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Research unit

Cystic fibrosis research program

A. Epigenetic regulation and cystic fibrosis: impact of DNA methylation on gene expression
Cystic fibrosis (CF) is an autosomal recessive disease affecting respiratory, digestive and reproductive tracts. It is caused by mutations in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene, coding for the CFTR protein, an ion channel implicated in chloride secretion. Dysfunction of the CFTR channel leads to chronic respiratory infections, increased inflammatory response and neutrophil accumulation in the lungs. Over time, cystic fibrosis destroys the lung tissue, leading to respiratory insufficiency. There is increasing evidence that tends to demonstrate the involvement of epigenetic regulation mechanisms affecting gene expression profiles in pulmonary diseases like asthma, COPD and lung cancer. These mechanisms can be defined as all processes leading to reversible and transmissible modifications of gene expression, without alteration of nucleotide sequences. Studies carried out in our laboratory have shown that modulation of promoters methylation patterns may regulate the expression of genes related to the disease like RGS2 and help to explain the variations in disease severity (Figure 1). Our project currently aims to better understand the impact of these methylation changes on inflammatory response and to explain the underlying mechanisms.


B. 
Serological markers and cystic fibrosis: impact of chitinase YKL-40 level on cystic fibrosis-related diabetes
The mutations in the CFTR gene leading to cystic fibrosis also reduce the insulin secretion by beta-cells in the pancreatic islets. About 10% of patients with cystic fibrosis of all ages will develop partially insulinopenic diabetes induced by pancreatic fibrosis, and one third of patients will have glucose intolerance. Diabetes occurs in one third of patients after 20 years and in half the patients after 30 years. Several endogenous molecules trigger initiation of inflammatory response by interacting with specific receptors in order to point out dangers for the cell. A study using DNA microarrays and carried out to compare the expression of all transcripts expressed in CF and non-CF human bronchial epithelial cells highlighted overexpression, in CF cells, of a transcript coding for chitinase-like protein YKL-40 implicated in many fundamental mechanisms like angiogenesis, apoptosis and inflammatory response. Dosing of biological markers (ELISA) in serum of a cohort of patients with CF and diabetes at various stages highlighted high serum levels of YKL-40 in CF patients having intolerance to insulin (Figure 2). We believe that the level of circulating YKL-40 in human plasma from patients with cystic fibrosis who developed cystic fibrosis-related diabetes is an excellent biological marker of inflammatory response of bronchial epithelial cells. Therefore, this project aims to identify the YKL-40 source in CF patients and to better understand the modulation of its expression and the role of YKL-40 in the inflammatory response and in the onset of diabetes in CF patients.

 

Research program on epithelial dysfunction in pulmonary lesions (acute lung injury)
Alveolar epithelial cells are covered with a fluid that is essential to pulmonary surfactant function, which in turn is responsible for the alveolar stability. Homeostasis of this fluid layer is ensured by ion transport, particularly Na+ transport. Na+ transport allows edema reabsorption in pathological conditions like acute respiratory distress syndrome (ARDS) or in cardiogenic edema. The molecular mechanisms implicated in this active transport are 1) the ENaC channel on the apical surface of epithelial cells and 2) the Na/K-ATPase pump found on the basolateral surface. We found that proinflammatory conditions observed in pneumonia or in some stresses affecting the lungs (TNF, LPS, calgranuline, TGF-β) reduce the expression and activity of ENaC in alveolar epithelial cells culture. We also found a correlation between the expression of αENaC mRNA and the Na+ transepithelial movement in these cells. In vivo, a chronic infection with Pseudomonas aeruginosa or intratracheal instillation of LPS reduce expression of ENaC subunits in mice. Lastly, in a dog model of pulmonary transplantation, a rapid reduction of ENaC mRNA was observed following reperfusion as well as a diminution of lung fluid clearance. This research program aims to better understand the molecular mechanisms implicated in the regulation of expression and activity of the ENaC channel as well as the role of alarmins and inflammatory molecules in this process.

A. Study of the modulation mechanisms of ENaC in alveolar epithelial cells
We study the mechanisms that downregulate the levels of αENaC mRNA in primary culture of alveolar epithelial cells under cellular stress. LPS from Pseudomonas aeruginosa and cycloheximide (CHX) have a similar effect on the expression level of mRNA coding for the alpha subunit of the ENaC channel (αENaC). In both cases, there is an approximate 65% decrease of the transcription level after 4 hours of treatment. Although the same signaling pathways are activated, there are notable differences between the two cases. CHX leads to sustained activation of the ERK and P38-MAPCK pathways while LPS activate these pathways on a transient basis only. Similarly, the pharmacologic inhibition of the ERK or P38­MAPCK pathway is sufficient to inhibit the effects of CHX on αENaC while the combined inhibition of these two pathways is necessary to inhibit the effects of LPS. The level of a messenger depends on its synthesis (transcription) level and its stability. While LPS reduce the transcriptional activity of the αENaC promoter, as measured by the expression of LUC gene cloned downstream of the promoter, this activity is not regulated by CHX. Thus, the response of alveolar epithelial cells to stress depends on the stress itself as it relates to the mechanisms that modulate ENaC expression. A mRNA stability is regulated by the three prime untranslated regions (3’UTR). The 3’UTR of αENaC mRNA is very long (907 bp). A computer analysis showed highly conserved sequence motifs between the 3’UTRs of different species, suggesting their selection to regulate the transcript stability. To gain a deeper understanding, we used the TET-Off expression system to study the half-life time of αENaC mRNA. The mRNAs showing 3’UTR domain integrity have a half-life time of ~1h. Without the 3’UTR sequences, the half-life falls to ~30 min. Therefore, αENaC/3’UTR are stabilizing sequences. Interestingly, in presence of cycloheximide, the transcript stability falls to ~30 min. The modulation of the αENaC transcript stability suggests that the modulation of the mRNA stability may play a role in some pathophysiological conditions. We are currently studying the nature of the Cis sequences that modulate this stability (Figure 3).

In parallel, we also study the regulation of the activity of this channel and the signaling mechanisms implicated in its activation in physiological and inflammatory conditions by measuring the electrical activity of monolayers of alveolar epithelial cells using a Ussing chamber.


B. 
Danger signaling and pulmonary epithelial lesions
Alarmins are endogenous molecules associated with cellular damages (Damage Associated Molecular Pattern or "DAMP"). They are expressed and excreted in non-programmed cell death and by some immune cells during inflammation, two constantly encountered conditions in pulmonary epithelial lesions. Thus, alarmins, including HMGB1 and calgranulins, have long been regarded as markers of tissue damage. However, several studies have shown their active participation in constitution of cellular damage. Our team works on the identification of signaling pathways related to alarmins in different models of pulmonary epithelial lesions (Figure 4). We are particularly interested in the impact of alarmins on the reabsorption of pulmonary edema via the ENaC epithelial sodium channel. The signaling pathways being studied include the interaction of calgranulins and HMGB1 with their receptor RAGE (Receptor for Advanced Glycation End products) as well as the interaction between alarmins and heme oxygenase. We also work on a clinical project on the expression profile of alarmins in pulmonary transplantation.

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