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1.
Nat Immunol ; 23(11): 1644-1652, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36271145

RESUMO

Interleukin-17A (IL-17A) is a key mediator of protective immunity to yeast and bacterial infections but also drives the pathogenesis of several autoimmune diseases, such as psoriasis or psoriatic arthritis. Here we show that the tetra-transmembrane protein CMTM4 is a subunit of the IL-17 receptor (IL-17R). CMTM4 constitutively associated with IL-17R subunit C to mediate its stability, glycosylation and plasma membrane localization. Both mouse and human cell lines deficient in CMTM4 were largely unresponsive to IL-17A, due to their inability to assemble the IL-17R signaling complex. Accordingly, CMTM4-deficient mice had a severe defect in the recruitment of immune cells following IL-17A administration and were largely resistant to experimental psoriasis, but not to experimental autoimmune encephalomyelitis. Collectively, our data identified CMTM4 as an essential component of IL-17R and a potential therapeutic target for treating IL-17-mediated autoimmune diseases.


Assuntos
Artrite Psoriásica , Encefalomielite Autoimune Experimental , Psoríase , Humanos , Camundongos , Animais , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Encefalomielite Autoimune Experimental/genética , Proteínas com Domínio MARVEL/genética
2.
Nature ; 593(7859): 424-428, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33767445

RESUMO

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/uso terapêutico , Peso Corporal , COVID-19/prevenção & controle , Dependovirus/genética , Modelos Animais de Doenças , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Tratamento Farmacológico da COVID-19
3.
Cell Mol Life Sci ; 79(8): 423, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35838828

RESUMO

In cancer, the activating transcription factor 2 (ATF2) has pleiotropic functions in cellular responses to growth stimuli, damage, or inflammation. Due to only limited studies, the significance of ATF2 in colorectal cancer (CRC) is not well understood. We report that low ATF2 levels correlated with worse prognosis and tumor aggressiveness in CRC patients. NanoString gene expression and ChIP analysis confirmed trophoblast cell surface antigen 2 (TROP2) as a novel inhibitory ATF2 target gene. This inverse correlation was further observed in primary human tumor tissues. Immunostainings revealed that high intratumoral heterogeneity for ATF2 and TROP2 expression was sustained also in liver metastasis. Mechanistically, our in vitro data of CRISPR/Cas9-generated ATF2 knockout (KO) clones revealed that high TROP2 levels were critical for cell de-adhesion and increased cell migration without triggering EMT. TROP2 was enriched in filopodia and displaced Paxillin from adherens junctions. In vivo imaging, micro-computer tomography, and immunostainings verified that an ATF2KO/TROP2high status triggered tumor invasiveness in in vivo mouse and chicken xenograft models. In silico analysis provided direct support that ATF2low/TROP2high expression status defined high-risk CRC patients. Finally, our data demonstrate that ATF2 acts as a tumor suppressor by inhibiting the cancer driver TROP2. Therapeutic TROP2 targeting might prevent particularly the first steps in metastasis, i.e., the de-adhesion and invasion of colon cancer cells.


Assuntos
Fator 2 Ativador da Transcrição , Antígenos de Neoplasias , Neoplasias Colorretais , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral/metabolismo , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Camundongos , Regulação para Cima
4.
BMC Gastroenterol ; 22(1): 186, 2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35413796

RESUMO

BACKGROUND: Ubiquitin ligases (Ub-ligases) are essential intracellular enzymes responsible for the regulation of proteome homeostasis, signaling pathway crosstalk, cell differentiation and stress responses. Individual Ub-ligases exhibit their unique functions based on the nature of their substrates. They create a complex regulatory network with alternative and feedback pathways to maintain cell homeostasis, being thus important players in many physiological and pathological conditions. However, the functional classification of Ub-ligases needs to be revised and extended. METHODS: In the current study, we used a novel semantic biclustering technique for expression profiling of Ub-ligases and ubiquitination-related genes in the murine gastrointestinal tract (GIT). We accommodated a general framework of the algorithm for finding tissue-specific gene expression clusters in GIT. In order to test identified clusters in a biological system, we used a model of epithelial regeneration. For this purpose, a dextran sulfate sodium (DSS) mouse model, following with in situ hybridization, was used to expose genes with possible compensatory features. To determine cell-type specific distribution of Ub-ligases and ubiquitination-related genes, principal component analysis (PCA) and Uniform Manifold Approximation and Projection technique (UMAP) were used to analyze the Tabula Muris scRNA-seq data of murine colon followed by comparison with our clustering results. RESULTS: Our established clustering protocol, that incorporates the semantic biclustering algorithm, demonstrated the potential to reveal interesting expression patterns. In this manner, we statistically defined gene clusters consisting of the same genes involved in distinct regulatory pathways vs distinct genes playing roles in functionally similar signaling pathways. This allowed us to uncover the potentially redundant features of GIT-specific Ub-ligases and ubiquitination-related genes. Testing the statistically obtained results on the mouse model showed that genes clustered to the same ontology group simultaneously alter their expression pattern after induced epithelial damage, illustrating their complementary role during tissue regeneration. CONCLUSIONS: An optimized semantic clustering protocol demonstrates the potential to reveal a readable and unique pattern in the expression profiling of GIT-specific Ub-ligases, exposing ontologically relevant gene clusters with potentially redundant features. This extends our knowledge of ontological relationships among Ub-ligases and ubiquitination-related genes, providing an alternative and more functional gene classification. In a similar way, semantic cluster analysis could be used for studding of other enzyme families, tissues and systems.


Assuntos
Semântica , Ubiquitina-Proteína Ligases , Animais , Análise por Conglomerados , Trato Gastrointestinal/metabolismo , Humanos , Camundongos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
6.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934845

RESUMO

Commensal microbiota contribute to gut homeostasis by inducing transcription of mucosal genes. Analysis of the impact of various microbiota on intestinal tissue provides an important insight into the function of this organ. We used cDNA microarrays to determine the gene expression signature of mucosa isolated from the small intestine and colon of germ-free (GF) mice and animals monoassociated with two E. coli strains. The results were compared to the expression data obtained in conventionally reared (CR) mice. In addition, we analyzed gene expression in colon organoids derived from CR, GF, and monoassociated animals. The analysis revealed that the complete absence of intestinal microbiota mainly affected the mucosal immune system, which was not restored upon monoassociation. The most important expression changes observed in the colon mucosa indicated alterations in adipose tissue and lipid metabolism. In the comparison of differentially expressed genes in the mucosa or organoids obtained from GF and CR mice, only six genes were common for both types of samples. The results show that the increased expression of the angiopoietin-like 4 (Angptl4) gene encoding a secreted regulator of lipid metabolism indicates the GF status.


Assuntos
Perfilação da Expressão Gênica , Vida Livre de Germes/genética , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Animais , Biomarcadores/metabolismo , Colo/metabolismo , Escherichia coli/fisiologia , Regulação da Expressão Gênica , Sistema Imunitário/metabolismo , Imunidade nas Mucosas , Camundongos Endogâmicos BALB C , Microbiota
7.
Genesis ; 49(3): 142-51, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21309068

RESUMO

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.


Assuntos
Alelos , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Fatores de Transcrição Kruppel-Like/genética , Animais , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Feminino , Deleção de Genes , Marcação de Genes , Genes Reporter , Integrases/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/genética
8.
Front Immunol ; 12: 618332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33986741

RESUMO

LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes. LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.


Assuntos
Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Colite/etiologia , Colite/metabolismo , Colite/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Genótipo , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação
9.
bioRxiv ; 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33501434

RESUMO

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) 1,2 . We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 3 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

10.
Mol Biol Cell ; 15(2): 497-505, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14595115

RESUMO

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.


Assuntos
Diferenciação Celular/fisiologia , Células Musculares/citologia , Músculo Esquelético/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Somatomedinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Morfolinas/farmacologia , Células Musculares/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Proteína MyoD/farmacologia , Fatores de Regulação Miogênica/metabolismo , Miogenina/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt , Fatores de Transcrição/metabolismo , Troponina T/metabolismo
11.
Int J Oncol ; 28(2): 559-65, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16391813

RESUMO

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.


Assuntos
Butiratos/farmacologia , Células HT29/patologia , Adenocarcinoma , Fosfatase Alcalina/metabolismo , Antígenos CD/metabolismo , Apoptose , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Clonais , Neoplasias Colorretais , Células HT29/efeitos dos fármacos , Células HT29/ultraestrutura , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Int J Mol Med ; 17(1): 69-75, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16328013

RESUMO

beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.


Assuntos
Adenocarcinoma/metabolismo , Butiratos/metabolismo , Neoplasias Colorretais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , beta Catenina/metabolismo , Fosfatase Alcalina/metabolismo , Androstadienos/metabolismo , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Wortmanina , beta Catenina/genética
13.
Int J Mol Med ; 15(2): 329-35, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15647851

RESUMO

The present study focuses on a putative regulation of PKCbetaII by phosphatidylinositol-3 kinase (PI 3-kinase) in colorectal carcinoma cells; little is known about the role and activity of PKCbetaII in these cells. We examined the activity of PI 3-kinase in two adenocarcinoma cell lines, HT29 cells that differentiate only after stimulation with appropriate agents, and Caco-2 cells that can differentiate spontaneously. The activity of PI 3-kinase as well as the activity of PKCbetaII appeared to decrease only in HT29 cells in which differentiation was induced by sodium butyrate. In HT29 cells infected with recombinant adenovirus encoding constitutively active PI 3-kinase, the activity of alkaline phosphatase was almost completely blocked, and this PI 3-kinase significantly potentiated the activity of PKCbetaII in HT29 cells despite the presence of NaBT in the culture medium. On the contrary, in differentiating Caco-2 cells, the activity of PI 3-kinase was not butyrate-sensitive. In agreement with these findings, the alkaline phosphatase activity was not affected by constitutively active PI 3-kinase overexpressed in Caco-2 cells. These observations suggest that PKCbetaII is regulated by PI 3-kinase in HT29 cells and that the mechanisms of spontaneous differentiation versus butyrate-induced differentiation of adenocarcinoma cells may be different.


Assuntos
Neoplasias Colorretais/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Oxibato de Sódio/farmacologia , Adenocarcinoma/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultura/farmacologia , Regulação para Baixo , Humanos , Immunoblotting , Íons , Proteína Quinase C beta , Fatores de Tempo
14.
Dev Cell ; 35(6): 713-24, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26702830

RESUMO

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.


Assuntos
Movimento Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Dente Molar/embriologia , Morfogênese/fisiologia , Dente/citologia , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Dente Molar/citologia , Dente Molar/metabolismo , Odontogênese/fisiologia , Dente/embriologia
15.
Mol Cancer Res ; 13(7): 1139-48, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25934696

RESUMO

UNLABELLED: Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses "danger" signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. IMPLICATIONS: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells.


Assuntos
Carcinogênese/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Azoximetano , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais , Técnicas de Silenciamento de Genes , Humanos , Intestinos/citologia , Camundongos , Camundongos Transgênicos , Regulação para Cima
16.
Transl Oncol ; 2(4): 281-90, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19956390

RESUMO

Migration is a complex process that, besides its various physiological functions in embryogenesis and adult tissues, plays a crucial role in cancer cell invasion and metastasis. The focus of this study is the involvement and collaboration of Akt, focal adhesion kinase (FAK), and Src kinases in migration and invasiveness of colorectal cancer cells. We show that all three kinases can be found in one protein complex; nevertheless, the interaction between Akt and Src is indirect and mediated by FAK. Interestingly, induced Akt signaling causes an increase in tyrosine phosphorylation of FAK, but this increase is attenuated by the Src inhibitor SU6656. We also show that active Akt strongly stimulates cell migration, but this phenomenon is fully blocked by FAK knockdown or partly by inhibition of Src kinase. In addition, we found that all three kinases were indispensable for the successful invasion of colorectal cancer cells. Altogether, the presented data bring new insights into the mechanism how the phosphatidylinositol-3-kinase (PI3-K)/Akt pathway can influence migration of colorectal adenocarcinoma cells. Because FAK is indispensable for cell movements and functions downstream of Akt, our results imply FAK kinase as a potential key molecule during progression of tumors with active PI3-K/Akt signaling.

17.
Neoplasia ; 10(2): 99-107, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18283331

RESUMO

Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV)-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB), phosphorylation of tuberin (TSC2), mammalian target of rapamycin (mTOR), S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.


Assuntos
Proteínas Quinases/metabolismo , Adenoviridae/genética , Animais , Linhagem Celular , Linhagem Celular Transformada , Cricetinae , Indóis/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Vírus do Sarcoma de Rous/genética , Transdução de Sinais , Sulfonamidas/farmacologia
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