RESUMO
Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.
Assuntos
Colo/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Interleucina-1/farmacologia , Regulação para Cima/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Adenocarcinoma/patologia , Benzoquinonas , Linhagem Celular , Colo/metabolismo , Neoplasias do Colo/patologia , Cicloeximida/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Humanos , Indóis/farmacologia , Lactamas Macrocíclicas , Maleimidas/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Quinonas/farmacologia , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Rifabutina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
12-o-Tetradecanoylphorbol-13-Acetate (TPA) down-regulates the expression of the gene responsible for cystic fibrosis (CFTR). To understand the mechanism by which TPA down-regulates CFTR, we decided to study genes specifically induced by this phorbol ester in T84 human colon carcinoma cells, which highly express CFTR, using differential display. Several strategies that allowed us to overcome false-positive reactions in differential displays are described. We have detected different cDNAs obtained from mRNAs specifically induced by TPA. A cDNA fragment corresponding to a mRNA of approximately 2.2 kb was sequenced. Part of this sequence has been reported by others in GenBank and corresponds to a cDNA from a human lung library. The function is unknown and does not have any significant homology with other sequences. It is expressed after two hrs in T84 cells treated with TPA, reaching a maximum response by four hrs. The dose-response curve shows increased mRNA levels starting at 10 ng/ml of TPA and reaching a maximum by 50 ng/ml TPA (10-fold stimulation over control). This mRNA shows a rapid and large response to TPA and it might be involved in the differentiation of T84 cells induced by TPA.
