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1.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924755

RESUMO

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/ß, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.


Assuntos
Caspase 12/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo/enzimologia , Neoplasias Nasofaríngeas/enzimologia , Linhagem Celular Tumoral , Ativação Enzimática , Humanos
2.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445676

RESUMO

The Yes-associated protein (YAP) is a transcriptional co-activator that plays critical roles in organ development and tumorigenesis, and is verified to be inhibited by the Hippo signaling pathway. In the present study, we show that the YAP 3'UTR is alternatively spliced to generate a novel 950 bp 3'UTR mRNA from the full length 3'UTR region (3483 bp) in human cancer cells. The ratio of full length 3'UTR YAP mRNA to alternatively spliced 3'UTR YAP mRNA is up-regulated by exposure of the cells to PKC inhibitor chelerythrine chloride. Further study using luciferase reporter assay showed that the expression of the alternatively spliced 3'UTR mRNA is much lower compared with the full length 3'UTR mRNA, suggesting that alternatively spliced 3'UTR YAP mRNA may have a shorter half-life than full length 3'UTR mRNA. Interestingly, PKC represses YAP 3'UTR-mediated mRNA stability is dependent on a splicing factor, hnRNP F. Activation of PKC induces nuclear translocation of cytosolic hnRNP F. Ectopic expression of hnRNP F enhances YAP 3'UTR splicing. Our results suggest that hnRNP F regulates YAP 3'UTR-mediated mRNA stability in an alternative splicing-dependent manner, and PKC regulated YAP expression is dependent on nuclear translocation of hnRNP F in human cancer cell lines.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Processamento Alternativo/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína Quinase C/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Citosol/metabolismo , Células Hep G2 , Humanos , Células PC-3 , Precursores de RNA/genética , Estabilidade de RNA/genética , Transdução de Sinais/genética , Ativação Transcricional/genética , Regulação para Cima/genética , Proteínas de Sinalização YAP
3.
Int J Mol Sci ; 23(1)2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-35008480

RESUMO

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.


Assuntos
Carcinoma Embrionário/genética , Células-Tronco de Carcinoma Embrionário/fisiologia , MicroRNAs/genética , Proteína Homeobox Nanog/genética , Interferência de RNA/fisiologia , Transcrição Gênica/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células-Tronco Embrionárias/fisiologia , Humanos , Mutação/genética , Regulação para Cima/genética
4.
Biochim Biophys Acta Gene Regul Mech ; 1862(1): 12-24, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30312683

RESUMO

The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/fisiologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/fisiologia , Humanos , Células PC-3 , Fosfoproteínas/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Fatores de Transcrição , Proteínas de Sinalização YAP
5.
Cell Death Dis ; 9(4): 414, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29549325

RESUMO

The overexpression of lumican has been found in lung cancer cells; however, the functional role of lumican in lung cancer cells remains unclear. In this study, we found lumican functioned as a tubulin-binding protein and the depletion of lumican by transfection with its specific shRNA increased lung cancer cell invasion. Such alterations led to morphological changes and actin cytoskeleton remodeling, including the induction of membrane ruffling or protrusion and stress fiber formation, correlated with the increased activities of Rac and Rho. The downregulation of lumican was also implicated in macrophage-conditioned media (maCM)-induced cell invasion. Immunofluorescence images and immunoprecipitation assays revealed the co-localization of p120-catenin (p120ctn) and lumican. Reduction in the levels of p120ctn induced membrane ruffling and the activation of the Rho family, which accelerated cell invasion. Our data indicated that lumican is associated with microtubule-modulated p120ctn signaling, providing important insights into lung cancer progression.


Assuntos
Cateninas/metabolismo , Lumicana/metabolismo , Citoesqueleto de Actina , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo , Humanos , Lumicana/antagonistas & inibidores , Lumicana/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , delta Catenina
6.
Oncotarget ; 8(20): 33515-33526, 2017 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-28380444

RESUMO

Caspase-12 (Casp12), an inflammatory caspase, functions as a dominant-negative regulator of inflammatory responses and is associated with the signaling of apoptosis. However, the physiological function of Casp12 presented in cancer cells is still unclear. This study demonstrated that overexpression of Casp12 mediated IκBα degradation and significantly increased NF-κB activity. Exposure of human nasopharyngeal carcinoma (NPC) cells to phorbol-12-myristate-13-acetate (PMA) increased the levels of Casp12 and MMP-9 resulting in NPC cell invasion. Target suppression of Casp12 by small interfering RNA (siRNA) or an inhibitor of Casp12 markedly decreased the level of PMA-induced MMP-9 protein and cell invasion. Moreover, suppression of Casp12 significantly inhibited the basal activity of NF-κB and decreased the PMA-induced NF-κB reporter activity. The effect of Casp12 on NF-κB activation was indicated via the post-translational degradation of IκB. This study revealed that a critical role of Casp12 on the activation of NF-κB via IκBα degradation which provides a link between inflammatory and aggressive invasion in NPC cells.


Assuntos
Carcinoma/genética , Carcinoma/metabolismo , Caspase 12/metabolismo , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/genética , Inibidor de NF-kappaB alfa/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Carcinoma/patologia , Caspase 12/genética , Linhagem Celular Tumoral , Movimento Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas I-kappa B/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Proteólise , Interferência de RNA
7.
BMC Cancer ; 16(1): 800, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27737648

RESUMO

BACKGROUND: N-cadherin is a trans-membrane adhesion molecule associated with advanced carcinoma progression and poor prognosis. The effect of N-cadherin on matrix metalloproteinase 9 (MMP-9) regulation is implicated in human nasopharyngeal carcinoma (NPC) cell invasion. METHODS AND RESULTS: Exposure of NPC cells to phorbol-12-myristate-13-acetate (PMA) or macrophage conditioned media (CM) upregulated MMP-9 and N-cadherin cleavage, which resulted in NPC cell invasion. MMP-9 cleaved the extracellular domain of N-cadherin, which was further cleaved by γ-secretase with PMA or macrophage-CM treatment. The extracellular cleavage of N-cadherin was inhibited with treatment with an MMP inhibitor and MMP-9 siRNA, whereas the intracellular cleavage of N-cadherin was inhibited by treatment with a γ-secretase inhibitor (γI), which resulted in enhanced accumulation of N-cadherin C-terminal fragment (CTF1, ~40 kDa). CTF2/N-cad (CTF2), a product of the γ-secretase cleavage of N-cadherin, was released and translocated into the nuclear compartment in PMA-treated cells. Moreover, CTF2 enhanced the effect of PMA-mediated MMP-9 gene expression as assessed by treatment with γI or overexpression with exogenous CTF2. Additionally, siRNA silencing of N-cadherin decreased PMA-mediated MMP-9 expression and cell invasion. The outside-in signaling effect of MMP-9 in macrophage CM- or PMA-treated cell cultures significantly enhanced NPC cell invasion via N-cadherin cleavage. CONCLUSION: Extracellular and intracellular cleavage of N-cadherin might be involved in elevated MMP-9 expression enhancing tumor cell invasion. Furthermore, N-cadherin-affected tumor progression might be via enhanced MMP-9 signaling in a cross-talk regulatory mechanism. N-cadherin might contribute to the invasive characteristics of carcinoma cells by upregulating MMP-9, thereby leading to increased aggressive metastasis.


Assuntos
Caderinas/metabolismo , Carcinoma/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular , Expressão Gênica , Humanos , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Invasividade Neoplásica , Ligação Proteica , Transporte Proteico , Proteólise , Transdução de Sinais
8.
Acta Biomater ; 31: 144-155, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26612415

RESUMO

The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin ß1, and integrin-linked kinase (ILK). Nuclear ß-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear ß-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin ß1/ILK, which indirectly activated Wnt/ß-catenin and subsequently deltaNp63α. Crosstalk between the integrin ß1/ILK and Wnt/ß-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. STATEMENT OF SIGNIFICANCE: We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin ß1 to the Wnt/ß-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin ß1, followed by activation of the ILK/Wnt/ß-catenin/p63 cascade.


Assuntos
Âmnio/metabolismo , Carbodi-Imidas/química , Células Epiteliais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Reagentes de Ligações Cruzadas/química , Regulação da Expressão Gênica , Humanos , Camundongos , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Células NIH 3T3 , Medicina Regenerativa/métodos
9.
Carcinogenesis ; 34(7): 1497-509, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23536578

RESUMO

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Carcinoma , Proliferação de Células , Progressão da Doença , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Células Hep G2 , Proteínas de Homeodomínio/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Mutagênese Sítio-Dirigida , Proteína Homeobox Nanog , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/enzimologia , Neoplasias Nasofaríngeas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica
10.
Invest Ophthalmol Vis Sci ; 52(7): 4685-93, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21447682

RESUMO

PURPOSE: To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. METHODS: siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin ß1, and involucrin. RESULTS: Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin ß1, and promotes the expression of involucrin. CONCLUSIONS: STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.


Assuntos
Diferenciação Celular/genética , DNA/genética , Epitélio Corneano/citologia , Regulação da Expressão Gênica , Limbo da Córnea/citologia , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Epitélio Corneano/metabolismo , Immunoblotting , Limbo da Córnea/metabolismo , Reação em Cadeia da Polimerase , Coelhos , Fator de Transcrição STAT3/biossíntese , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor/biossíntese
11.
J Cell Physiol ; 219(1): 117-22, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19089994

RESUMO

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The DeltaNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. DeltaNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of DeltaNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a DeltaNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of DeltaNp63 in cell proliferation and cell-cycle regulation. We found that the proliferation of NPC-076 cell is greatly suppressed when the overexpressed DeltaNp63 is silenced by specific DeltaNp63 siRNA. Further studies show that DeltaNp63 silencing results in the upregulation of CKIs, including p27(kip1) and p57(kip2) in both mRNA and protein levels. Cell-cycle analysis shows that DeltaNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that DeltaNp63 plays important roles in the regulation of NPC-076 cell-cycle progression, and may play a role in the maintenance of NPC-076 tumor cell phenotype.


Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27 , Inibidor de Quinase Dependente de Ciclina p57/genética , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Nasofaríngeas/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
12.
J Cell Biochem ; 105(2): 447-53, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18615589

RESUMO

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.


Assuntos
Quinase 3 da Glicogênio Sintase/fisiologia , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Transdução de Sinais , Transativadores/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genética , beta Catenina/metabolismo , Linhagem Celular , Glicogênio Sintase Quinase 3 beta , Humanos , Cloreto de Lítio/farmacologia , Regiões Promotoras Genéticas , Elementos de Resposta , Fatores de Transcrição , Ativação Transcricional
13.
J Biol Chem ; 283(12): 7328-37, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18198175

RESUMO

The N terminus-truncated splicing variant of TAp63 is known as DeltaNp63. DeltaNp63 lacks transactivation function and is thought to antagonize the transcriptional regulation of the p53 and TAp63 target genes. Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role in carcinogenesis. In the present study we present data showing that the DeltaNp63 gene promoter activity is positively regulated by DeltaNp63alpha, and such positive autoregulation is mediated via activation of STAT3 activity. We show that expression of DeltaNp63alpha in Hep3B cells induces Stat3 phosphorylation on Tyr-705 and Ser-727. A putative STAT3-responsive element (STAT3-RE) is identified in the DeltaNp63 promoter region. Electrophoretic mobility shift and avidin biotin-Conjugated DNA assays show direct binding of STAT3 to STAT3-RE of the DeltaNp63 promoter, and such binding is stimulated by DeltaNp63alpha. Binding of the endogenous STAT3 to the DeltaNp63 promoter in Hep3B cells was demonstrated by a chromatin immunoprecipitation assay. The stimulation of the DeltaNp63 transcriptional activity by DeltaNp63alpha is abolished by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490, dominant-negative STAT3, STAT3 small interfering RNA, and deletion of the STAT3-RE sequence from DeltaNp63 promoter. Taken together these observations clearly indicated that autoregulation of DeltaNp63 gene transcription is mediated through activation of STAT3 and its subsequent binding to the STAT3RE. Because the activation of STAT3 by interleukin-6 also leads to DeltaNp63 up-regulation and the blockade of DeltaNp63 or STAT3 expression by siRNA leads to repression of the cell growth, the identified regulatory pathway is presumably of cell physiological significance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Elementos de Resposta , Fator de Transcrição STAT3/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-6/farmacologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Elementos de Resposta/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transativadores/genética , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética , Tirfostinas/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
14.
Biochem Biophys Res Commun ; 342(4): 1356-60, 2006 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-16516862

RESUMO

p63 splicing variants lacking NH(2)-terminal transactivating domain, known as DeltaNp63, are thought to antagonize p53 and p63 functions and are suggested to play roles in keratinocyte differentiation. Here, we show that DeltaNp63 has a dual-regulatory effect on the activity of its own promoter in NPC-076 cell. Down-regulation of the transcriptional activity is observed when DeltaNp63 is present in low levels. In contrast, up-regulation of DeltaNp63 transcriptional activity is observed when DeltaNp63 is expressed at higher levels. The down-regulation effect is abolished when the p53-binding site of the DeltaNp63 promoter is mutated. In sharp contrast, similar mutation does not affect the up-regulation of the DeltaNp63 transcriptional activity under the same experimental conditions. Further study shows that the up-regulation is correlated with the activation of the STAT3, as the blockade of STAT3 nuclear translocation abolishes the up-regulation by DeltaNp63. Thus, DeltaNp63 exerts a bidirectional regulation of the DeltaNp63 transcriptional activity in NPC-076 cell.


Assuntos
Carcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição STAT3/genética , Transativadores/genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Humanos , Neoplasias Nasofaríngeas , Fatores de Transcrição , Proteínas Supressoras de Tumor
15.
J Biomed Sci ; 11(6): 773-80, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15591774

RESUMO

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IkappaB-alpha-NF-kappaB-TNF-alpha (tumor necrosis factor-alpha) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-alpha expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-alpha protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IkappaB-alpha phosphorylation and NF-kappaB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-kappaB activation and subsequently reduced TNF-alpha expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.


Assuntos
Regulação para Baixo , Isquemia , Músculos/patologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Capilares , Movimento Celular , Proteínas I-kappa B/metabolismo , Imuno-Histoquímica , Inflamação , Precondicionamento Isquêmico , Leucócitos/metabolismo , Macrófagos , Microcirculação , Microscopia de Vídeo , Músculos/metabolismo , Miocárdio/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Br J Pharmacol ; 135(7): 1627-33, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11934802

RESUMO

1. Dietary antioxidants are thought to be beneficial in reducing the incidence of coronary heart disease. In this study, we compared resveratrol and analogues on their antioxidation and free radical scavenging activities to their protective effects on ischaemia-reperfusion induced injuries of rat hearts. 2. Astringinin (3,3',4',5-tetrahydroxystilbene) was shown to be a more potent inhibitor than other analogues against Cu(2+)-induced LDL (low-density lipoprotein) oxidation, as measured by the formation of conjugated diene and TBARS (thiobarbituric acid-reactive substance) and by the electrophoretic mobility of the oxidized LDL. 3. Resveratrol (trans-3,4',5-trihydroxystilbene) and astringinin scavenged the stable free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) with an IC(0.200) of 7.1 and 4.3 microM, respectively. 4. Astringinin has a superoxide anion scavenging activity about 160 fold more potent than resveratrol. 5. After a 30 min global ischemia followed by 2 h reperfusion, astringinin (10 microM) significantly reduced infarct size, superoxide anion production and increased functional recovery of the coronary flow in Langendorff-perfused rat hearts. 6. The result showed there is a positive correlation between the anti-oxidation and cardioprotective activities among these phenolic compounds. Our finding together with the fact that astringinin is more water-soluble than resveratrol suggest that astringinin could potentially be used as an anti-oxidant and cardioprotective agent in biological systems.


Assuntos
Isquemia Miocárdica/prevenção & controle , Substâncias Protetoras/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Estilbenos/uso terapêutico , Animais , Compostos de Bifenilo , Modelos Animais de Doenças , Sequestradores de Radicais Livres/farmacologia , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Isquemia Miocárdica/metabolismo , Oxirredução , Picratos/farmacologia , Ratos , Ratos Endogâmicos WKY , Traumatismo por Reperfusão/metabolismo , Resveratrol , Superóxidos/metabolismo
17.
In Vitro Cell Dev Biol Anim ; 38(6): 320-5, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12513119

RESUMO

Oxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Proteínas ras/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Humanos , Mitógenos/farmacologia , Proteínas ras/fisiologia
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