Tyrosine kinase c-Src constitutes a bridge between cystic fibrosis transmembrane regulator channel failure and MUC1 overexpression in cystic fibrosis

Fil: González Guerrico, Anatilde M. Instituto de Investigaciones Bioquı́micas Fundación Campomar (UBA, CONICET), 1405 Buenos Aires; Argentina.Fil: Cafferata, Eduardo. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Radrizzani, Martín. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Marcucci, Florencia. Instituto de Investigaciones Bioquı́micas Fundación Campomar (UBA, CONICET), 1405 Buenos Aires; Argentina.Fil: Gruenert, Dieter. Human Molecular Genetics Unit, Department of Medicine, University of Vermont, Burlington; Estados Unidos.Fil: Pivetta, Omar H. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Favaloro, Roberto R. Fundación Favaloro, 1093 Buenos Aires; Argentina.Fil: Laguens, Rubén. Fundación Favaloro, 1093 Buenos Aires; Argentina.Fil: Perrone, Sergio V. Fundación Favaloro, 1093 Buenos Aires; Argentina.Fil: Gallo, Guillermo C. Hospital de Pediatrı́a Prof. Dr. Juan P. Garrahan, 1425 Buenos Aires; Argentina.Fil: Santa-Coloma, Tomás A. Instituto de Investigaciones Bioquı́micas Fundación Campomar (UBA, CONICET), 1405 Buenos Aires; Argentina.Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection

Interleukin-1β regulates CFTR expression in human intestinal T84 cells

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-1β (IL-1β) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1β (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1β (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 μM) and GF109203X (1 μM) inhibited the stimulatory effect of IL-1β. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 μM) and herbymicin A (2 μM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1β. The repression of CFTR up-regulation by cycloheximide (35.5 μM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 μM), suggest that the increased mRNA levels seen after IL-1β treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1β, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.Fil: Cafferata, Eduardo Gustavo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; ArgentinaFil: González Guerrico, Anatilde M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giordano, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pivetta, Omar Hilario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; ArgentinaFil: Santa Coloma, Tomás Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

NF-κB Activation Is Involved in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Interleukin-1β

Interleukin-1 beta (IL-1b) regulates the levels of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein in the T84 human carcinoma cell line. Here, we studied the role of the transcription factor NF-kB in this regulation. Initially, T84 cells were pretreated with the NF-kB inhibitor pyrrolidine dithiocarbamate. Cells were then stimulated with IL-1b, and CFTR mRNA levels were determined after 4 h by Northern blot analysis. As a result of PDTC treatment, IL-1b stimulation of CFTR mRNA was blocked. On the other hand, daunorubicin, an NF-kB activator, increased the steady-state levels of CFTR mRNA. Furthermore, after treatment with IL-1b for 1 h, cytoplasmic IkBa degradation occurred simultaneously with translocation of p65 into the nucleus. The T84 cells were also transduced with an adenoviral vector expressing a dominant negative form of IkBa, which prevents IkBa phosphorylation and the subsequent nuclear translocation of NF-kB. After viral transduction, the cells were stimulated with IL-1b for 4 h, and CFTR mRNA levels were measured by Northern blot analysis. The stimulation of CFTR, induced by IL-1b, was also blocked in the presence of the dominant negative mutant. These results indicate that NF-kB is involved in the pathway by which IL-1b regulates CFTR.Fil: Cafferata, Eduardo Gustavo Alfredo. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: González Guerrico, Anatilde M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pivetta, Omar Hilario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán". Centro Nacional de Genética Médica; ArgentinaFil: Santa Coloma, Tomás Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin