RESUMO
Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4+ but not in CD8+ cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.
Assuntos
Proteínas Aviárias/genética , Linfócitos T CD4-Positivos/imunologia , Galinhas/imunologia , Macrófagos/imunologia , Receptores do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Animais , Proteínas Aviárias/metabolismo , Células Cultivadas , Clonagem Molecular , Corvos/imunologia , Sequência Rica em GC/genética , Humanos , Mamíferos/imunologia , NF-kappa B/metabolismo , Paleógnatas/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Alinhamento de SequênciaRESUMO
Activation of the Wnt/ß-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant ß-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-ß-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33(ΔN-Bcat) mice showed an increase in the constitutive Wnt/ß-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33(ΔN-Bcat) mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7 and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33(ΔN-Bcat) mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33(ΔN-Bcat) mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-ß-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/ß-catenin pathway to facilitate and promote tumorigenesis.
Assuntos
Pólipos Adenomatosos/genética , Transformação Celular Neoplásica/genética , Colo , Neoplasias do Colo/genética , Pólipos do Colo/genética , Mutação , Via de Sinalização Wnt/genética , beta Catenina/genética , Pólipos Adenomatosos/induzido quimicamente , Pólipos Adenomatosos/metabolismo , Pólipos Adenomatosos/patologia , Animais , Azoximetano , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Pólipos do Colo/induzido quimicamente , Pólipos do Colo/metabolismo , Pólipos do Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes APC , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estadiamento de Neoplasias , Neovascularização Patológica , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Fenótipo , Tenascina/genética , Tenascina/metabolismo , Microambiente Tumoral , beta Catenina/metabolismoRESUMO
Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.
Assuntos
Capilares/crescimento & desenvolvimento , Inibição de Contato , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Morfogênese , Receptores Acoplados a Proteínas G/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Capilares/citologia , Capilares/enzimologia , Proliferação de Células , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Solubilidade , Regulação para CimaRESUMO
The sodium iodide symporter (NIS) mediates iodide uptake into the thyroid. Because of this mechanism, differentiated thyroid cancer is susceptible for radioiodine therapy. Functional NIS expression in extrathyroidal tumors has been reported mainly in breast cancer. We screened colorectal tumors for NIS expression and investigated the mechanisms regulating NIS activity. Cell lines were screened for iodide uptake in vitro and NIS expression was evaluated by real-time RT-PCR, immunocytochemistry and immunoblotting. Iodide and pertechnetate uptake were evaluated in allograft tumors by biodistribution studies and scintigraphy. Tumors of transgenic mouse models for colorectal cancer harboring mutations in the oncogenes KRAS, beta-catenin or the tumor-suppressor gene adenomatous-polyposis coli (APC) were screened for NIS expression by RT-PCR. In vitro, functional NIS activity was detected in murine CMT93 rectal carcinoma cells and NIS expression was verified on mRNA and protein level. Inhibition of tyrosine kinases increased iodide uptake. Inhibition of tyrosine phosphatases decreased iodide uptake. In vivo, functional NIS expression was preserved in CMT93 tumors and tumor uptake could be enhanced by treatment of mice with tyrosine kinase inhibitors. In transgenic murine models of colorectal cancer, 14% of endogenous tumors expressed elevated levels of NIS mRNA. We conclude that NIS is functionally expressed in a subset of murine colorectal tumors and its activity is regulated by tyrosine phosphorylation. Therefore, with specific tyrosine kinase inhibition, these tumors might be susceptible for radioiodine treatment. Further studies are justified to identify the specific pathways regulating NIS activity and to transfer these findings to human cell lines and tissues.
Assuntos
Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Iodetos/metabolismo , Simportadores/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Genes APC/fisiologia , Humanos , Técnicas Imunoenzimáticas , Radioisótopos do Iodo/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores/genética , Distribuição Tecidual , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Osteopontin (OPN) is a secreted phosphoprotein, which has been reported to be associated with tumor progression in numerous solid tumors. In a previous transcriptome study on colorectal cancer, we identified the gene OPN among the most strongly up-regulated transcripts. OPN has been suggested as a putative target of Wnt signaling, but the molecular mechanism responsible for its aberrant transcription is not fully understood. We analyzed 13 normal colon tissues, 9 adenomas, 120 primary colon tumors, and 10 liver metastases by quantitative reverse-transcription PCR. OPN expression was strongly elevated in primary colon cancer and liver metastasis, but not in pre-cancerous lesions and UICC stage I tumors. Multivariate analysis established OPN expression as an independent prognostic parameter for overall survival. Moreover, high OPN expression identified a subgroup of patients with bad prognosis. Next, we determined immunohistochemically a correlation of OPN expression with aberrant beta-catenin staining, which is indicative of Wnt activation. Elevated expression of OPN was significantly correlated with increased cytoplasmic and nuclear beta-catenin staining. The in vivo role of Wnt signaling for the expression of OPN was tested in genetically defined mouse models with (Apc(1638N)) or without (pvillin-KRAS(V12G)) Wnt activating mutations. Mutation of the tumor suppressor APC was necessary for upregulation of OPN expression in the murine tumors on transcript and on protein levels. Thus, OPN is a transcriptional target of aberrant Wnt signaling, and OPN expression alone predicts survival in human colon cancer.