Chantal Massé

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

Phloretin inhibits Na+ and K+ uptake in cultured alveolar type II cells by reduction of cellular ATP content.

A number of Na+ and K+ transport pathways have been identified in the alveolar epithelium and multiple inhibitors have been used to uncover these mechanisms. However, the effect of phloretin, a small dipolar nonelectrolyte compound which exerts many effects on membrane transport on Na+ and K+ uptake in alveolar epithelial cells, is not known. The purpose of this study was then to determine the impact of phloretin in Na+ and K+ uptake in cultured rat alveolar type II cells. Phloretin at a dose of 250 microM decreased Na+ uptake by 80% and K+ uptake by 90%. This decrease in Na+ and K+ uptake was not associated with a cytotoxic effect of phloretin, but this treatment did decrease ATP levels in the cells to 80% of the control cells value by 5 minutes and to 95% by 10 minutes. Our study demonstrates that phloretin is a nonspecific inhibitor of ions transport in alveolar type II cells. This inhibition is probably mediated by a reduction of intracellular ATP content.A number of Na+ and K+ transport pathways have been identified in the alveolar epithelium and multiple inhibitors have been used to uncover these mechanisms. However, the effect of phloretin, a small dipolar nonelectrolyte compound which exerts many effects on membrane transport on Na+ and K+ uptake in alveolar epithelial cells, is not known. The purpose of this study was then to determine the impact of phloretin in Na+ and K+ uptake in cultured rat alveolar type II cells. Phloretin at a dose of 250 muM decreased Na+ uptake by 80% and K+ uptake by 90%. This decrease in Na+ and K+ uptake was not associated with a cytotoxic effect of phloretin, but this treatment did decrease ATP levels in the cells to 80% of the control cells value by 5 minutes and to 95% by 10 minutes. Our study demonstrates that phloretin is a nonspecific inhibitor of ions transport in alveolar type II cells. This inhibition is probably mediated by a reduction of intracellular ATP content.

Alveolar liquid clearance in lung injury: Evaluation of the impairment of the β2-adrenergic agonist response in an ischemia-reperfusion lung injury model

While alveolar liquid clearance (ALC) mediated by the β2-adrenergic receptor (β2-AR) plays an important role in lung edema resolution in certain models of lung injury, in more severe lung injury models, this response might disappear. Indeed, we have shown that in an ischemia-reperfusion-induced lung injury model, β2-agonists do not enhance ALC. The objective of this study was to determine if downregulation of the β2-AR could explain the lack of response to β2-agonists in this lung injury model. In an in vivo canine model of lung transplantation, we observed no change in β2-AR concentration or affinity in the injured transplanted lungs compared to the native lungs. Furthermore, we could not enhance ALC in transplanted lungs with dcAMP + aminophylline, a treatment that bypasses the β2-adrenergic receptor and is known to stimulate ALC in normal lungs. However, transplantation decreased αENaC expression in the lungs by 50%. We conclude that the lack of response to β2-agonists in ischemia-reperfusion-induced lung injury is not associated with significant downregulation of the β2-adrenergic receptors but is attributable to decreased expression of the ENaC channel, which is essential for sodium transport and alveolar liquid clearance in the lung.

DNA Methylation Regulates RGS2-induced S100A12 Expression in Airway Epithelial Cells

&NA; RGS2 is a key modulator of stress in human airway epithelial cells, especially of hyperresponsiveness and mucin hypersecretion, both of which are features of cystic fibrosis (CF). Because its expression can be modulated through the DNA methylation pathway, we hypothesize that RGS2 is downregulated by DNA hypermethylation in CF airway epithelial cells. This downregulation would then lead to an enhanced inflammatory response. We demonstrated RGS2 transcript and protein downregulation in cultured airway epithelial cells from patients with CF and validated our findings in two CF epithelial cell lines. A methylated DNA immunoprecipitation array showed the presence of methylated cytosine on 13 gene promoters in CF. Among these genes, we confirmed that the RGS2 promoter was hypermethylated by using bisulfite conversion coupled with a methylation‐specific PCR assay. Finally, we showed that downregulation of RGS2 in non‐CF cells increased the expression of S100A12, a proinflammatory marker. These results highlight the importance of epigenetic regulation in gene expression in CF and show that RGS2 might modulate the inflammatory response in CF through DNA methylation control.