RESUMEN
This study examined the role of the protein kinase C (PKC) signalling pathway in the regulation of expression of human complement factor I (CFI) gene. The production of CFI by Hep G2 cells was enhanced in a dose- and time-dependent fashion by 12-O-tetradecanoyl-1,2-phorbol 13-acetate (TPA), a potent PKC activator. 4Alpha-phorbol didecanoate, an inactive phorbol ester, had no effect on CFI synthesis. The TPA-dependent increase in CFI secretion was correlated with an increase in CFI mRNA levels. Forskolin, a cAMP-inducing agent, augmented the TPA response. W7, an inhibitor of protein kinase A and genistein, an inhibitor of protein tyrosine kinase(s) both did not prevent the increase in CFI expression mediated by TPA. However, calphostin C, a specific inhibitor of PKC, abolished the TPA-induced increase in CFI mRNA levels. Down regulation of intracellular PKC levels by prior exposure of Hep G2 cells to a high concentration of TPA also blocked the increase in CFI mRNA levels induced by TPA suggesting that the TPA effects were mediated via activation of PKC. mRNA decay studies indicated that the half-life of CFI mRNA in TPA-induced cells was not significantly different from control. Nuclear run-on transcriptional assays on the other hand demonstrated that whereas the CFI gene is transcribed under basal conditions in Hep G2 cells, TPA induced a 3-4 fold increase in the transcription rate of CFI gene in 24 h. The transcription rate of GAPDH gene did not change, indicating that the effects were not general on gene transcription. Transient transfections of Hep G2 cells with chloramphenicol acetyltransferase reporter gene (CAT) constructs containing a series of sequential 5' deletions of the CFI promoter and CAT assays showed that the sequence between -136 and -130, containing an AP-1 consensus sequence (TGAGTCA) was required for the TPA response. This observation was substantiated by the finding that mutation of this AP-1 site to TttaTCA or TtAtcCA abolished the TPA responsiveness. The enhancement of the activity of transfected chimeric CAT constructs by TPA was abrogated by calphostin C and by pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB and AP-1 transactivation). These results indicate that TPA regulation of CFI gene requires PKC signalling and is mediated by via a TPA response element (TRE) in the CFI promoter region located at -136/-130 and involves the transactivation of AP-1 and NF-kappaB transcription factors. We suggest that PKC may be one of the intracellular pathways that control CFI gene expression and that cellular processes (involving growth factors, hormones, cytokines etc.) that activate PKC may upregulate the expression of the CFI gene.
Asunto(s)
Carcinoma Hepatocelular/genética , Factor I de Complemento/genética , Proteína Quinasa C/metabolismo , Transcripción Genética , Carcinoma Hepatocelular/enzimología , Factor I de Complemento/efectos de los fármacos , Factor I de Complemento/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mutagénesis Sitio-Dirigida , Naftalenos/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteína Quinasa C/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Pirrolidinas/farmacología , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tiocarbamatos/farmacología , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that appear to be related to its ability to inhibit complement factor I, thereby activating the complement cascade. Using X-ray crystal structure information, we have designed compounds that have greater selectivity for thrombin over factor I and that have reduced tendency to produce these side effects.