RESUMEN
Trefoil factor family protein 3 (Tff3) protects the gastrointestinal mucosa and has a complex mode of action in different tissues. Here, we aimed to determine the effect of Tff3 deficiency on intestinal tissues in a long-term high-fat-diet (HFD)-fed model. A novel congenic strain without additional metabolically relevant mutations (Tff3-/-/C57Bl6NCrl strain, male and female) was used. Wild type (Wt) and Tff3-deficient mice of both sexes were fed a HFD for 36 weeks. Long-term feeding of a HFD induces different effects on the intestinal structure of Tff3-deficient male and female mice. For the first time, we found sex-specific differences in duodenal morphology. HFD feeding reduced microvilli height in Tff3-deficient females compared to that in Wt females, suggesting a possible effect on microvillar actin filament dynamics. These changes could not be attributed to genes involved in ER and oxidative stress, apoptosis, or inflammation. Tff3-deficient males exhibited a reduced cecal crypt depth compared to that of Wt males, but this was not the case in females. Microbiome-related short-chain fatty acid content was not affected by Tff3 deficiency in HFD-fed male or female mice. Sex-related differences due to Tff3 deficiency imply the need to consider both sexes in future studies on the role of Tff in intestinal function.
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Dieta Alta en Grasa , Proteínas , Ratones , Masculino , Animales , Femenino , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos , Duodeno , Ratones Endogámicos C57BL , Factor Trefoil-3/genéticaRESUMEN
Chronic rhinosinusitis (CRS) is a prevalent, multifaceted inflammatory condition affecting the nasal cavity and the paranasal sinuses, frequently accompanied by formation of nasal polyps (CRSwNP). This apparently uniform clinical entity is preceded by heterogeneous changes in cellular and molecular patterns, suggesting the presence of multiple CRS endotypes and a diverse etiology. Alterations of the upper airway innate defense mechanisms, including antimicrobial and antioxidant capacity, have been implicated in CRSwNP etiology. The aim of this study was to investigate mRNA expression patterns of antioxidative enzymes, including superoxide dismutase (SOD) and peroxiredoxin-2 (PRDX2), and innate immune system defense players, namely the bactericidal/permeability-increasing fold-containing family A, member 1 (BPIFA1) and PACAP family members, particularly adenylate-cyclase-activating polypeptide receptor 1 (ADCYAP1) in nasal mucosa and nasal polyps from CRSwNP patients. Additional stratification based on age, sex, allergic comorbidity, and disease severity was applied. The results showed that ADCYAP1, BPIFA1, and PRDX2 transcripts are differentially expressed in nasal mucosa and scale with radiologically assessed disease severity in CRSwNP patients. Sinonasal transcriptome is not associated with age, sex, and smoking in CRSwNP. Surgical and postoperative corticosteroid (CS) therapy improves endoscopic appearance of the mucosa, but variably reverses target gene expression patterns in the nasal cavity of CRSwNP patients. Transcriptional cross-correlations analysis revealed an increased level of connectedness among differentially expressed genes under inflammatory conditions and restoration of basic network following CS treatment. Although results of the present study imply a possible engagement of ADCYAP1 and BPIFA1 as biomarkers for CRSwNP, a more profound study taking into account disease severity and CRSwNP endotypes prior to the treatment would provide additional information on their sensitivity.
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Pólipos Nasales , Rinitis , Sinusitis , Enfermedad Crónica , Humanos , Inflamación/metabolismo , Mucosa Nasal/metabolismo , Pólipos Nasales/complicaciones , Pólipos Nasales/genética , Estrés Oxidativo/genética , Rinitis/complicaciones , Rinitis/genética , Sinusitis/complicaciones , Sinusitis/genéticaRESUMEN
Trefoil family factor (TFF) proteins contribute to antimicrobial defense and the maintenance of sinonasal epithelial barrier integrity. Dysregulation of TFF expression may be involved in the development of chronic inflammation and tissue remodeling characteristically found in chronic rhinosinusitis with nasal polyposis (CRSwNP). Expressions of TFF1 and TFF3 were determined in specimens of middle nasal turbinate (MNT-0), bulla ethmoidalis (BE), and nasal polyps (NP) from CRSwNP patients (n = 29) and inferior nasal turbinate from a group of control patients (underwent nasal septoplasty, n = 25). An additional MNT sample was collected 6 months after functional endoscopic sinus surgery (FESS, MNT-6). TFF1 mRNA levels were significantly reduced in all specimens by approximately three- to five-fold, while TFF3 was increased in MNT-0, as compared with controls. Six months after surgery their levels were reversed to control values. CRSwNP patients with S. epidermidis isolated from sinus swabs showed upregulation of TFF3 in MNT and NP as compared with patients with sterile swabs. Target gene regulation was not affected by the presence of type 2 inflammation in patients with confirmed allergy. Results of this study imply participation of TFFs genes in the development of CRSwNP.
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Pólipos Nasales/genética , Rinitis/genética , Sinusitis/genética , Factor Trefoil-1/genética , Factor Trefoil-3/genética , Adulto , Anciano , Enfermedad Crónica , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pólipos Nasales/complicaciones , Pólipos Nasales/cirugía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rinitis/complicaciones , Rinitis/cirugía , Sinusitis/complicaciones , Sinusitis/cirugía , Factor Trefoil-1/metabolismo , Factor Trefoil-3/metabolismo , Adulto JovenRESUMEN
Endoplasmic reticulum (ER) stress, a cellular condition caused by the accumulation of unfolded proteins inside the ER, has been recognized as a major pathological mechanism in a variety of conditions, including cancer, metabolic and neurodegenerative diseases. Trefoil factor family (TFFs) peptides are present in different epithelial organs, blood supply, neural tissues, as well as in the liver, and their deficiency has been linked to the ER function. Complete ablation of Tff3 expression is observed in steatosis, and as the most prominent change in the early phase of diabetes in multigenic mouse models of diabesity. To elucidate the role of Tff3 deficiency on different pathologically relevant pathways, we have developed a new congenic mouse model Tff3-/-/C57BL6/N from a mixed background strain (C57BL6/N /SV129) by using a speed congenics approach. Acute ER stress was evoked by tunicamycin treatment, and mice were sacrificed after 24 h. Afterwards the effect of Tff3 deficiency was evaluated with regard to the expression of relevant oxidative and ER stress genes, relevant proinflammatory cytokines/chemokines, and the global protein content. The most dramatic change was noticed at the level of inflammation-related genes, while markers for unfolded protein response were not significantly affected. Ultrastructural analysis confirmed that the size of lipid vacuoles was affected as well. Since the liver acts as an important metabolic and immunological organ, the influence of Tff3 deficiency and physiological function possibly reflects on the whole organism.
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Estrés del Retículo Endoplásmico/genética , Hígado/metabolismo , Factor Trefoil-3/deficiencia , Animales , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Hígado/patología , Hígado/ultraestructura , Ratones , Ratones Noqueados , Estrés Oxidativo/genética , Proteoma , Proteómica/métodosRESUMEN
Trefoil factor family 3 (Tff3) peptide is present during intrauterine endochondral ossification in mice, and its deficiency affects cancellous bone quality in secondary ossification centers of mouse tibiae. The aim of this study was to quantitatively analyze parameters describing the growth plate and primary ossification centers in tibiae of 1-month-old wild-type and Tff3 knock-out mice (n=5 per genotype) by using free and open-source software. Digital photographs of the growth plates and trabecular bone were processed by open-source computer programs GIMP and FIJI. Histomorphometric parameters were calculated using measurements made with FIJI. Tff3 knock-out mice had significantly smaller trabecular number and significantly larger trabecular separation. Trabecular bone volume, trabecular bone surface, and trabecular thickness showed no significant difference between the two groups. Although such histomorphological differences were found in the cancellous bone structure, no significant differences were found in the epiphyseal plate histomorphology. Tff3 peptide probably has an effect on the formation and quality of the cancellous bone in the primary ossification centers, but not through disrupting the epiphyseal plate morphology. This work emphasizes the benefits of using free and open-source programs for morphological studies in life sciences.
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BACKGROUND: Dupuytren's disease (DD) is a nodular palmar fibromatosis that causes irreversible permanent contracture of fingers and results in the loss of hand function. Surgery still remains the only available solution for DD patients but cannot permanently cure the disease nor reduce high recurrence rates. With this rationale, we designed a study aimed at an improved understanding of the molecular mechanisms underlying DD. Our major focus was an analysis of the global gene expression profile and signalling pathways in DD cells with the aim of identifying novel biomarkers and/or therapeutic targets. METHODS: Primary cells were cultured from surgically removed diseased and healthy tissue. Microarray expression analysis (HG-U133A array, Affymetrix) and qPCR was performed with total RNA isolated from primary DD cells. Mechanistic studies involving inhibition of p38 phosphorylation were performed on normal human fibroblasts' and primary DD cells' in vitro models. Expression of stem cell markers in primary fibroblasts/myofibroblasts was assessed as well. RESULTS: We identified 3 p38MAPK signalling pathway regulatory genes, THBS1, GADD45α and NUAK1, all involved in cellular proliferation and production of the extracellular matrix proteins. Inhibition of the p38MAPK signalling pathway induced down-regulation of myofibroblast markers, α-smooth muscle actin and palladin. A stem-cell like subpopulation positive for CD90 marker was identified among primary DD cells. CONCLUSION: The study reveals involvement of the p38 MAPK pathway as a possible signalling cascade in the pathogenesis of Dupuytren's disease. Moreover, a particular stem cell-like CD90(+) subpopulation was identified that might contribute to DD development.
Asunto(s)
Contractura de Dupuytren/genética , Contractura de Dupuytren/metabolismo , Perfilación de la Expresión Génica , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Contractura de Dupuytren/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Miofibroblastos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Antígenos Thy-1/análisis , Antígenos Thy-1/metabolismoRESUMEN
TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica.
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Técnicas de Inactivación de Genes , Intestinos/microbiología , Mucinas/genética , Proteínas Musculares/genética , Péptidos/genética , Ganglios Linfáticos Agregados/microbiología , Yersiniosis/genética , Yersiniosis/transmisión , Yersinia enterocolitica/patogenicidad , Animales , Femenino , Inmunidad Innata , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Intestinos/patología , Antígeno de Macrófago-1/análisis , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Noqueados , Mucinas/inmunología , Proteínas Musculares/inmunología , Péptidos/inmunología , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Ganglios Linfáticos Agregados/patología , Bazo/inmunología , Bazo/metabolismo , Bazo/microbiología , Bazo/patología , Factor Trefoil-2 , Yersiniosis/inmunología , Yersiniosis/patología , Yersinia enterocolitica/inmunologíaRESUMEN
New N-1-sulfonylpyrimidines showed potent growth inhibitory activity against human and mouse tumour cells of different origin. 1-(p-toluenesulfonyl)cytosine (TsC) and 1-(p-toluenesulfonyl)cytosine hydrochloride (TsC × HCl) inhibited the growth of human cervical carcinoma cells (HeLa), and had no significant cytotoxic effects on normal human foreskin fibroblasts (BJ). TsC and TsC × HCl interfered with the HeLa cell cycle progression bringing about the accumulation of G1 phase cells and the induction of apoptosis. Antiproliferative effects of TsC and TsC × HCl were additionally confirmed by investigating de novo synthesis of RNA, DNA and proteins in HeLa cells. Monitoring gene expression using DNA Chip Analysis and quantitative PCR showed that TsC × HCl affects the expression of several cell-cycle regulating genes implying that cell cycle arrest and DNA damage-induced apoptosis might account for the observed cellular effects. In vivo experiments revealed low toxicity of TsC × HCl, as demonstrated by unaltered haematological and metabolic blood parameters. In conclusion, potent antitumour efficacy and low toxicity of new compounds in comparison with the common chemotherapy drug 5-FU make them promising anticancer agents. Additional pre-clinical and clinical studies are warranted to illuminate the mode of action of these newly synthesized compounds in vivo, which would lay the groundwork for their further optimization.
Asunto(s)
Antineoplásicos/farmacología , Citosina/análogos & derivados , Citosina/farmacología , Animales , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citosina/toxicidad , Daño del ADN , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Prepucio/citología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Pruebas Hematológicas , Humanos , Masculino , Ratas Wistar , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Trefoil factor 3 (Tff3) protein is a small secretory protein expressed on various mucosal surfaces and is involved in proper mucosal function and recovery via various mechanisms, including immune response. However, Tff3 is also found in the bloodstream and in various other tissues, including the liver. Its complete attenuation was observed as the most prominent event in the early phase of diabetes in the polygenic Tally Ho mouse model of diabesity. Since then, its role in metabolic processes has emerged. To elucidate the complex role of Tff3, we used a new Tff3-deficient mouse model without additional metabolically relevant mutations (Tff3-/-/C57BL/6NCrl) and exposed it to a high-fat diet (HFD) for a prolonged period (8 months). The effect was observed in male and female mice compared to wild-type (WT) counter groups (n = 10 animals per group). We monitored the animals' general metabolic parameters, liver morphology, ultrastructure and molecular genes in relevant lipid and inflammatory pathways. Tff3-deficient male mice had reduced body weight and better glucose utilization after 17 weeks of HFD, but longer HFD exposure (32 weeks) resulted in no such change. We found a strong reduction in lipid accumulation in male Tff3-/-/C57BL/6NCrl mice and a less prominent reduction in female mice. This was associated with downregulated peroxisome proliferator-activated receptor gamma (Pparγ) and upregulated interleukin-6 (Il-6) gene expression, although protein level difference did not reach statistical significance due to higher individual variations. Tff3-/-/C57Bl6N mice of both sex had reduced liver steatosis, without major fatty acid content perturbations. Our research shows that Tff3 protein is clearly involved in complex metabolic pathways. Tff3 deficiency in C57Bl6N genetic background caused reduced lipid accumulation in the liver; further research is needed to elucidate its precise role in metabolism-related events.
RESUMEN
Genetic impairment of the genes coding for three mammalian trefoil peptides resulted in severe gastrointestinal malfunctions. The trefoil peptides also appear involved in caloric metabolism. Monitoring global miRNA expression of Tff3 deficient mice points to an interplay of Tff3 with a miRNA regulatory network. We identified 21 miRNAs that were deregulated when compared to the wild type strain. In silico evaluation indicated that the majority of the 21 miRNA were connected with the metabolic pathway "glycolysis/gluconeogenesis'' (p=0.032), a signaling pathway including nine target genes Aldh9a1, Aldh2, Pck1, Aldoc, Pgam2, Pck2, Adh4, Adh5, and Fbp1. Association of Tff3 with this metabolic pathway is further supported by the observation that the body mass of adult Tff3 KO mice (five months) showed a clearly reduced weight. Furthermore, the majority of the identified 21 miRNA genes are localized on murine chromosomes 2 and 5 in three clusters (2A1, 2B, 5B3) suggesting a coordinated expression control and function.
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Enfermedades Gastrointestinales/genética , MicroARNs/metabolismo , Mucinas/metabolismo , Transducción de Señal , Animales , Peso Corporal , Cromosomas de los Mamíferos/genética , Modelos Animales de Enfermedad , Ingestión de Energía , Enfermedades Gastrointestinales/metabolismo , Enfermedades Gastrointestinales/fisiopatología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Gluconeogénesis , Glucólisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/sangre , MicroARNs/genética , Factor Trefoil-3RESUMEN
The roles of phenolics from olive oils as effective anticancer agents have been documented in various in vitro studies of different cancer cells lines, but the relationship between the phenolic profile of olive oil and its biological activity needs more elucidation. In this study, we analysed phenolic profiles of extra virgin olive oils (EVOOs) from different autochthonous cultivars from Croatia (Oblica, Bjelica, Buza, Zizolera) and investigated the biological effect of EVOO phenolic extracts (EVOO-PEs) on human cervical (HeLa) and human colon (SW48) cancer cell lines alone and in combination with cisplatin (cDDP), carboplatin (CBP), 5-fluorouracil (5-FU) and irinotecan. The quantitative evaluation of olive oil polyphenols was performed by HPLC-DAD and spectrophotometric analysis. The biological effect of EVOO-PEs alone and in combination with anticancer drugs was measured by MTT assay. Analysed EVOO-PEs differ in phenolic profile and inhibited HeLa and SW48 cells in a dose-dependent manner. Further, it is shown that EVOO-PEs (Oblica-Sea, Buza and Zizolera), in combination with anticancer drugs, increase the metabolic activity of HeLa and SW48 cells and have a protective role. These data imply careful consummation of olive oil during chemotherapy of cancer patients.
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Tff peptides are secreted mainly by the gastrointestinal epithelial cells and their primary role is maintaining normal structure and function of mucous epithelia. Ongoing studies on their expression pattern have disclosed other sites of their synthesis thus revealing additional physiological functions in the organism. Here we present new data about Tff3 expression in the cochlea of the rodent inner ear. On the basis of RT-PCR we describe the presence of Tff3 transcripts in both, a mouse cDNA library isolated from whole cochleae from postnatal days 3-15 (P3-P15), and also in cochlear tissue. By using a riboprobe for the fragment containing exon 1, 2 and 3 of Tff3, in situ hybridization, localized Tff3 signals in neurons of spiral ganglion and vestibular organ. We did not observe any abnormalities in the middle ear of Tff3 knock-out mice, neither did histological examination of the inner ear indicate any gross morphological changes in the cochlea. However, ABR (auditory evoked brain stem responses) audiograms revealed that the Tff3 knock-out animals show an accelerated presbyacusis and a hearing loss of about 15 dB at low frequencies increasing to 25 dB loss at higher frequencies. These findings suggest that Tff3 could play a role in neurosensory signaling. Further studies are needed to clarify this new function in the auditory system.
Asunto(s)
Pérdida Auditiva/metabolismo , Pérdida Auditiva/fisiopatología , Mucinas/deficiencia , Mucinas/metabolismo , Presbiacusia/metabolismo , Presbiacusia/fisiopatología , Animales , Progresión de la Enfermedad , Oído Interno/citología , Oído Interno/metabolismo , Oído Medio/citología , Oído Medio/metabolismo , Regulación de la Expresión Génica , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Ratones , Ratones Noqueados , Mucinas/genética , Presbiacusia/genética , Presbiacusia/patología , ARN Mensajero/genética , Factor Trefoil-3RESUMEN
The objective of this study was to determine differential expression of TFF1, TFF2 and TFF3 genes and proteins in breast tumor subtypes. In addition, we investigated the correlation between TFF genes within tumor subgroups, and TFF genes with clinical and pathologic characteristics of the tumor. Study group included 122 patients with surgically removed breast tumors. Samples were investigated using qRT-PCR and immunohistochemistry. TFF1 and TFF3 genes and proteins were expressed in breast tumors, while the levels of TFF2 gene and protein expression were very low or undetectable. TFF1 was significantly more expressed in benign tumors, while TFF3 was more expressed in malignant tumors. Gene and protein expression of both TFF1 and TFF3 was greater in lymph node-negative tumors, hormone positive tumors, tumors with moderate levels of Ki67 expression, and in grade II tumors. A strong positive correlation was found between TFF1 and TFF3 genes, and the expression of both negatively correlated with Ki67 and the level of tumor histologic differentiation. Our results suggest that TFF1 and TFF3, but not TFF2, may have a role in breast tumor pathogenesis and could be used in the assessment of tumor differentiation and malignancy.
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Neoplasias de la Mama , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Biomarcadores de Tumor , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Mucinas , Proteínas Musculares , Péptidos , Factor Trefoil-1/metabolismo , Factor Trefoil-2/metabolismo , Factor Trefoil-3/metabolismoRESUMEN
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.
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Células Epiteliales/metabolismo , Epitelio/metabolismo , Estratos Germinativos/metabolismo , Factor Trefoil-3/metabolismo , Animales , Apoptosis , Diferenciación Celular , Movimiento Celular , Desarrollo Embrionario , Células Epiteliales/inmunología , Epitelio/inmunología , Femenino , Estratos Germinativos/citología , Ratones , Membrana Mucosa/citología , Membrana Mucosa/metabolismo , Embarazo , Sistema Respiratorio/metabolismo , Sistema Urinario/metabolismoRESUMEN
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.
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Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Mucinas/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Péptidos/metabolismo , Animales , Citoplasma/metabolismo , Desarrollo Embrionario/genética , Femenino , Ganglión/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Ratones , Mucinas/genética , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Péptidos/genética , Embarazo , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
OBJECTIVES: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. METHODS: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-ß1 and/or inhibitor of p38 phosphorylation. RESULTS: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. CONCLUSIONS: TGF-ß1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence.
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Lack of regular cell-cell interaction is one major cause for neoplastic growth and metastasis. In head and neck squamous cell carcinomas a 10-fold down-regulation of connexin31.1 (GJB5) as well as mutations in the TGF-beta-receptor-II were reported. We performed mutation screenings in GJB5 and the TGF-beta-receptor-II poly(10)adenine hot spot employing larynx cancer samples of 10 patients. Variable length of the TGF-beta-receptor-II adenine homopolymer in controls and tumours indicate a high slippage error rate of the DNA polymerases rendering mutational analyses inconsistent. Lack of GJB5 mutations in the entire tumour collection suggests that this gene is not primarily involved in laryngeal tumorigenesis.
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Carcinoma de Células Escamosas/genética , Conexinas/biosíntesis , Neoplasias Laríngeas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Comunicación Celular , Análisis Mutacional de ADN , ADN Polimerasa Dirigida por ADN/farmacología , Regulación hacia Abajo , Femenino , Humanos , Neoplasias Laríngeas/patología , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador betaRESUMEN
The acute phase response is strictly connected with modulation of gene expression. Transcriptional control of many genes is mediated by binding of diverse transcription factors to cis-acting DNA motifs in the respective promoter sequence. We now describe such regulatory elements for the TFF3 gene coding for a peptide involved in response to gut irritation. TNF-alpha stimulation, which induces NF-kappaB activation, evoked up to 10-fold reduction of TFF3 expression in the colon tumour cell line HT-29. Several consensus binding sites for members of the NF-kappaB transcription factor subunits are located in the 5'-flanking region of TFF3. Mutating these sites was aimed at discovering which one is responsible for NF-kappaB binding and thus regulation of TFF3 expression.
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Mucinas/genética , Proteínas Musculares/genética , FN-kappa B/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/genética , Ensayo de Cambio de Movilidad Electroforética , Células HT29 , Humanos , Mutación/genética , Péptidos , Factor de Transcripción ReIA , Factor Trefoil-3 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We have recently demonstrated that human TFF2 inhibits apoptosis in the non-TFF2 expressing breast adenocarcinoma cell line MCF-7. In this study we examined the impact of TFF2 and an anti-TFF2 antibody (hSP3) on the survival of other human adenocarcinoma cell lines; TFF2-positive (LS174T and SW480) and TFF2-negative (MCF-7 and T47D). Addition of TFF2 protected the (TFF2-) lines but had no effect on those constitutively expressing TFF2. Blocking with hSP3 significantly increased apoptosis in the (TFF2+) cell lines with minimal effect on the (TFF2-) cells. Our results show that the cytoprotective effect of TFF2 seen in MCF-7 cells is not cell line-specific and can be abrogated by inhibition of its expression.
Asunto(s)
Anticuerpos/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/metabolismo , Mucinas/farmacología , Proteínas Musculares/farmacología , Péptidos/farmacología , Humanos , Proteínas Recombinantes/farmacología , Factor Trefoil-2 , Células Tumorales CultivadasRESUMEN
Trefoil factor family protein 3 (TFF3) is found in cartilage affected by osteoarthritis and septic arthritis, whereas no TFF3 presence is observed in healthy cartilage. During endochondral ossification, bone tissue replaces degenerating cartilage. There is no data about the role of TFF3 in this process. Our aim was to study the localization of TFF3 in cartilage during endochondral ossification in the mouse fetus. CD1 mouse fetuses, days 14-17, were isolated, fixed, and paraffin embedded. Fetuses were cut into 6µm sections, and processed for immunohistochemical staining with affinity purified polyclonal rabbit anti-TFF3 antibody. TFF3 was present in cartilage chondrocytes undergoing endochondral ossification, particularly in zone of proliferation, hypertrophy and calcification as well as in zone of cartilage degeneration during the monitored fetal period. Resting cartilage showed no presence of TFF3, while during endochondral ossification TFF3 localization showed an analogous pattern to that reported in cartilage affected by osteoarthritis and septic arthritis. Our data indicate that the role of TFF3 in these pathological conditions is similar to its role in the physiological process of endochondral ossification.