RESUMO
Macrophage inhibitory cytokine-1 (MIC-1) is a cytokine with pleotropic actions and its expression is markedly increased by inflammation and cardiac injury and in cancers. In particular, MIC-1 production after cardiac ischemia injury is associated with enhanced cardiac angiogenesis as well as myocardial protection. However, it remains uncertain whether MIC-1 itself has proangiogenic activity. In this study, we tried to determine the precise role of MIC-1 in physiological and pathological angiogenesis. Human microvessel endothelial cells responded to MIC-1 with enhanced angiogenic behaviors. Employing various angiogenesis assays, MIC-1 was found to promote vessel formation and development with a potency similar to that of vascular endothelial growth factor (VEGF). MIC-1 transgenic (Tg) mice also displayed enhanced neovascularization in both developing embryos and neonatal mouse retinas, compared with wild-type mice. Furthermore, endothelial cells (ECs) isolated from MIC-1 Tg mouse lung exhibited higher angiogenic potential than ECs from wild-type lung. MIC-1-induced angiogenesis was also observed in the recovery or healing processes of injuries such as hindlimb ischemia and skin wounds in mice. However, unlike VEGF, MIC-1 induced neither endothelial inflammation nor increased vascular permeability. In ECs, the MIC-1 signal exerted proangiogenic actions via the MEK/extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Notably, these MIC-1 signaling events in ECs were abrogated by small interfering RNA-mediated knockdown of GFRAL, suggesting that GFRAL is an EC receptor for MIC-1. In summary, we here show a novel role of MIC-1 as a potent EC activator, which promotes both normal and injury-related angiogenesis.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Animais , Embrião de Mamíferos/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Humanos , Inflamação/patologia , Isquemia/patologia , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/citologia , Permeabilidade , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/fisiologia , Retina/metabolismo , Pele/patologia , CicatrizaçãoRESUMO
BACKGROUND: The epithelial-to-mesenchymal transition (EMT) is closely associated with cancer invasion and metastasis. Since the transforming growth factor ß (TGF-ß) and Wnt signals induce EMT in various epithelial cell types, we examined whether and how the CD82/KAI1 metastasis suppressor affects the TGF-ß and Wnt signal-dependent EMT in human prostate cancer cells. METHODS: The invasiveness of cancer cells was evaluated by examining their ability to pass through the basement membrane matrigel. The subcellular localizations of Smad4 and ß-catenin proteins were respectively examined by confocal microscopy following immunofluorescence antibody staining and immunoblotting analysis following subcellular fractionation. The transcriptional activities of the TGF-ß1 -responsive TRE and Wnt-responsive Tcf/Lef promoters were determined by a luciferase reporter assay following transfection of the recombinant reporter vector into the cell. RESULTS: TGF-ß1 and Wnt3a treatments of human prostate cancer cells without CD82 expression resulted in not only increased invasiveness but also EMT involving the development of motile structures, downregulation of E-cadherin, and upregulation of the mesenchymal proteins. However, in the cells with high levels of CD82, the TGF-ß1 and Wnt3a stimulations neither elevated invasiveness nor induced EMT. Furthermore, the TGF-ß1 signaling events occurring in the CD82-deficient cells, such as phosphorylation of Smad2, nuclear translocation of Smad4, and transactivation of the TRE promoter, did not take place in the high CD82-expressing cells. Further, high CD82 expression interfered with the Wnt signal-dependent alterations in the phosphorylation pattern of glycogen synthase kinase 3ß (GSK-3ß) in prostate cancer cells, which allowed GSK-3ß to continue phosphorylating ß-catenin, thereby attenuating the Wnt signaling effects on the nuclear translocation of ß-catenin and subsequent transactivation of the Tcf/Lef promoter. CONCLUSIONS: The results of the present study suggest that CD82/KAI1 functions in suppressing TGF-ß1 - and Wnt-induced EMT in prostate cancer cells by inhibiting the TGF-ß1 /Smad and Wnt/ß-catenin pathways. Therefore, loss or decrease of CD82 expression is likely to render prostate cancer cells prone to respond to the TGF-ß1 and Wnt signals with EMT, resulting in the development of a motile and invasive mesenchymal phenotype related to the initiation of the metastatic cascade.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Proteína Kangai-1/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Próstata/metabolismo , Proteína Smad2/metabolismo , Via de Sinalização WntRESUMO
Solution processed barium titanate (BTO) was used to fabricate an Al/BaTiO3/p-Si metal-insulator-semiconductor (MIS) structure, which was used as a gate insulator. Changes in the electrical characteristics of the film were investigated as a function of the film thickness and post deposition annealing conditions. Our results showed that a thickness of 5 layers and an annealing temperature of 650 °C produced the highest electrical performance. BaxTi1-xO3 was altered at x = 0.10, 0.30, 0.50, 0.70, 0.90, and 1.0 to investigate changes in the electrical properties as a function of composition. The highest dielectric constant of 87 was obtained for x = 0.10, while the leakage current density was suppressed as Ba content increased. The lowest leakage current density was 1.34×10-10 A/cm2, which was observed at x = 0.90. The leakage current was related to the resistivity of the film, the interface states, and grain densification. Space charge limited current (SCLC) was the dominant leakage mechanism in BTO films based on leakage current analysis. Although a Ba content of x = 0.90 had the highest trap density, the traps were mainly composed of Ti-vacancies, which acted as strong electron traps and affected the film resistivity. A secondary phase, Ba2TiO4, which was observed in cases of excess Ba, acted as a grain refiner and provided faster densification of the film during the thermal process. The absence of a secondary phase in BaO (x = 1.0) led to the formation of many interface states and degradation in the electrical properties. Overall, the insulator properties of BTO were improved when the composition ratio was x = 0.90.
RESUMO
BACKGROUND: Repression of the KAI1 metastasis suppressor gene is closely associated with malignancy and poor prognosis in many human cancer types including prostate cancer. Since gene repression in human cancers frequently results from epigenetic alterations by DNA methylation and histone modifications, we examined whether the KAI1 gene becomes silenced through these epigenetic mechanisms in prostate cancer. METHODS: KAI1 mRNA and protein levels were determined by RT-PCR and immunoblotting analyses, respectively. Methylation status of the KAI1 promoter DNA in prostate cancer cell lines and tissues was evaluated by methylation-specific PCR analysis of bisulfite-modified genomic DNAs. Methylated CpG sites in the KAI1 promoter were identified by sequencing the PCR clones of the bisulfite-modified KAI1 promoter DNA. KAI1 protein levels in human prostate cancer tissue samples were examined by immunofluorescence staining of the tissues with an anti-KAI1 antibody. RESULTS: Among the three human prostate cancer cell lines examined, PC3 and DU145 cells exhibited markedly decreased levels of KAI1 mRNA and protein as compared to LNCaP cells, even though the exogenous KAI1 promoter not being methylated was normally functional in all these cell lines. Treatment of the low KAI1-expressing cell lines with a demethylating agent, 5'-aza-2'-deoxycytidine, significantly elevated KAI1 expression levels, implicating the involvement of DNA methylation in KAI1 downregulation. Methylation of CpG islands within the KAI1 promoter region was observed in the low KAI1-expressing cells, but not in the high KAI1-expressing cells. Also, methyl CpG-binding proteins such as MBD2 and MeCP2 were complexed to the KAI1 promoter in the low KAI1-expressing cells. Bisulfite sequencing analysis identified the intensively methylated CpG residues in the KAI1 promoter clones derived from prostate cancer cells and tissues with no or low KAI1 expression. As in prostate cancer cell lines, prostate cancer tissues from patients also displayed a negative association between KAI1 expression levels and methylation status of the KAI1 promoter. CONCLUSIONS: The present data suggest that the KAI1 gene might be repressed by epigenetic alterations through the promoter CpG-site methylation during prostate cancer progression. This epigenetic mechanism could provide a clue for understanding how the KAI1 gene was silenced in metastatic prostate cancers. Prostate 77: 350-360, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Ilhas de CpG/fisiologia , Repressão Epigenética/fisiologia , Genes Supressores de Tumor/fisiologia , Proteína Kangai-1/metabolismo , Regiões Promotoras Genéticas/fisiologia , Neoplasias da Próstata/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Proteína Kangai-1/genética , Masculino , Neoplasias da Próstata/genéticaRESUMO
Despite the importance of multiple tetraspanin proteins in cancer invasion and metastasis, little is known about the role and significance of tetraspanin CD81 in these processes. In the present study, we examined CD81 effects on melanoma cell invasiveness and metastasis. Transfection of CD81 into melanoma cells lacking endogenous CD81 expression significantly enhanced the migrating, invasive, and metastatic abilities of melanoma cells. Interestingly, membrane type 1 matrix metalloproteinase (MT1-MMP) expression was found in CD81-expressing melanoma cells but not in CD81-deficient cells. siRNA knockdown of CD81 in melanoma cells with endogenous CD81 demonstrated decreased MT1-MMP levels and cell motility. Notably, CD81-induced cell migration was abrogated by antibody blocking and siRNA knockdown of MT1-MMP, indicating that MT1-MMP is responsible for CD81-stimulated melanoma cell migration. Promoter analysis revealed an essential role of the Sp1 transcription factor in CD81-induced MT1-MMP transcription. We also demonstrate that the Sp1-activating Akt pathway is involved in adhesion-dependent CD81 signaling to induce MT1-MMP expression and cell motility. Importantly, human skin cancer tissue specimens displayed a positive correlation of CD81 with MT1-MMP expression levels and a close association of CD81 with malignant melanomas. Taken together, these results strongly suggest that CD81 stimulates melanoma cell motility by inducing MT1-MMP expression through the Akt-dependent Sp1 activation signaling pathway, leading to increased melanoma invasion and metastasis.
Assuntos
Metaloproteinase 14 da Matriz/genética , Melanoma/enzimologia , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/enzimologia , Tetraspanina 28/metabolismo , Carcinoma Basocelular/enzimologia , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Melanoma/secundário , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Neoplasias Cutâneas/patologia , Fator de Transcrição Sp1/metabolismo , Tetraspanina 28/genética , Regulação para Cima/fisiologiaRESUMO
Macrophage inhibitory cytokine-1 (MIC-1) has been reported to be elevated in various human cancers including melanoma; however, the function of MIC-1 in cancer remains unclear. In this study, we attempt to clarify the role of MIC-1 in tumor pathogenesis by employing the orthotopic B16F1 melanoma mouse model in which serum MIC-1 levels are positively correlated with tumor size. By stably transfecting a MIC-1 expression construct into B16F1 melanoma cells, we increased the expression and secretion levels of MIC-1. This increase in MIC-1 expression significantly enhanced the growth of tumors derived from B16F1 cells in vivo, despite not affecting in vitro cell growth. The elevated MIC-1 expression in B16F1 cells also resulted in lymph node metastasis in B16F1 tumor-bearing mice, significantly increasing mortality. Interestingly, among small melanoma tumors of similar size, tumors derived from the MIC-1-transfected B16F1 cells exhibited enhanced blood vessel formation compared with those of mock transfectant cells. Also, more MIC-1 was found in well-vascularized tumor regions than in poorly vascularized tumor regions. Moreover, conditioned medium (CM) of the MIC-1-transfected melanoma cells enhanced the angiogenic properties of endothelial cells more than CM of mock transfectant cells. Notably, hypoxic culture conditions forced parental B16F1 cells to secrete more endothelial cell-stimulating factors, among which the function of MIC-1 was confirmed by blocking the effects with an anti-MIC-1 antibody. Taken together, these results suggest that the MIC-1 produced by melanoma cells in response to oxygen deprivation promotes tumor vascularization during melanoma development in vivo, leading to enhanced tumor growth and metastasis.
Assuntos
Fator 15 de Diferenciação de Crescimento/metabolismo , Melanoma/genética , Neoplasias Cutâneas/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Melanoma/patologia , Camundongos , Metástase Neoplásica , Neovascularização Patológica , Neoplasias Cutâneas/patologia , TransfecçãoRESUMO
In TNF signaling, ubiquitination of RIP1 functions as an early cell-death checkpoint, which prevents the spatial transition of the signaling complex from complex-I to death-inducing complex-II. Here, we report that ankyrin repeat domain 13a (ANKRD13a) acts as a novel component of complex-II to set a higher signal threshold for the cytotoxic potential of TNF. ANKRD13a deficiency is sufficient to turn the response to TNF from survival to death by promoting the formation of complex-II without affecting NF-κB activation. ANKRD13a binds to ubiquitinated-RIP1 via its UIM, and subsequently limits the association of FADD and caspase-8 with RIP1. Moreover, high ANKRD13a expression is inversely correlated with apoptotic phenotypes in ovarian cancer tissues and is associated with poor prognosis. Our work identifies ANKRD13a as a novel gatekeeper of the early cell-death checkpoint, which may function as part of an escape mechanism from cell death in some cancers.
Assuntos
Proteínas de Membrana , NF-kappa B , Complexo de Proteínas Formadoras de Poros Nucleares , Neoplasias Ovarianas , Proteínas de Ligação a RNA , Fator de Necrose Tumoral alfa , Apoptose/fisiologia , Caspase 8/metabolismo , Morte Celular/fisiologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , UbiquitinaçãoRESUMO
We have previously demonstrated that albumin is directly involved in the formation of cytoplasmic lipid droplets in pancreatic stellate cells and may act as a downstream effector of adipogenic transcription factors, PPAR-gamma and C/EBP-alpha. Here, we investigated the role of albumin in adipocyte differentiation using 3T3-L1 cells. Albumin expression was significantly increased at later stages of adipocyte differentiation, which was accompanied with increased C/EBP-beta binding to albumin promoter. Suppression of albumin expression using short-hairpin RNA (shRNA) during differentiation led to a considerable reduction in lipid droplet formation, whereas albumin overexpression was stimulatory. Furthermore, point mutation in its fatty acid-binding sites inhibited lipid droplet formation. Consistent with these in vitro finding, Nagase analbuminemic rats displayed reduced fat accumulation. Therefore, our findings suggest that albumin may play a distinct role in adipocyte differentiation by promoting lipid accumulation.
Assuntos
Adipócitos/fisiologia , Adipogenia , Albuminas/fisiologia , Metabolismo dos Lipídeos , Células 3T3-L1 , Adipócitos/citologia , Albuminas/genética , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ácidos Graxos , Regulação da Expressão Gênica , Masculino , Camundongos , Mutação Puntual , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-DawleyRESUMO
Tetraspanin membrane proteins form physical complexes with signaling molecules and have been suggested to influence the signaling events of associated molecules. Of the tetraspanin proteins, CD82 has been shown to promote homotypic cell-cell adhesion, which partially accounts for its role in suppressing cancer invasion and metastasis. We found here that CD82-induced cell-cell adhesion is attributed to increased E-cadherin expression through CD82-mediated downregulation of the E-cadherin repressor Snail. The Snail repression by CD82 resulted from the reduced binding of the Sp1 transcription factor to the Snail gene promoter. Notably, high CD82 expression did not allow the fibronectin matrix to induce Sp1 phosphorylation, implicating CD82 inhibition of the fibronectin-integrin signaling-dependent Sp1 activation. Meanwhile, E-cadherin upregulated by CD82 pulled ß-catenin up to the membrane region, and consequently reduced the amount of cytoplasmic ß-catenin that was able to move into to the nucleus. The Wnt signal-induced nuclear translocation of ß-catenin was also inhibited by the CD82 function of upregulating E-cadherin. Overall, high CD82 expression was likely to suppress fibronectin adhesion-induced Sp1 activation signaling for Snail expression, resulting in continuous E-cadherin expression, which contributed not only to the maintenance of strong cell-cell adhesion but also to the blockage of nuclear ß-catenin signaling.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Adesão Celular , Proteína Kangai-1/fisiologia , Neoplasias da Próstata/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator de Transcrição Sp1/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Masculino , Via de Sinalização WntRESUMO
The transmembrane protein CD82/KAI1 suppresses the metastatic potential of various cancer cell types. Moreover, decrease or loss of CD82 expression is closely associated with malignancy and poor prognosis in many human cancers including prostate cancer. Despite intense scrutiny, the mechanisms underlying the metastasis-suppressing role of CD82 are still not fully understood. Here, we found that a fibronectin matrix induced mesenchymal phenotypes in human prostate cancer cells with no or low CD82 expression levels. However, high CD82 expression rendered prostate cancer cells to have intensified epithelial characteristics upon fibronectin engagement, along with decreased cell motility and invasiveness. The CD82 function of inhibiting fibronectin-induced epithelial-to-mesenchymal transition (EMT) was dependent not only on CD82 interactions with fibronectin-binding α3ß1/α5ß1 integrins but also on the integrin-mediated intracellular signaling events. Notably, CD82 attenuated the FAK-Src and ILK pathways downstream of the fibronectin-receptor integrins. Immunofluorescence staining of human prostate cancer tissue specimens illustrated a negative association of CD82 with EMT-related gene expression as well as prostate malignancy. Altogether, these results suggest that CD82 suppresses EMT in prostate cancer cells adhered to the fibronectin matrix by repressing adhesion signaling through lateral interactions with the associated α3ß1 and α5ß1 integrins, leading to reduced cell migration and invasive capacities.