Targeting transcription factor activity as a strategy to inhibit pro-inflammatory genes involved in cystic fibrosis: decoy oligonucleotides and low-molecular weight compounds.

The development of drugs able to inhibit the expression of pro-inflammatory genes is of great interest in the treatment of cystic fibrosis (CF). Chronic pulmonary inflammation in the lungs of patients affected by CF is characterized by massive intra-bronchial infiltrates of neutrophils. This process is initiated upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells. Consequently, they release cytokines, the most represented being the potent neutrophilic chemokine Interleukin (IL)-8 and the pro-inflammatory cytokine IL-6. The chronic inflammatory process is crucial, since it leads to progressive tissue damage and severe respiratory insufficiency. In order to reduce the adverse effects of the excessive inflammatory response, one of the approaches leading to inhibition of IL-8 and IL-6 gene expression is the transcription factor (TF) decoy approach, based on intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of TFs and causing inhibition of binding of TF-related proteins to regulatory sequences identified in the promoters of specific genes. Since the promoters of IL-8 and IL-6 contain consensus sequences for NF-κ B and Sp1, double stranded TF "decoy" ODNs targeting NF-κB and Sp1 can be used. Alternatively, screening of drugs targeting relevant TFs can be performed using drug cocktails constituted by extracts from medicinal plants inhibiting TF/DNA interactions. Finally, virtual screening might lead to identification of putative bioactive molecules to be validated using molecular and cellular approaches. By these means, low-molecular drugs targeting NF-κB and inhibiting IL-8 gene expression are available for pre-clinical testing using experimental systems recapitulating chronic pulmonary inflammation of patients affected by CF.

Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5.

Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by alpha(v) integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382-390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 microg/ml) and sCAR-D1 (200 microg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [(3)H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm(2)) with respect to that of cells grown to confluence (200 to 300,000 cells/cm(2)), in parallel with increased expression of HS GAGs. [(3)H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [(3)H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.

Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions.

Gene therapy vectors derived from subgroup C adenoviruses of the serotype 5 (Ad5) and 2 (Ad2) resulted in inefficient infection of well differentiated respiratory cells, both in vitro and in vivo. The level of expression and localization of the primary receptor for Ad5 and Ad2, termed CAR, do not completely explain why the infection efficiency varies greatly in different experimental conditions. The possibility that additional receptors like proteoglycans are involved in the infection of Ad5 and Ad2 was investigated, because several pathogenic microorganisms use heparan sulfate-glycosaminoglycans (HS-GAGs) as coreceptors for multistep attachment to target cells. The HS-GAG analog heparin decreased Ad5- and Ad2-mediated infection and binding starting from the concentration of 0.1 microgram/ml, up to a maximum of 50%. A similar reduction in Ad5 binding and infection was obtained by treatment of cells with heparin lyases I, II, and III but not with chondroitin ABC lyase. The effect of heparin on Ad5 binding has not been observed in surface GAG-defective Raji cells and after treating A549 cells with heparin lyases I, II,and III. The binding of Ad5 was completely abolished when both CAR was blocked with RmcB antibody and HS-GAGs were competitively inhibited by heparin. Parallel experiments demonstrate that HS-GAGs are irrelevant to binding and infection of the subgroup B adenovirus type 3. Collectively, these results demonstrate for the first time that HS-GAGs expressed on the cell surface are involved in the binding of Ad5 and Ad2 to host cells.

Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene.

The regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator (CFTR) is phosphorylated by protein kinase A and protein kinase C (PKC) in vivo (Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267, 12742-12752), but so far the functional effect of the PKC-dependent phosphorylation has not been clarified. We investigated the effect of PKC on the CFTR-mediated Cl- transport by treating with phorbol 12-myristate 13-acetate (PMA), the cell line C127i stably expressing CFTR wild type (C127 CFTRw/t), or CFTR bearing the most common mutation deltaF508 (C127 CFTRdF508). We show that PMA activates Cl- efflux in C127 CFTRw/t, but not in C127 CFTRdF508 and C127i. The PMA-dependent activation of CFTR is not mediated by increase of intracellular [cAMP] and is not the result of a primary activation of a K+ conductive pathway. These results strongly suggest that PKC activates directly CFTR-mediated Cl- transport.

Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells.

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.

Prenatal diagnosis of cystic fibrosis. Analytical evaluation of microvillar enzyme determinations in amniotic fluid.

The activity of gamma-glutamyltranspeptidase and total alkaline phosphatase and its isoenzymes has been determined in 261 amniotic fluid samples taken from pregnant women with known normal outcome and in 30 amniotic fluid samples from pregnant women with a 1:4 risk for cystic fibrosis (CF). Preliminarily, 114 amniotic fluid samples were assayed in parallel in three different laboratories, and a good correlation was found even though different assays were used. From the results obtained in control amniotic fluids, normal range and CF-predictive cutoff values were established. No false-negative results were found in this study. Among the predicted affected pregnancies 7 were terminated, and 3 went to term: 1 resulting in a CF-affected child and the other 2 in healthy children. CF was confirmed in all the aborted fetuses. In 1 case the results were inconclusive. In this study numerical results obtained for samples with a 1:4 risk of CF analyzed in the three laboratories were always virtually identical.

Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis.

Previous evidence suggests that the molecular defect in cystic fibrosis (CF) could reside in an altered chloride conductance of epithelial tissues. Since the brush border of the syncytiotrophoblast of the chorionic villi of human placenta is an abundant source of epithelial membranes and it is unaltered by secondary pathology or treatment we chose to characterize its chloride conductance and to compare it in normal and CF membranes. Chloride transport was studied in microvillar vesicles (MVV) by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Chloride conductance at 23 degrees C: (a) increased by 39% under a membrane potential change of 70 mV; (b) was inhibited by diphenylamine 2-carboxylate (Ki = 150 microM); (c) displayed an activation energy of 3.5 kcal.mol-1. The comparison of the chloride conductance for an inwardly directed gradient of 150 mM Cl- at 23 degrees C (membrane potential set at 0 mV) between CF and control membranes was not significantly different. These findings demonstrate the presence of a chloride conductive pathway in microvillar vesicles from human placenta and preliminary results exclude major differences in the conductance of CF derived material in the absence of neurohormonal stimuli.

The Km of NADH dehydrogenase is decreased in mitochondria of cystic fibrosis cells.

The kinetic properties of the NADH dehydrogenase of the mitochondrial respiratory chain, assayed as NADH-dependent rotenone-sensitive cytochrome c reductase have been studied in mitochondria isolated from mononuclear white blood cells in patients affected by cystic fibrosis. Data reported here show that the apparent Km of the enzyme for NADH is significantly decreased in cystic fibrosis mitochondria. These findings are independent of the age or the clinical state of the disease and have also been obtained with mitochondria isolated from cultured skin fibroblasts. These observations support the notion that cystic fibrosis is possibly accompanied by alterations of intracellular membranes and these are evident also in circulating cells and cultured fibroblasts.