RESUMO
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene, expression of which is frequently suppressed in human cancers. Very little is known about the molecular basis of HIC1 in antagonizing oncogenic pathways. Here, we report that HIC1 forms complexes with the signal transducers and activators of transcription 3 (STAT3) and attenuates STAT3-mediated transcription. STAT3 was identified as a HIC1-interacting protein by affinity capture and followed by mass spectrometry analysis. Overexpression or depletion of HIC1 resulted in decreased or increased levels of interleukin-6 (IL-6)/oncostatin M (OSM)-induced STAT3-mediated reporter activity and expression of target genes such as VEGF and c-Myc, respectively. Furthermore, HIC1 suppressing the VEGF and c-Myc promoter activity and the colony formation of MDA-MB 231 cells were STAT3-dependent. Further studies showed that HIC1 interacts with the DNA binding domain of STAT3 and suppresses the binding of STAT3 to its target gene promoters. Domain mapping study revealed that HIC1 C-terminal domain binds to STAT3. HIC1 mutant defective in STAT3 interaction reduced its repressive effect on STAT3 DNA binding activity, the reporter activity and gene expression of the VEGF and c-Myc genes, and cell growth in MDA-MB 231 cells. Altogether, our findings not only provide a novel role of HIC1 in antagonizing STAT3-mediated activation of VEGF and c-Myc gene expression and cell growth, but also elucidate a molecular basis underlying the inhibitory effect of HIC1 on STAT3 transcriptional potential.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Transcrição STAT3/genética , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genética , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Genes Reporter , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/genética , Dados de Sequência Molecular , Oncostatina M/genética , Oncostatina M/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The present study investigates the receptor and mechanisms involved in ATP-stimulated transforming growth factor-beta 1 (TGF-beta 1) mRNA expression of a type-2 astrocyte cell line, RBA-2. RT-PCR analysis revealed that RBA-2 type-2 astrocytes possess abundant P2X(4) and P2X(7) receptors. ATP and P2X(7) receptor-sensitive agonist, BzATP, both stimulated TGF-beta 1 mRNA expression in a time and dose-dependent manner. The stimulation required a minimum of 500 muM ATP; BzATP was much more potent that ATP, and P2X(7)-selective antagonist, oATP, inhibited the effects. In addition, ATP metabolites ADP, AMP and adenosine were ineffective in stimulation of TGF-beta 1 mRNA expression. Thus, the effect of ATP was mediated through the P2X(7) receptors. To investigate further the mechanisms by which the P2X(7) receptor mediated the TGF-beta 1 mRNA expression, the cells were treated with inhibitors for mitogen-activated kinase (MAPK) or protein kinase C (PKC), PD98059 or GF109203X, respectively. Both PD98059 and GF109203X inhibited the ATP-stimulated TGF-beta 1 mRNA expression. Furthermore, ATP and BzATP stimulated ERK1/2 activation and the activation was inhibited by PKC inhibitors, GF109203X and Gö6976. In conclusion, activation of P2X(7) receptors enhanced TGF-beta 1 mRNA expression and the effect involved PKC/MAPK signalling pathway in RBA-2 type-2 astrocytes.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Astrócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/fisiologia , Fator de Crescimento Transformador beta/genética , Adenosina/farmacologia , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Northern Blotting , Western Blotting , Encéfalo/citologia , Carbazóis/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Expressão Gênica , Indóis/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases/fisiologia , Maleimidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/genética , Ratos , Receptores Purinérgicos P2/análise , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta1RESUMO
The present study demonstrated that endotheline-1 (ET-1) stimulated a biphasic (transient and sustained) increase in [Ca(2+)](i) and signaling was blocked by BQ123 and inhibited by BQ788. RT-PCR analysis revealed that ET(A) was expressed more than ET(B) mRNA-suggesting that ET(A) is the major receptor. Simply reintroducing Ca(2+) in the buffer stimulated a sustained increase in [Ca(2+)](i) and the effect was inhibited by U73122, thapsigargin (TG), miconazole and SKF96365. When measured in Ca(2+)-free buffer, the ET-1-stimulated Ca(2+) transient decreased by 73% and the reintroduction of Ca(2+) induced a large sustained increase in [Ca(2+)](i). These effects were not affected by nifedipine, but were inhibited by miconazole and SKF96365-indicating that the sustained increase in [Ca(2+)](i) mediated by ET-1 was mostly due to capacitative Ca(2+) entry (CCE). The ET-1-induced CCE was inhibited by phorbol ester (PMA) but was enhanced by GF109203X; it was also enhanced by 8-bromo-cyclic AMP (8-Br-cAMP) but was inhibited by H89. Thus, protein kinase C (PKC) negatively regulated and cAMP-dependent protein kinase (PKA) positively regulated the ET-1-mediated CCE in these cells.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cálcio/farmacocinética , Endotelina-1/farmacologia , Receptores de Endotelina/metabolismo , Aminoácidos Cíclicos/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Astrócitos/citologia , Encéfalo/citologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Capacitância Elétrica , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Peptídeos Cíclicos/farmacologia , Proteína Quinase C/metabolismo , Ratos , Receptor de Endotelina A , Receptor de Endotelina BRESUMO
ATP is an important signaling molecule in the nervous system and it's signaling is mediated through the metabotropic P2Y and ionotropic P2X receptors. ATP is known to stimulate Ca(2+) influx and phospholipase D (PLD) activity in the type-2 astrocyte cell line, RBA-2; in this study, we show that the release of preloaded [(3)H]GABA from RBA-2 cells is mediated through the P2X(7) receptors. ATP and the ATP analogue 3'-O-(4-benoylbenoyl)-adenosine-5'-triphosphate (BzATP) both stimulated [(3)H]GABA release in a concentration dependent manner, while the nonselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the P2X(7)-sensitive antagonist oxidized ATP (oATP), and high extracellular Mg(2+) all inhibited the ATP-stimulated [(3)H]GABA release. The ATP-stimulated [(3)H]GABA release was not affected neither by removing extracellular Na(+) nor by changes in the intracellular or extracellular Ca(2+) concentration. The GABA transporter inhibitors nipecotic acid and beta-alanine also had no effect. The ATP-stimulated [(3)H]GABA release was blocked, however, when media Cl(-) was replaced with gluconate and when extracellular HCO(3)(-) was removed. The Cl(-) channel/exchanger blockers 4,4'-diisothiocyanatostilbene-2',2'-disulfonic acid (DIDS) and 4-acetamido-4'- isothiocyanatostilbene-2',2'-disulfonic acids (SITS), but not diphenylamine-2-carboxylic acid (DPC) and furosemide, blocked the ATP-stimulated [(3)H]GABA release. The anionic selectivity of the process was F(-) > Cl(-) > Br(-) which is the same as that reported for volume-sensitive Cl(-) conductance. Treating cells with phorbol-12-myristate 13-acetate (PMA), forskolin, dibutyryl-cAMP, PD98059, neomycin, and D609 all inhibited the ATP-stimulated [(3)H]GABA release. We concluded that in RBA-2 cells, ATP stimulates [(3)H]GABA release through the P2X(7) receptors via a Cl(-)/HCO(3)(-)-dependent mechanism that is regulated by PKC, PKA, MEK/ERK, and PLD.
