No abstract

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.

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.

The profile of mutations in the CFTR gene, which reflects the heterogeneity of its inhabitants, shows the complexity of the molecular diagnosis of CF mutations in most of the Latin American countries.

Interleukin-1 beta (IL-1␤) 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-B in this regulation. Initially, T84 cells were pretreated with the NF-B inhibitor pyrrolidine dithiocarbamate. Cells were then stimulated with IL-1␤, and CFTR mRNA levels were determined after 4 h by Northern blot analysis. As a result of PDTC treatment, IL-1␤ stimulation of CFTR mRNA was blocked. On the other hand, daunorubicin, an NF-B activator, increased the steady-state levels of CFTR mRNA. Furthermore, after treatment with IL-1␤ for 1 h, cytoplasmic IB␣ 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 IB␣, which prevents IB␣ phosphorylation and the subsequent nuclear translocation of NF-B. After viral transduction, the cells were stimulated with IL-1␤ for 4 h, and CFTR mRNA levels were measured by Northern blot analysis. The stimulation of CFTR, induced by IL-1␤, was also blocked in the presence of the dominant negative mutant. These results indicate that NF-B is involved in the pathway by which IL-1␤ regulates CFTR. Cystic fibrosis (CF)1 is an inherited disorder associated with severe inflammation, repeated infection, and colonization by Pseudomonas aeruginosa and Staphylococcus aureus (1). Before lung infection, an excessive release of proinflammatory cytokines and an increased number of neutrophils has been reported in the bronchoalveolar lavage fluids of CF patients (2), which also contain high levels of mucus.We have recently reported that the proinflammatory cytokine, interleukin-1 beta (IL-1␤), regulates the steady-state levels of CFTR mRNA and protein in the T84 human carcinoma cell line in a biphasic way: stimulatory at 0.5 ng/ml and inhibitory at higher doses (3). However, the mechanism involved in this response of CFTR to IL-1␤ is essentially unknown. In most cells, IL-1␤ causes translocation of the transcription factor NF-B into the nucleus (4). This transcription factor is a heterodimer (composed most commonly of p65/p50), the constituents of which are members of the Rel family of transcription factors (5). NF-B resides in the cytoplasm as an inactive complex, bound to the endogenous cytoplasmic inhibitors known as IBs. The best characterized and most extensively studied NF-B inhibitor is IB␣. In response to extracellular stimuli, such as IL-1␤, the IB␣ protein is rapidly phosphorylated by the IB kinase complex and targeted for proteolysis (6). This exposes the nuclear localization sequence of NF-B and facilitates its translocation into the nucleus with subsequent initiation of gene transcription (4, 5). It has been established that IB kinase phosphorylates IB␣ on serine residues 32 and 36 and that this phosphorylation is the prerequisite for ubiquitination and proteosome-dependent degradation of IB␣. This, in turn, causes the liberation of NF-B ...