decarboxylase activity following standard endoscopy preparation regimens.J Surg Oncol. 1989; 42: 150-153Crossref PubMed Scopus (13) Google Scholar Such specimens should ideally be taken using no bowel preparation and minimal instrumentation to avoid altering gene expression. The results for SERT mRNA levels are quite different from the earlier Coates papers and the reasons for these differences are hard to explain. IBS is heterogeneous, so there may be variations between the populations studied, although the discrepancies seem too large for this to be the only explanation. A significant proportion of patients were taking serotonin-modulating drugs, which did not appear to explain the differences; however, the numbers are too small to be certain about this. Investigators have all become increasingly reliant on preassembled “validated” kits and also tend to accept results such as output from real-time PCR machines at face value; however, this study suggests we should look more critically at these assumptions. The methodologies used in both studies are based on quantitative real-time PCR. Coates et al8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar used a proprietary assay in which SYBR green was used to detect PCR products generated using gene-specific primers. The current study used a primer and fluorescent probe-based method purchased from a commercial source. The fluorescent probe method tends to give greater specificity because the probe only binds to the DNA amplified from the gene of interest, whereas SYBR green dye binds to any amplified DNA. Thus, if the primers used in the SYBR green-based assay are not sufficiently gene specific and can amplify more than one mRNA species, the mRNA levels may appear to be higher than they actually are. These differences in experimental approach are unlikely to be the cause of the differences in the results obtained, however, and both techniques have been widely reported in the literature. The methods used to calculate SERT gene expression also differ. Coates et al8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar present their data as fg SERT mRNA per pg β-actin mRNA, whereas Camilleri et al7Camilleri M. Andrews C.N. Bharucha A.E. Carlson P.J. Ferber I. Stephens D. Smyrk T.C. Urrutia R. Aerssens J. Thielemans L. Göhlmann H. Van Den Wyngaert I. Coulie B. Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome.Gastroenterology. 2007; 132: 17-25Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar use the relative fold difference method, in which the relative fold difference between the threshold value of the gene of interest (SERT) from that of the control gene (β-actin) is calculated for each sample. Again, both methods are robust and it is improbable that the different methods used to express the data have led to the differences in results observed. In the case of real-time PCR, as with most scientific procedures, the devil is in the details. Because of space constraints in most scientific journals, much experimental detail and information relating to the validation of assays are now omitted. It is only in cases such as this, where widely varying results are obtained from 2 similar studies, that experimental methodologic details become essential. In both the Coates et al8Coates M.D. Mahoney C.R. Linden D.R. Sampson J.E. Chen J. Blaszyk H. Crowell M.D. Sharkey K.A. Gershon M.D. Mawe G.M. Moses P.L. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome.Gastroenterology. 2004; 126: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar and Camilleri et al7Camilleri M. Andrews C.N. Bharucha A.E. Carlson P.J. Ferber I. Stephens D. Smyrk T.C. Urrutia R. Aerssens J. Thielemans L. Göhlmann H. Van Den Wyngaert I. Coulie B. Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome.Gastroenterology. 2007; 132: 17-25Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar studies, certain information that would allow the reader to discriminate between possible causes of the difference between the 2 sets of data has been omitted. In real-time PCR experiments, the control or reference gene is used to normalize the results obtained for the gene of interest based on the assumption that the control gene will not vary in expression levels between experimental groups. In both studies, it is not explicit that there were no statistically significant differences between the patient groups in terms of control gene expression. Although it is highly probable that both groups carried out this analysis, this is not included in the text. It is not clear from the methods in either study whether the real-time PCR assays for the gene of interest and reference gene were carried out simultaneously (“multiplexing” in the same tube) or in separate assays. Multiplexing has the advantage that the PCR conditions are identical for both gene of interest and reference; however, depending on primer sequences, carrying out 2 PCR reactions in the same tube can lead to profoundly different results than those obtained when assays are carried out separately. Real-time PCR is based on the premise that each PCR cycle leads to a doubling of the amount of amplified DNA. In practice, this rarely happens and the PCR efficiency can vary dramatically from one target mRNA to another. If the efficiency of amplification of the reference gene is different from the gene of interest, results can be highly misleading. No data relating to the calculated PCR efficiencies of the assays used are included in either manuscript. Commercial real-time PCR assays are often sold as “validated,” suggesting that they are ready to use and require no quality assurance. In practice, no PCR-based assay can be relied on to perform reliably under all experimental conditions. Given the variation in both the number of genes expressed and their level of expression in different tissues and under varying pathologic or experimental conditions, it is essential that commercial assays be tested for both efficiency and specificity of PCR amplification. This point is highlighted by the results of the p11 mRNA assays in the current manuscript, in which 2 commercially available assays for the same mRNA gave statistically differing results (see Figure 5B in Camilleri et al). This is not to suggest that data using commercially produced kits are unreliable, but that such assays should be validated by the user. In the absence of these technical details, it is not clear whether differences in methodology are responsible for the differences in results obtained; however, it is probable that both groups carried out all the necessary checks and balances required to ensure that their methodology was robust. One further point that should be considered is simple variation between human subjects. When GAPDH, a commonly used housekeeper gene, mRNA levels in normal colonic epithelium taken from 51 individuals13Bustin S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.J Mol Endocrinol. 2000; 25: 169-193Crossref PubMed Scopus (3036) Google Scholar were measured by real-time PCR the levels expressed as Log mRNA copy number varied from 1 × 105 to 1 × 1010. Thus, when the numbers of individuals within a study are small, the potential for a few individuals to skew results is great and the differences between the 2 studies may be due to heterogeneity within IBS. Given these caveats, it is worth considering whether mRNA is the most useful measure to make when what one is really interested in is actual function. Although convenient, SERT mRNA may not be the best way of assessing function. Thus, selective serotonin reuptake inhibitors induce a marked decrease in SERT function as assessed by 5HT clearance and imipramine binding to SERT with no significant change in SERT mRNA.14Benmansour S. Owens W.A. Cecchi M. Morilak D.A. Frazer A. Serotonin clearance in vivo is altered to a greater extent by antidepressant-induced downregulation of the serotonin transporter than by acute blockade of this transporter.J Neurosci. 2002; 22: 6766-6772PubMed Google Scholar Recent work suggests that the critical factor in SERT function is whether the transporter is located on the cell membrane or recycled within an endosome, a feature also true of many receptors. Ramamoorthy and Blakely et al15Ramamoorthy S. Bauman A.L. Moore K.R. Han H. Yang-Feng T. Chang A.S. Ganapathy V. Blakely R.D. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization.Proc Natl Acad Sci U S A. 1993; 90: 2542-2546Crossref PubMed Scopus (780) Google Scholar have recently shown that phosphorylation is a key step here, so assaying phosphorylated SERT might be a closer approximation to function than measuring mRNA (Figure 1). A true measure of function, namely the uptake of H3-5HT by enterocytes in biopsies is confounded by the very high nonspecific binding of serotonin to matrix proteins which can only be avoided by using isolated enterocytes.16Linden D.R. Foley K.F. McQuoid C. Simpson J. Sharkey K.A. Mawe G.M. Serotonin transporter function and expression are reduced in mice with TNBS-induced colitis.Neurogastroenterol Motil. 2005; 17: 565-574Crossref PubMed Scopus (114) Google Scholar This could be done in IBS patients, although of course the rather radical processing required to dissociate enterocytes may alter membrane function in an unknown way. H3-5HT binding to platelets has been used as a measure of SERT expression and in one study, yet to be repeated, this correlated very well with symptoms in IBS-D.17Bellini M. Rappelli L. Blandizzi C. Costa F. Stasi C. Colucci R. Giannaccini G. Marazziti D. Betti L. Baroni S. Mumolo M.G. Marchi S. Del Tacca M. Platelet serotonin transporter in patients with diarrhea-predominant irritable bowel syndrome both before and after treatment with alosetron.Am J Gastroenterol. 2003; 98: 2705-2711Crossref PubMed Scopus (60) Google Scholar Although this is not the gut, it is a true functional assay and might be an alternative route to pursue, especially if it could be shown to correlate with gut SERT function. The increase in p11 was most obvious in IBS-C, but was also seen in some IBS-D patients, suggesting that p11 may be an indicator of a more general abnormality, rather than one specific to any particular bowel pattern. The findings in the rectum were less conclusive, but given the poor reproducibility already discussed, perhaps this is not surprising. p11, also known as (calpactin 1 light chain or annexin II ligand) is a calcium-binding protein that, with annexin II (p36), forms a heterotetrameric protein complex (p36)2(p11)2 called calpactin I. p11 modulates annexin, causing it to translocate to the surface, a process that depends on tyrosine phosphorylation.18Deora A.B. Kreitzer G. Jacovina A.T. Hajjar K.A. An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface.J Biol Chem. 2004; 279: 43411-43418Crossref PubMed Scopus (189) Google Scholar p11 is stabilized by annexin II, which increases p11’s half life 6-fold; thus, the relationship between p11 mRNA and its protein level is unlikely to be simple. p11 also increases the cell surface localization of 5HT1B receptors.18Deora A.B. Kreitzer G. Jacovina A.T. Hajjar K.A. An annexin 2 phosphorylation switch mediates p11-dependent translocation of annexin 2 to the cell surface.J Biol Chem. 2004; 279: 43411-43418Crossref PubMed Scopus (189) Google Scholar, 19Svenningsson P. Chergui K. Rachleff I. Flajolet M. Zhang X. El Yacoubi M. Vaugeois J.M. Nomikos G.G. Greengard P. Alterations in 5-HT1B receptor function by p11 in depression-like states.Science. 2006; 311: 77-80Crossref PubMed Scopus (453) Google Scholar These inhibitory receptors are important because their desensitization is thought to underlie the effect of selective serotonin reuptake inhibitors in depression. p11’s role in the intestine is unknown, but in sensory nerves it increases surface expression of Nav1.8,20Okuse K. Malik-Hall M. Baker M.D. Poon W.Y. Kong H. Chao M.V. Wood J.N. Annexin II light chain regulates sensory neuron-specific sodium channel expression.Nature. 2002; 417: 653-656Crossref PubMed Scopus (233) Google Scholar TRVP5&6,21van de Graaf S.F. Hoenderop J.G. Gkika D. Lamers D. Prenen J. Rescher U. Gerke V. Staub O. Nilius B. Bindels R.J. Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.EMBO J. 2003; 22: 1478-1487Crossref PubMed Scopus (245) Google Scholar and TASK-1,22Girard C. Tinel N. Terrenoire C. Romey G. Lazdunski M. Borsotto M. p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1.EMBO J. 2002; 21: 4439-4448Crossref PubMed Scopus (135) Google Scholar all ion channels important in neural excitability. Increased surface expression of these receptors might contribute to visceral hypersensitivity, one of the most consistent findings in IBS. This novel finding opens up a new field, perhaps demonstrating yet another way in which brain and gut function can be mutually disturbed. It offers exciting new possibilities for treatment that might address both central and peripheral issues in IBS. Alterations in Expression of p11 and SERT in Mucosal Biopsy Specimens of Patients With Irritable Bowel SyndromeGastroenterologyVol. 132Issue 1PreviewBackground & Aims: The pathophysiology of irritable bowel syndrome (IBS) remains enigmatic; abnormalities in serotonin metabolism have been implicated. Two proteins that influence the function of serotonin and serotonergic receptors are serotonin transporter protein (SERT or soluble carrier protein, SLC6A4) and p11 (S-100A10, or calpactin I light chain). Both proteins are reported to be associated with depression-like states, a frequent comorbid condition in IBS. We explored the hypothesis that expression of these 2 proteins in colonic and rectal mucosa is abnormal in patients with IBS as compared with healthy controls. Full-Text PDF