The trefoil factor family – small peptides with multiple functionalities

The trefoil factor family (TFF) comprises a group of small peptides which are highly expressed in tissues containing mucus-producing cells – especially in the mucosa lining the gastrointestinal tract. The peptides seem crucial for epithelial restitution and may work via other pathways than the conventional factors involved in restitution. In vitro studies have shown that the TFFs promote restitution using multiple mechanisms. The peptides also have other functionalities including interactions with the immune system. Moreover, therapeutic effects of the TFFs have been shown in several animal models of gastrointestinal damage. Still it is not clear which of their in vitro properties are involved in the in vivo mode of action. This review describes the TFF family with emphasis on their biological properties and involvement in mucosal protection and repair. Received 10 October 2008; received after revision 07 November 2008; accepted 10 November 2008     

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.     

Trefoil factors

Trefoil Factor 1 (TFF1), the first member of the trefoil factor family, is normally expressed in the stomach mucosa. Ectopic expression is also observed in various human pathological conditions, notably in numerous carcinomas and gastrointestinal acute inflammatory disorders. In vivo experimental data using TFF1-deficient mice highlight the pleiotropic functions of TFF1: (i) it is a gastric tumor suppressor gene involved in gastric ontogenesis and homeostasis; (ii) it protects gut mucosa from aggression; (iii) it participates in folding secreted proteins inside the endoplasmic reticulum. At the cellular level, it limits cell proliferation and apoptosis, and favors cell differentiation. Collectively, these data suggest that TFF1 may provide an alternative pharmacological tool for the prevention and treatment of human gastrointestinal diseases.     

Trefoil factors

The present review will include the mammalian trefoil factors, TFF1, TFF2 and TFF3. It will summarise the amino acid sequences from different species, their posttranslational modifications and their structures determined by X-ray analysis and nuclear magnetic resonance studies. Trefoil factors all have a well-defined, structurally conserved trefoil domain. The trefoil domain consists of 42 or 43 amino acid residues and contains 6 cysteine residues that form disulphide bonds in a 1-5, 2-4 and 3-6 configuration. By the establishment of an additional intra-molecular disulphide bond at the C-terminal end, TFF1 and TFF3 form homodimers or heterodimers. This dimer formation of TFF1 and TFF3 will be discussed, and the possible implications for biological activity will be reviewed. The physicochemical characteristics including protease stability of trefoil factors will be summarised. The biological implications of different molecular forms of trefoil factors and their interaction with mucins will be discussed together with other functional insights.     

The role of innate immune-stimulated epithelial apoptosis during gastrointestinal inflammatory diseases

The maintenance of mucosal barrier equilibrium in the intestine requires a delicate and dynamic balance between enterocyte loss by apoptosis and the generation of new cells by proliferation from stem cell precursors at the base of the intestinal crypts. When the balance shifts towards either excessive or insufficient apoptosis, a broad range of gastrointestinal diseases can manifest. Recent work from a variety of laboratories has provided evidence in support of a role for receptors of the innate immune system, including Toll-like receptors 2, 4, and 9 as well as the intracellular pathogen recognition receptor NOD2/CARD15, in the initiation of enterocyte apoptosis. The subsequent induction of enterocyte apoptosis in response to the activation of these innate immune receptors plays a key role in the development of various intestinal diseases, including necrotizing enterocolitis, Crohn’s disease, ulcerative colitis, and intestinal cancer. This review will detail the regulatory pathways that govern enterocyte apoptosis, and will explore the role of the innate immune system in the induction of enterocyte apoptosis in gastrointestinal disease.     

The trefoil protein TFF1 is bound to MUC5AC in humangastric mucosa

The trefoil protein TFF1 is expressed principally in the superficial cells of the gastric mucosa. It is a small protein and forms homo- and hetero-dimers via a disulphide bond through Cys58 which is located three amino acids from the C terminus. TFF1 is co-expressed with the secreted mucin MUC5AC in superficial cells of the gastric mucosa suggesting that it could be involved in the packaging or function of gastric mucus. We have previously shown that TFF1 co-sediments with mucin glycoproteins on caesium chloride gradients. To extend this observation we have now used gel filtration under physiological conditions, immunoprecipitation and Western transfer analysis to characterise the interaction of TFF1 with gastric mucin glycoproteins. We show that TFF1 co-elutes with MUC5AC but not MUC6 on gel filtration and that immunoprecipitation and Western transfer analysis confirms that TFF1 interacts with MUC5AC. We also demonstrate that the TFF1 dimer is the predominant molecular form bound to MUC5AC. Salt and chelators of divalent cations such as EDTA and EGTA disrupted the TFF1- MUC5AC interaction and increased the degradation of MUC5AC, whereas calcium increased the amount of TFF1 bound to MUC5AC. These data support the contention that TFF1 is pivotal in the packaging and function of human gastric mucusa.Received 24 March 2004; received after revision 14 May 2004; accepted 7 June 2004     

Trefoil factors

Trefoil factor family (TFF) peptides have many in vivo and in vitro effects on restitution, wound healing, apoptosis, cell motility, adhesion and vectorial ion pumping, amongst others. (125)I-TFF peptides bind to cell membranes with classical saturable ability. It would be surprising if there were not TFF-protein interactions that would explain these actions, but to date no convincing TFF-binding partner has been shown which unambiguously takes part in any of these functions. Nevertheless, several TFF-binding proteins exist, including the small intestinal CRP-ductin (muclin), which binds TFF2, and the recently described gastric foveolar proteins TFIZ1 (TFF1-binding) and blottin (TFF2-binding), any of which may yet interact in novel ways to elicit TFF-mediated events. This review describes the expression and, where known, the functions of such proteins.     

Trefoil factors

Rapid repair of mucous epithelia is essential for preventing inflammation which is a critical component of cancer progression. 'Restitution' is an early repair process which can begin within minutes and is achieved via the migration of neighbouring cells into the wounded area. Mucosal restitution is a multistep process which requires continuous blood flow and includes at least (i) the reduction of cell-cell contacts and a shift in the cell shape towards a migratory phenotype (characteristics of the epithelial-mesenchymal transition), (ii) migration of cells, (iii) repolarization and formation of tight junctions (morphological restitution) and (iv) restoration of barrier function (transmucosal epithelial resistance, functional restitution). Secretory TFF (trefoil factor family) peptides TFF1, TFF2 and TFF3 are well known for their potent protective and healing effects after mucosal damage (function as 'luminal surveillance peptides'). Here, the contributions of the TFFs during the different steps of mucosal restitution are discussed, i. e. the modulation of cell-cell contacts, their motogenic activity and synergy with epidermal growth factor, their anti-apoptotic and pro-angiogenic effects. Special emphasis has been given to discussion of the various signal transduction networks triggered by TFFs. It is becoming increasingly clear that these pathways differ depending on the respective TFF.     

Activation of protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by human TFF2

Trefoil factors (TFFs) promote epithelial cell migration to reseal superficial wounds after mucosal injury, but their receptors and the molecular mechanisms underlying this process are poorly understood. In this study, we showed that frog TFF2 activates protease-activated receptor (PAR) 1 to induce human platelet aggregation. Based on this result, we further tested the involvement of PARs in human TFF2 (hTFF2)-promoted mucosal healing. hTFF2-stimulated migration of epithelial HT-29 cells was largely inhibited by PAR4 depletion with small interfering RNAs but not by PAR1 or PAR2 depletion. The PAR4-negative epithelial cell lines AGS and LoVo were highly responsive to hTFF2 as assessed by phosphorylation of ERK1/2 and cell migration upon PAR4 expression. Our findings suggest that hTFF2 promotes cell migration via PAR4. These findings will be helpful in further investigations into the functions and molecular mechanisms of TFFs and PARs in physiology and disease.     

The enteric nervous system in gastrointestinal disease etiology

A highly conserved but convoluted network of neurons and glial cells, the enteric nervous system (ENS), is positioned along the wall of the gut to coordinate digestive processes and gastrointestinal homeostasis. Because ENS components are in charge of the autonomous regulation of gut function, it is inevitable that their dysfunction is central to the pathophysiology and symptom generation of gastrointestinal disease. While for neurodevelopmental disorders such as Hirschsprung, ENS pathogenesis appears to be clear-cut, the role for impaired ENS activity in the etiology of other gastrointestinal disorders is less established and is often deemed secondary to other insults like intestinal inflammation. However, mounting experimental evidence in recent years indicates that gastrointestinal homeostasis hinges on multifaceted connections between the ENS, and other cellular networks such as the intestinal epithelium, the immune system, and the intestinal microbiome. Derangement of these interactions could underlie gastrointestinal disease onset and elicit variable degrees of abnormal gut function, pinpointing, perhaps unexpectedly, the ENS as a diligent participant in idiopathic but also in inflammatory and cancerous diseases of the gut. In this review, we discuss the latest evidence on the role of the ENS in the pathogenesis of enteric neuropathies, disorders of gut–brain interaction, inflammatory bowel diseases, and colorectal cancer.

     

Intestinal epithelial barrier and mucosal immunity

The mucosal immune system maintains a delicate balance between providing robust defense against infectious pathogens and, at the same time, regulating responses toward innocuous environmental and food antigens and commensal microbes. The Peyer's patch (PP) has been studied in detail as a major inductive site for mucosal immunity within the small intestine. While the mechanisms responsible for the induction of mucosal immunity versus tolerance are not yet fully understood, recent studies have highlighted mucosal dendritic cells (DCs) as regulators of the immune responses to orally administered antigens. Here we discuss recent studies that describe the role of PP DCs in immune induction and speculate on the mechanism by which the resident DCs regulate T cell and immunoglobulin A (IgA) responses in the gastrointestinal mucosa.     

Tachykinins and their functions in the gastrointestinal tract

In the gastrointestinal tract, tachykinins are peptide neurotransmitters in nerve circuits that regulate intestinal motility, secretion, and vascular functions. Tachykinins also contribute to transmission from spinal afferents that innervate the gastrointestinal tract and have roles in the responses of the intestine to inflammation. Tachykinins coexist with acetylcholine, the primary transmitter of excitatory neurons innervating the muscle, and act as a co-neurotransmitter of excitatory neurons. Excitatory transmission is mediated through NK1 receptors (primarily on interstitial cells of Cajal) and NK2 receptors on the muscle. Tachykinins participate in slow excitatory transmission at neuro-neuronal synapses, through NK1 and NK3 receptors, in both ascending and descending pathways affecting motility. Activation of receptors (NK1 and NK2) on the epithelium causes fluid secretion. Tachykinin receptors on immune cells are activated during inflammation of the gut. Finally, tachykinins are released from the central terminals of gastrointestinal afferent neurons in the spinal cord, particularly in nociceptive pathways. Received 24 March 2007; received after revision 30 August 2007; accepted 14 September 2007     

Nematodes and the spleen: An immunological relationship

Despite being a major organ of the immune system, the spleen's role in resisting, controlling or simply ameliorating nematode infections has been neglected. A review of both filarial and gastrointestinal nematodes suggests that though it is difficult to fully assess or quantify the organ's importance in vivo, the spleen is prominent in acting against nematode parasites in mammals. One manifestation of this is that transfer of lymphocytes from the spleen of immunised individuals can protect recipients against the disease. Expansion of splenic lymphoid tissue also alludes to its activity during nematode infection. There is a considerable need for investigation of the spleen under natural conditions as well as much more rigorously controlled experiments even in mammals besides birds and other vertebrates.     

Nematodes and the spleen: An immunological relationship

Despite being a major organ of the immune system, the spleen's role in resisting, controlling or simply ameliorating nematode infections has been neglected. A review of both filarial and gastrointestinal nematodes suggests that though it is difficult to fully assess or quantify the organ's importance in vivo, the spleen is prominent in acting against nematode parasites in mammals. One manifestation of this is that transfer of lymphocytes from the spleen of immunised individuals can protect recipients against the disease. Expansion of splenic lymphoid tissue also alludes to its activity during nematode infection. There is a considerable need for investigation of the spleen under natural conditions as well as much more rigorously controlled experiments even in mammals besides birds and other vertebrates.     

The therapeutic potential of GPR43: a novel role in modulating metabolic health

GPR43 is a receptor for short-chain fatty acids. Preliminary data suggest a putative role for GPR43 in regulating systemic health via processes including inflammation, carcinogenesis, gastrointestinal function, and adipogenesis. GPR43 is involved in secretion of gastrointestinal peptides, which regulate appetite and gastrointestinal motility. This suggests GPR43 may have a role in weight control. Moreover, GPR43 regulates plasma lipid profile and inflammatory processes, which further indicates that GPR43 could have the ability to modulate the etiology and pathogenesis of metabolic diseases such as obesity, type 2 diabetes mellitus, and cardiovascular disease. This review summarizes the current evidence regarding the ability of GPR43 to mediate both systemic and tissue specific functions and how GPR43 may be modulated in the treatment of metabolic disease.     

Emerging topics and new perspectives on HLA-G

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.     

Identification of the bioactive peptide PEC-60 in brain

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60may play a role in the central nervous system disorders involving dopamine dysregulation. Received 6 December 2002; received after revision 10 December 2002; accepted 11 December 2002 RID="*" ID="*"Corresponding author.     

Cationic host defence peptides: Innate immune regulatory peptides as a novel approach for treating infections

An increase in antibiotic resistance and the emergence of new pathogens has led to an urgent need for alternative approaches to infection management. Immunomodulatory molecules that do not target the pathogen directly, but rather selectively enhance and/or alter host defence mechanisms, are attractive candidates for therapeutic development. Natural cationic host defence peptides represent lead molecules that boost innate immune responses and selectively modulate pathogen-induced inflammatory responses. This review discusses recent evidence exploring the mechanisms of cationic host defence peptides as innate immune regulators, their role in the interface of innate and adaptive immunity, and their potential application as beneficial therapeutics in overcoming infectious diseases. Received 3 November 2006; received after revision 14 December 2006; accepted 22 January 2007     

Human adenosine deaminases ADA1 and ADA2 bind to different subsets of immune cells

At sites of inflammation and tumor growth, the local concentration of extracellular adenosine rapidly increases and plays a role in controlling the immune responses of nearby cells. Adenosine deaminases ADA1 and ADA2 (ADAs) decrease the level of adenosine by converting it to inosine, which serves as a negative feedback mechanism. Mutations in the genes encoding ADAs lead to impaired immune function, which suggests a crucial role for ADAs in immune system regulation. It is not clear why humans and other mammals possess two enzymes with adenosine deaminase activity. Here, we found that ADA2 binds to neutrophils, monocytes, NK cells and B cells that do not express CD26, a receptor for ADA1. Moreover, the analysis of CD4+ T-cell subset revealed that ADA2 specifically binds to regulatory T cells expressing CD39 and lacking the receptor for ADA1. Also, it was found that ADA1 binds to CD16? monocytes, while CD16+ monocytes preferably bind ADA2. A study of the blood samples from ADA2-deficient patients showed a dramatic reduction in the number of lymphocyte subsets and an increased concentration of TNF-α in plasma. Our results suggest the existence of a new mechanism, where the activation and survival of immune cells is regulated through the activities of ADA2 or ADA1 anchored to the cell surface.     

Role of bile in intestinal absorption of 203Pb in rats.

The author studied absorption of 203Pb after administering intraduodenally 203Pb eliminated with bile. The results obtained were compared with absorption of 203Pb administered into the duodenum as 203PbCl2 in rats forming 2 groups, one with bile ducts cannulated, the other intact. It was found that bile played an important role in absorption of Pb from the gastrointestinal tract. Absorption of Pb203 is significantly reduced if the bile is drained off by means of a canula.