Trefoil Factor Family Peptides Research Articles

Obstructive sleep apnea and rhonchopathy are associated with downregulation of trefoil factor family peptide 3 (TFF3)-Implications of changes in oral mucus composition.

Study objectivesTrefoil factor family (TFF) peptides belong to the family of mucin-associated peptides and are expressed in most mucosal surfaces. TFF peptides carry out functions such as proliferation and migration enhancement, anti-apoptosis, and wound healing. Moreover, TFFs are associated with mucins and interact with them as “linker peptides”, thereby influencing mucus viscosity.To test the hypothesis that in rhonchopathy and obstructive sleep apnea (OSA) changes occur in the expression of TFF3 and -2 that could contribute to changes in mucus viscosity, leading to an increase in upper airway resistance during breathing.MethodsRT-PCR, Western-blot, immunohistochemistry and ELISA were performed to detect and quantify TFF3 and -2 in uvula samples. In addition, 99 saliva samples from patients with mild, moderate or severe OSA, as well as samples from rhonchopathy patients and from healthy volunteers, were analyzed by ELISA.ResultsTFF3 was detected in all uvula samples. Immunohistochemistry revealed a subjectively decreasing antibody reactivity of the uvula epithelia with increasing disease severity. ELISA demonstrated significantly higher TFF3 saliva protein concentrations in the healthy control group compared to cases with rhonchopathy and OSA. Predisposing factors of OSA such as BMI or age showed no correlation with TFF3. No significant changes were observed with regard to TFF2.ConclusionsThe results suggest the involvement of TFF3 in the pathogenesis of rhonchopathy and OSA and lead to the hypothesis that reduction of TFF3 production by the epithelium and subepithelial mucous glands of the uvula contribute to an increase in breathing resistance due to a change in mucus organization.

Localization of trefoil factor family peptide 3 (TFF3) in epithelial tissues originating from the three germ layers of developing mouse embryo

Trefoil factor family (TFF) peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old) were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3,3'-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation.

Proteolytic effects of gingipains on trefoil factor family peptides.

The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis. Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides. MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin. Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides. The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.

Reduction of the tumorigenic potential of human retinoblastoma cell lines by TFF1 overexpression involves p53/caspase signaling and miR-18a regulation.

Trefoil factor family (TFF) peptides have been shown to play a pivotal role in oncogenic transformation, tumorigenesis and metastasis by changing cell proliferation, apoptosis, migration and invasion behavior of various cancer cell lines. In the study presented, we investigated the effect of TFF1 overexpression on cell growth, viability, migration and tumorigenicity of different retinoblastoma (RB) cell lines. Transient TFF1 overexpression significantly increases RB cell apoptosis levels. Stable, lentiviral TFF1 overexpression likewise decreases RB cell viability, proliferation and growth and significantly increases apoptosis as revealed by WST-1 assays, BrdU and DAPI cell counts. TFF1-induced apoptosis is executed via cleaved caspase-3 activation as revealed by caspase blockage experiments and caspase-3 immunocytochemistry. Results from pG13-luciferase reporter assays and Western blot analyses indicate that TFF1-induced apoptosis is mediated through transcriptional activity of p53 with concurrently downregulated miR-18a expression. In ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF1 overexpression significantly decreases the size of tumors forming from Y79 and RB355 cells and reduces the migration potential of RB355 cells. Differentially expressed genes and pathways involved in cancer progression were identified after TFF1 overexpression in Y79 cells by gene expression array analysis, underlining the effects on reduced tumorigenicity. TFF1 knockdown in RBL30 cells revealed caspase-3/7-independent apoptosis induction, but no changes on cell proliferation level. In summary, the in vitro and in vivo data demonstrate for the first time a tumor suppressor function of TFF1 in RB cells which is at least partly mediated by p53 activation and miR-18a downregulation.

Increased trefoil factor 2 levels in patients with chronic kidney disease

In chronically damaged tissue, trefoil factor family (TFF) peptides ensure epithelial protection and restitution. In chronic kidney disease (CKD), TFF1 and TFF2 are reported to be upregulated. Especially in the early phase, CKD is associated with silently ongoing renal damage and inflammation. Moreover, many patients are diagnosed late during disease progression. We therefore sought to investigate the potential of TFF2 as biomarker for CKD. We followed 118 patients suffering from predialysis CKD and 23 healthy volunteers. TFF2 concentrations were measured using ELISA. Our results showed, that median TFF2 serum levels were significantly higher in patients with later CKD stages as compared to healthy controls (p < 0.001) or early stages (p < 0.001). In patients with mid CKD stages TFF2 serum levels were significantly higher than in healthy controls (p = 0.002). Patients with early or mid CKD stages had significantly higher TFF2 urine concentrations than later CKD stages (p < 0.001 and p = 0.009, respectively). Fractional TFF2 excretion differed significantly between early CKD stages and healthy controls (p = 0.01). ROC curve showed that TFF2 levels can predict different CKD stages (AUC > 0.75). In conclusion, urine and serum TFF2 levels of CKD patients show a different profile dependent on CKD stages. Whereas TFF2 urine levels continuously decreased with disease progression, TFF2 serum concentrations progressively increased from the early to later CKD stages, indicating changes in renal function and offering the potential to examine the course of CKD.

Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway.

Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors. We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3-CXCR4/CXCR7 interaction were accomplished by cell culture-based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade. We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture-based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity. Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.

Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation

Background/Aims: The trefoil factor family (TFF) peptide TFF1 is a typical secretory product of the gastric mucosa and a very low level of expression occurs in nearly all regions of the murine brain. TFF1 possesses a lectin activity and binding to a plethora of transmembrane glycoproteins could explain the diverse biological effects of TFF1 (e.g., anti-apoptotic effect). It was the aim to test whether TFF expression is changed during neuroinflammation. Methods: Expression profiling was performed using semi-quantitative RT-PCR analyses in two murine models of neuroinflammation, i.e. Toxoplasma gondii-induced encephalitis and experimental autoimmune encephalomyelitis (EAE), the latter being the most common animal model of multiple sclerosis. Tff1 expression was also localized using RNA in situ hybridization histochemistry. Results: We report for the first time on a significant transcriptional induction in cerebral Tff1 expression in both T. gondii-induced encephalitis and EAE. In contrast, Tff2 and Tff3 expression were not altered. Tff1 transcripts were predominantly localized in the internal granular layer of the cerebellum indicating neuronal expression. Furthermore, also glial cells are expected to express Tff1. Characterization of both experimental models by expression profiling (e.g., inflammasome sensors, inflammatory cytokines, microglial marker Iba1, ependymin related protein 1) revealed differences concerning the expression of the inflammasome sensor Nlrp1 and interleukin 17a. Conclusion: The up-regulated expression of Tff1 is probably the result of a complex inflammatory process as its expression is induced by tumor necrosis factor α as well as interleukins 1β and 17. However on the transcript level, Tff1^KO mice did not show any significant signs of an altered immune response after infection with T. gondii in comparison with the wild type animals.

Trefoil factor family peptides--friends or foes?

Trefoil factor family (TFF) peptides are a group of molecules bearing a characteristic three-loop trefoil domain. They are mainly secreted in mucous epithelia together with mucins but are also synthesized in the nervous system. For many years, TFF peptides were only known for their wound healing and protective function, e.g. in epithelial protection and restitution. However, experimental evidence has emerged supporting a pivotal role of TFF peptides in oncogenic transformation, tumorigenesis and metastasis. Deregulated expression of TFF peptides at the gene and protein level is obviously implicated in numerous cancers, and opposing functions as oncogenes and tumor suppressors have been described. With regard to the regulation of TFF expression, epigenetic mechanisms as well as the involvement of various miRNAs are new, promising aspects in the field of cancer research. This review will summarize current knowledge about the expression and regulation of TFF peptides and the involvement of TFF peptides in tumor biology and cancerogenesis.

Trefoil Factor 1 Excretion Is Increased in Early Stages of Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with high morbidity and mortality. In many patients CKD is diagnosed late during disease progression. Therefore, the implementation of potential biomarkers may facilitate the early identification of individuals at risk. Trefoil factor family (TFF) peptides promote restitution processes of mucous epithelia and are abundant in the urinary tract. We therefore sought to investigate the TFF peptide levels in patients suffering from CKD and their potential as biomarkers for CKD. We analysed TFF1 and TFF3 in serum and urine of 115 patients with CKD stages 1–5 without dialysis by ELISA. 20 healthy volunteers served as controls. Our results showed, that urinary TFF1 levels were significantly increased with the onset of CKD in stages 1–4 as compared to controls and declined during disease progression (p = 0.003, < 0.001, 0.005, and 0.007. median concentrations: 3.5 pg/mL in controls vs 165.2, 61.1, 17.2, and 15.8 pg/mL in CKD 1–4). TFF1 and TFF3 serum levels were significantly elevated in stages 3–5 as compared to controls (TFF1: p < 0.01; median concentrations: 12.1, 39.7, and 34.5 pg/mL in CKD 3–5. TFF3: p < 0.001; median concentrations: 7.1 ng/mL in controls vs 26.1, 52.8, and 78.8 ng/mL in CKD 3–5). TFF3 excretion was increased in stages 4 and 5 (p < 0.001; median urinary levels: 65.2 ng/mL in controls vs 231.5 and 382.6 ng/mL in CKD 4/5; fractional TFF3 excretion: 6.4 in controls vs 19.6 and 44.1 in CKD 4/5). ROC curve analyses showed, that monitoring TFF peptide levels can predict various CKD stages (AUC urinary/serum TFF > 0.8). In conclusion our results show increased levels of TFF1 and TFF3 in CKD patients with a pronounced elevation of urinary TFF1 in lower CKD stages. Furthermore, TFF1 and TFF3 seems to be differently regulated and show potential to predict various CKD stages, as shown by ROC curve analysis.

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae)

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation.

Trefoil factor family peptides TFF1 and TFF3 in the nervous tissues of developing mouse embryo.

Trefoil factor family peptides (TFF1, TFF2, and TFF3) are predominantly found in mucous epithelia of various organs. However, they have also been reported in the nervous tissue, particularly mouse, rat, porcine, and human brain. The aim of this research was to determine the presence of TFF1 and TFF3 in the nervous system of developing mouse embryo. Mouse embryos, at the stages E15 to E17 were isolated, fixed in 4% paraformaldehyde and embedded in paraffin blocks. Sagittal 6µm sections were made, processed for immunohistochemistry, and incubated with anti-TFF1 or anti-TFF3 primary polyclonal rabbit antibodies. Labeled streptavidin-biotin method was used for TFF detection. TFF1 and 3 were found in the cytoplasm of ganglion cell somata, while TFF3 staining was also visible in the cytoplasm of neurons in different areas and nuclei of brain and medulla oblongata. Neurons in the gray matter of spinal cord were also TFF1 and TFF3 positive, and signal for both peptides was found in the choroid plexus. TFF peptides might be involved in the complex processes of nervous system development and differentiation and brain plasticity.

Trefoil Factor Family Peptide 3 (TFF3) is Upregulated Under Experimental Conditions Similar to Dry Eye Disease and Supports Corneal Wound Healing Effects In Vitro

To elucidate the role of trefoil family peptide (TFF) 3 at the ocular surface under conditions similar to dry eye disease (DED) and in tears of patients suffering from DED. Trefoil family peptide 3 levels in tear samples from non-Sjögren's DED patients with moderate dry eye were analyzed by ELISA and compared with tears from healthy volunteers. A human corneal epithelial (HCE) cell line was treated with proinflammatory cytokines IL-1β and TNF-α, hyperosmolar medium, or scratching for up to 24 hours. Trefoil family peptide 3 gene expression and protein biosynthesis were analyzed by RT-PCR, immunofluorescence, and ELISA. Migration and proliferation of HCE cells under recombinant (r) human (h) trefoil factor family peptide 3 (TFF3) stimulation were investigated by scratching and bromodeoxyuridine (BrdU) proliferation assays. Tears of patients suffering from DED contained significantly higher TFF3 levels than tears from healthy volunteers. Stimulation of HCE cells with proinflammatory cytokines, culture under hyperosmolar conditions, or scratching resulted, with the exception of hyperosmolar conditions, in an increase in TFF3 expression and elevated secretion level of TFF3. Cell proliferation decreased and cell migration increased after 24-hours stimulation with rhTFF3. These results suggest that inflammatory factors or ocular surface damage as they occur in DED, lead to an increase of TFF3 tear film concentration, whereas hyperosmolarity does not. Our data underline a potential role for TFF3 as a candidate therapeutic for the ocular surface damage observed in DED.

Chronic rhinosinusitis without nasal polyps is associated with increased expression of trefoil factor family peptides

Trefoil factor family (TFF) peptides are mucin-associated secretory products that are produced in the airways and gastrointestinal tract. These peptides appear to play an important role in mucosal healing and epithelial protection and are overexpressed in chronically inflamed gastrointestinal tissues. We hypothesize that TFF peptides may also be differentially expressed in the sinonasal tissue of patients with and without chronic rhinosinusitis (CRS). Ethmoid sinus tissue was obtained from patients with CRS without nasal polyps (CRSsNP) (n = 12), CRS with nasal polyps (CRSwNP) (n = 12), and nondiseased controls (n = 7). Messenger RNA (mRNA) and protein were extracted from samples and expression of TFF1, TFF2, and TFF3 were assessed using quantitative real-time polymerase chain reaction qRT-PCR and Western blots, respectively. Tissue localization of TFF expression was confirmed using immunohistochemistry. TFF1 and TFF3 were both highly expressed in sinonasal tissue, while TFF2 was expressed at near-undetectable levels. CRSsNP tissue had a statistically significant increase in the expression of both TFF1 and TFF3. No difference in TFF expression was found between control and CRSwNP patients. TFF1 and TFF3 are overexpressed in CRSsNP. The role of TFF peptides in mucosal protection and repair suggests a possible important physiologic role in maintaining the sinonasal epithelial barrier and modulating innate immunity in the sinonasal tract.

Epigenetic Control of Trefoil Factor Family (TFF) Peptide Expression in Human Retinoblastoma Cell Lines

Background: Recent studies demonstrated that epigenetic mechanisms are involved in the regulation of trefoil factor family (TFF) peptide expression in cancer. In human tissues with endogenous TFF1, TFF2 or TFF3 gene expression, the corresponding promoter is unmethylated and in organs without TFF expression, the promoter of the three genes is highly methylated. Methods: Retinoblastoma (Rb) cell lines were treated with the DNA methyltransferase inhibitor 5-Aza-2`deoxycytidine (5-Aza-dC), the histone deacetylase inhibitor 4-Phenylbutyric acid (PBA) or both and analyzed for changes (i) in TFF mRNA expression by Real-time PCR and (ii) in the methylation status of the TFF promoters by genomic bisulfite sequencing. Results: The degree of promoter methylation correlates with endogenous TFF expression in the retinoblastoma cell lines analyzed. Nearly all Rb cell lines exhibiting high endogenous TFF1 expression displayed low methylation of the CpGs in the corresponding promoter region. Low expression of TFF3 in Rb cell lines is linked with high density methylation of the TFF3 promoter. 5-Aza-dC treatment induced TFF1 and TFF3 expression in nearly all cell lines investigated and combined treatment with PBA further increased this effect. The number of methylated CpG dinucleotides of the TFF promoter is clearly reduced upon treatment with 5-Aza-dC and combined treatment with PBA further extended the degree of demethylation. Conclusion: Our data clearly show that the expression of TFF3 in retinoblastoma cell lines is epigenetically regulated, whereas the level of TFF1 and TFF2 seems to be regulated by other or additional mechanisms.

Tff3 is Expressed in Neurons and Microglial Cells

Background/Aims: The trefoil factor family (TFF) peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

Quantitative measurements of trefoil factor family peptides: possibilities and pitfalls

The trefoil factor family (TFF) peptides TFF1, TFF2, and TFF3 are produced and secreted by mucous membranes throughout the body. Their importance for the protection and repair of epithelial surfaces is well established, and the three peptides are present in various amounts in mucosal secretions as well as in the circulation. They have been linked to both inflammatory diseases and to various types of cancer, and serum concentrations of TFF3 show a more than 47-fold increase during pregnancy. Several both commercial and in-house immunoassays exist, but a number of methodological issues remain unresolved. This review describes methodological challenges in the measurement of the peptides in humans, and summarizes current knowledge concerning the occurrence and possible significance of the peptides in human health and disease.

TFF1 is Differentially Expressed in Stationary and Migratory Rat Gastric Epithelial Cells (RGM-1) after in Vitro Wounding: Influence of TFF1 RNA Interference on Cell Migration

Background/Aims: The trefoil factor family (TFF) peptide TFF1 is typically secreted by gastric surface mucous cells. These cells are also the major players in gastric restitution, i.e., repair of the stomach mucosa by cell migration after injury. Methods: An established in vitro model of gastric restitution, i.e., migration of the non-transformed rat gastric cell line RGM-1 after scratch wounding, was investigated by expression profiling of selected genes from separated stationary and migratory cells. Also semi-quantitative immunocytochemistry was performed. Furthermore, RGM-1 cells were transfected with stealth RNAi™ duplexes targeting Tff1 and relative cell migration rates were analyzed. Results: Surprisingly, Tff1 expression was up-regulated in migratory cells. No unequivocal signs of the epithelial-mesenchymal transition were detectable in migratory cells. Transfection of RGM-1 cells with Tff1-siRNAi duplexes negatively influenced migration of these cells. Conclusion: This clearly points to a function of Tff1 as a motogen. A possible up-regulation of TFF1 synthesis in migratory surface mucous cells also in vivo would be an ideal mechanism to specifically enhance gastric restitution only where topologically needed and to minimize eventual negative side effects of TFF1 such as cell scattering and invasiveness.

Expression of Trefoil factor family peptides in the nasal allergic mucosa

Trefoil factor family peptides (TFFs) are the secretory products of mucous cells and are closely associated with mucins. TFFs appear to be important in mucosal healing processes. Although TFF1/3 are expressed in the human respiratory tract, their role in the nasal mucosa is not thoroughly understood. We investigated the association between TFFs and mucins and the role TFFs in the human nasal mucosa. Patients undergoing turbinectomy were included and it was determined whether patients had nasal allergies or not. The localization of TFF1/3, MUC5AC/5B expression was investigated using immunohistochemistry. The levels of the mRNA transcripts were examined using quantitative real-time PCR. TFF1/3 had a similar pattern of localization in epithelial goblet cells and submucosal glandular cells. TFF1/3 co-localized with MUC5AC in the epithelium, and co-localized with MUC5B in the epithelium and the submucosal glandular cells. The levels of TFF1/3 and MUC5B mRNA in allergic patients were significantly increased. Our results suggest that TFF1/3 may associate with MUC5AC and MUC5B in the nasal mucosa, and that up-regulation of TFF1/3 and MUC5B may play an important role in the clinical condition of the nasal allergic mucosa.

High trefoil factor 1 (TFF1) expression in human retinoblastoma cells correlates with low growth kinetics, increased cyclin-dependent kinase (CDK) inhibitor levels and a selective down-regulation of CDK6

Trefoil factor family (TFFs) peptides facilitate epithelial restitution, but also effect cell proliferation and apoptosis of normal and various cancer cell lines. In a recent study by our group, TFF2 expression was demonstrated in the murine retina, where it exhibits pro-proliferative and pro-apoptotic effects. In the present study, we investigated the expression and function of TFF peptides in eight human retinoblastoma cell lines. TFF1 was the only TFF peptide expressed at detectable levels in immunoblots of retinoblastoma cells. TFF1 expression levels were highly variable in different retinoblastoma cell lines and negatively correlated with cell growth curves. Recombinant human TFF1 had a negative effect on cell viability and caused a reduction in cell proliferation. Retinoblastoma cell lines with high TFF1 expression levels exhibited a selective down-regulation of cyclin-dependent kinase (CDK) 6, whereas CDK4 and CDK2 seem to be unaffected by TFF1 expression. In immunocytochemical studies, we observed a nuclear co-localization of TFF1 and CDK2 in Cajal bodies (CBs). In high TFF1 expressing human retinoblastoma cell lines CBs were smaller and higher in number compared to retinoblastoma lines with low TFF1 expression, indicating differences in cell cycle status between the different retinoblastoma cell lines. Our data further support the notion for a potential tumor suppressor function of TFF1. The nuclear localization of TFF1 in CBs--considered to play a role in cell cycle progression, potentially acting as a platform for CDK-cyclin function-offers a new impetus in the ongoing search for potential TFF1 interacting proteins.