cDNA cloning and expression of a hamster α-thrombin receptor coupled to Ca2+ mobilization

AbstractThe serine protease α-thrombin (thrombin) potently stimulates G-protein-coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G-protein-coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C-coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N-terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N-terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion-binding exosite of thrombin

Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene

AbstractBased on the knowledge that expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be modulated at the transcriptional level, and that the CFTR gene promoter contains sequences homologous to elements in other promoters that respond to tumor necrosis factor-α (TNF), we evaluated the hypothesis that TNF might modulate CFTR gene expression in epithelial cells. Studies with HT-29 cells, a colon epithelium-derived tumor cell line known to express the CFTR gene, demonstrated that TNF downregulated CFTR mRNA transcript levels in a dose- and time-dependent fashion. Interestingly, nuclear run-on analyses demonstrated that TNF did not affect the rate of transcription of CFTR gene, but exposure of the cells to TNF did modify the stability of CFTR mRNA transcripts, resulting in a mRNA half-life that was reduced to 65% of the resting level. These observations suggest that CFTR gene expression can be modulated by TNF, at least in part, at the post-transcriptional level

Cystic Fibrosis Transmembrane Conductance Regulator Does Not Affect Neutrophil Migration across Cystic Fibrosis Airway Epithelial Monolayers

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF15 cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF15 cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the ΔF508 CFTR cDNA, or the β-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF15 monolayers expressing β-galactosidase was similar to that seen across CF15 monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF15 cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl− secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection

Regulation of Gap Junctional Communication by a Pro-Inflammatory Cytokine in Cystic Fibrosis Transmembrane Conductance Regulator-Expressing but Not Cystic Fibrosis Airway Cells

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-κB (NF-κB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-α (TNF-α). TNF-α induced maximal translocation of NF-κB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-α progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-α remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-α-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-α. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functionnal human protein variant in transgenic mouse milk

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