Proteases in Irritable Bowel Syndrome: A Lot More Than Just Digestive Enzymes

Abstract

Patients with irritable bowel syndrome (IBS) continue to represent a significant challenge in daily clinical practice. A prodigious amount of research has clearly demonstrated the relevance of psychological, gastrointestinal motor, and visceral perception factors in IBS pathophysiology.1Drossman D.A. Camilleri M. Mayer E.A. et al.AGA technical review on irritable bowel syndrome.Gastroenterology. 2002; 123: 2108-2131Abstract Full Text Full Text PDF PubMed Scopus (1192) Google Scholar Recently, a new fertile area of research has provided a growing body of evidence indicating that subtle, but potentially relevant, biologic abnormalities could be identified in subgroups of IBS patients.2Barbara G. De Giorgio R. Stanghellini V. et al.New pathophysiological mechanisms in irritable bowel syndrome.Aliment Pharmacol Ther. 2004; 20: 1-9Crossref PubMed Scopus (178) Google Scholar Examples of these changes include cytokine gene polymorphisms,3Gonsalkorale W.M. Perrey C. Pravica V. et al.Interleukin 10 genotypes in irritable bowel syndrome: evidence for an inflammatory component?.Gut. 2003; 52: 9Google Scholar, 4van der Veek P.P. van den Berg M. de Kroon Y.E. et al.Role of tumor necrosis factor-alpha and interleukin-10 gene polymorphisms in irritable bowel syndrome.Am J Gastroenterol. 2005; 100: 2510-2516Crossref PubMed Scopus (178) Google Scholar abnormal serotonergic signaling,5Coates M.D. Mahoney C.R. Linden D.R. et al.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 (623) Google Scholar, 6Camilleri M. Andrews C.N. Bharucha A.E. et al.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 (113) Google Scholar mucosal immune activation,2Barbara G. De Giorgio R. Stanghellini V. et al.New pathophysiological mechanisms in irritable bowel syndrome.Aliment Pharmacol Ther. 2004; 20: 1-9Crossref PubMed Scopus (178) Google Scholar and altered host-bacterial interactions (eg, small intestinal bacterial overgrowth and infectious gastroenteritis).7Pimentel M. Chow E.J. Lin H.C. Eradication of small intestinal bacterial overgrowth reduces symptoms of irritable bowel syndrome.Am J Gastroenterol. 2000; 95: 3503-3506Crossref PubMed Google Scholar, 8Spiller R.C. Postinfectious irritable bowel syndrome.Gastroenterology. 2003; 124: 1662-1671Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar These studies are promising; however, much work has yet to be done to define whether these newly identified biologic abnormalities yield pathogenetic, diagnostic, and therapeutic value for IBS patients. The article by Roka et al,9Roka R. Rosztoczy A. Leveque M. et al.Fecal serine-protease activity: a pathophysiological factor in diarrhea-predominant irritable bowel syndrome—a pilot study.Clin Gastroenterol Hepatol. 2007; 5: 550-555Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar published in this issue of Clinical Gastroenterology and Hepatology, adds further evidence to support the presence of biologic abnormalities in IBS. In their article, the authors described a significant 3-fold increase in fecal concentration of proteases of the serine type (serine is the functional group present at the active site of catalytic activity) in 17 patients with diarrhea-predominant IBS (D-IBS) compared with 15 symptom-free controls. Samples from 14 patients with constipation-predominant and 7 patients with alternating-predominant IBS did not show those differences, suggesting that fecal protease changes were selective for D-IBS. However, the detection of increased proteases in 15 patients with active ulcerative colitis implies a lack of specificity of this marker. It could also be argued that accelerated gastrointestinal transit and increased fecal output in D-IBS might explain per se the higher fecal protease activity. However, the lack of increased fecal proteases in 5 patients with acute infectious gastroenteritis (which is also associated with increased fecal output) and the absence of correlation between protease activity and the number of bowel movements make that hypothesis less likely. Gastrointestinal proteases are involved in digestion of food proteins (eg, gastric pepsin or pancreatic trypsin). However, the discovery of protease-activated receptors (PARs) in the gastrointestinal tract (as well as in other organs and tissues) suggested that in addition to their role as agents of protein breakdown, proteases also might act as signaling molecules. PARs are G protein coupled receptors that are activated by a peculiar mechanism. Activation is, in fact, elicited by proteolytic cleavage of the N terminal domain of the receptor. This cleavage releases a new motif of amino acids on the N terminal tail, which then binds the receptor like an agonist to induce intracellular signaling.10Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (908) Google Scholar So far, 4 PARs (PAR1–4) have been identified, and they are differentially activated by a number of proteases.10Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (908) Google Scholar Little is known about the distribution and function of PARs in the human alimentary canal. However, studies in rodents have shown that PAR1 and PAR2, the 2 main receptor subtypes expressed in the gastrointestinal tract, are present in a wide array of cells (eg, endothelial, epithelial, smooth muscle, and immune cells, as well as enteric neurons). The finding reported in the article by Roka et al9Roka R. Rosztoczy A. Leveque M. et al.Fecal serine-protease activity: a pathophysiological factor in diarrhea-predominant irritable bowel syndrome—a pilot study.Clin Gastroenterol Hepatol. 2007; 5: 550-555Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar raises the question about the potential gastrointestinal effects of excessive luminal serine protease activity and related PAR activation. Exposure of naïve, noninflamed gastrointestinal tissue to small synthetic peptides, which can be used as PAR agonists, has shown important effects of PAR1 and PAR2 on intestinal ion transport, motility, permeability, immune function, and sensory perception.10Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (908) Google Scholar, 11Vergnolle N. Clinical relevance of proteinase activated receptors (pars) in the gut.Gut. 2005; 54: 867-874Crossref PubMed Scopus (134) Google Scholar The majority of these studies have been carried out in laboratory animals, and limited data are available in humans. Activation of PARs induces chloride secretion in isolated gastrointestinal tissues or epithelial cells. This seems to be a direct stimulatory effect of PAR1 on human epithelial cells, and it is at least partly mediated by the enteric nervous system for PAR2, although contradictory results have also been provided.11Vergnolle N. Clinical relevance of proteinase activated receptors (pars) in the gut.Gut. 2005; 54: 867-874Crossref PubMed Scopus (134) Google Scholar, 12Buresi M.C. Schleihauf E. Vergnolle N. et al.Protease-activated receptor-1 stimulates Ca(2+)-dependent Cl(-) secretion in human intestinal epithelial cells.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G323-G332PubMed Google Scholar, 13Green B.T. Bunnett N.W. Kulkarni-Narla A. et al.Intestinal type 2 proteinase-activated receptors: expression in opioid-sensitive secretomotor neural circuits that mediate epithelial ion transport.J Pharmacol Exp Ther. 2000; 295: 410-416PubMed Google Scholar In addition, data in rats have shown that intracolonic administration of PAR2 agonists induced visceral hypersensitivity to colonic distention14Coelho A.M. Vergnolle N. Guiard B. et al.Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats.Gastroenterology. 2002; 122: 1035-1047Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar and increased colonic paracellular permeability.15Cenac N. Coelho A.M. Nguyen C. et al.Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2.Am J Pathol. 2002; 161: 1903-1915Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar PAR2-evoked mucosal barrier defects might lead to excessive passage of luminal antigens and toxins with consequent mucosal immune activation, which, in turn, is thought to contribute to visceral hypersensitivity.15Cenac N. Coelho A.M. Nguyen C. et al.Induction of intestinal inflammation in mouse by activation of proteinase-activated receptor-2.Am J Pathol. 2002; 161: 1903-1915Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 16Cenac N. Garcia-Villar R. Ferrier L. et al.Proteinase-activated receptor-2-induced colonic inflammation in mice: possible involvement of afferent neurons, nitric oxide, and paracellular permeability.J Immunol. 2003; 170: 4296-4300PubMed Google Scholar Taken together, these experimental data support the hypothesis that the excessive luminal serine protease activity described in IBS patients might lead to PAR-induced increased secretion, visceral hypersensitivity, and increased mucosal permeability, which have been claimed to participate in the phenotypic expression of D-IBS.2Barbara G. De Giorgio R. Stanghellini V. et al.New pathophysiological mechanisms in irritable bowel syndrome.Aliment Pharmacol Ther. 2004; 20: 1-9Crossref PubMed Scopus (178) Google Scholar, 17Barbara G. Mucosal barrier defects in irritable bowel syndrome: who left the door open?.Am J Gastroenterol. 2006; 101: 1295-1298Crossref PubMed Scopus (24) Google Scholar However, a number of issues still remain to be clarified before considering proteases as relevant biologic factors in IBS patients. Indeed, to activate PARs, proteases must be released in sufficient concentrations, might require the presence of accessory cofactors, and their activity could be significantly inhibited by the presence of protease inhibitors.10Ossovskaya V.S. Bunnett N.W. Protease-activated receptors: contribution to physiology and disease.Physiol Rev. 2004; 84: 579-621Crossref PubMed Scopus (908) Google Scholar Finally, the development of good PAR antagonists for human use could be an excellent opportunity to clarify the importance of PARs in IBS pathophysiology and symptom generation. The increased fecal protease activity described by Roka et al9Roka R. Rosztoczy A. Leveque M. et al.Fecal serine-protease activity: a pathophysiological factor in diarrhea-predominant irritable bowel syndrome—a pilot study.Clin Gastroenterol Hepatol. 2007; 5: 550-555Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar is in line with recent data showing increased tryptase and trypsin activity in colonic mucosal biopsies obtained from patients with IBS18Barbara G. Stanghellini V. De Giorgio R. et al.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 (1126) Google Scholar, 19Barbara G. Wang B. Stanghellini V. et al.Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome.Gastroenterology. 2007; 132: 26-37Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 20Cenac N. Andrews C.N. Holzhausen M. et al.Role for protease activity in visceral pain in irritable bowel syndrome.J Clin Invest. 2007; : 636-647Crossref PubMed Scopus (483) Google Scholar and ulcerative colitis.20Cenac N. Andrews C.N. Holzhausen M. et al.Role for protease activity in visceral pain in irritable bowel syndrome.J Clin Invest. 2007; : 636-647Crossref PubMed Scopus (483) Google Scholar However, substantial differences with these previous studies exist. Increased tryptase activity detected in mucosal biopsies of IBS patients has been suggested to originate from infiltration and activation of mast cells in the colonic mucosa.18Barbara G. Stanghellini V. De Giorgio R. et al.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 (1126) Google Scholar In this context, proteases have been suggested to be involved in the excitation of sensory fibers conveying nociceptive information to the central nervous system.18Barbara G. Stanghellini V. De Giorgio R. et al.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 (1126) Google Scholar, 19Barbara G. Wang B. Stanghellini V. et al.Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome.Gastroenterology. 2007; 132: 26-37Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar Conversely, fecal proteases detected by Roka et al were neither of mast cell (ie, tryptase) or of pancreatic (ie, trypsin) origin. The authors postulate that luminal bacteria of the intestinal microflora could be a potential source of these proteases. This hypothesis is in line with the knowledge that nonmammalian cells (eg, bacteria, fungi, and viruses) can produce large amounts of proteases. Bacteria are so efficient in production of proteases that bacterial-derived enzymes are widely used in a variety of commercial applications (eg, detergent, food, and pharmaceutical industry), accounting for approximately 40% of the total worldwide enzyme sales.21Godfrey T. West S. Industrial enzymology. 2nd ed. Macmillan Publishers Inc, New York1996: 3Google Scholar An interesting observation that might bear significance to the results provided by Roka et al is that protease-producing bacteria have been demonstrated to signal to mammalian cells by activating PARs and induce certain diseases. For example, Porphyromonas ginigivalis, a bacterial pathogen found in the oral microflora of patients with periodontitis, produces the proteases “gingipains” that activate PAR2, leading to the progression of the periodontal disease.22Holzhausen M. Spolidorio L.C. Ellen R.P. et al.Protease-activated receptor-2 activation: a major role in the pathogenesis of Porphyromonas gingivalis infection.Am J Pathol. 2006; 168: 1189-1199Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar The hypothesis of a bacterial origin of the proteases found in D-IBS is interesting but needs further confirmation. Nonetheless, this hypothesis is in line with the evidence that subsets of IBS patients have positive lactulose breath testing suggestive of small intestinal bacterial overgrowth7Pimentel M. Chow E.J. Lin H.C. Eradication of small intestinal bacterial overgrowth reduces symptoms of irritable bowel syndrome.Am J Gastroenterol. 2000; 95: 3503-3506Crossref PubMed Google Scholar and, with the long-lasting theory, although not yet supported by consistent evidence, of qualitative alterations in the composition of intestinal microflora.23Malinen E. Rinttila T. Kajander K. et al.Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with real-time PCR.Am J Gastroenterol. 2005; 100: 373-382Crossref PubMed Scopus (576) Google Scholar Future research should demonstrate clear pathophysiologic implications of the identified biologic abnormalities in IBS, including the proteases, assess the diagnostic power of these markers, and provide novel sites for therapeutic intervention. Proteases and PARs could be promising targets. It is likely that translation of this novel information into clinical application will be hampered by several obstacles and might require a long time and substantial efforts. Nonetheless, for the time being, the results by Roka et al,9Roka R. Rosztoczy A. Leveque M. et al.Fecal serine-protease activity: a pathophysiological factor in diarrhea-predominant irritable bowel syndrome—a pilot study.Clin Gastroenterol Hepatol. 2007; 5: 550-555Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar like others in the field, suggest that this research is fast moving and holds promise for patients with IBS. I thank Drs R. De Giorgio and C. Cremon (University of Bologna, Italy) for helpful discussions.