Final Activity Report Summary - FPMLRCFTRPPAR (Functional proteomics of membrane lipid rafts associated with CFTR in a human airway epithelial cell model of cystic fibrosis)
Cystic fibrosis (CF) is a genetic disease attributed to mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, coding for a chloride channel. Excessive inflammation in the airways is one of the most prominent manifestations of the disease. The link between genotype of patients and phenotypic expression of CF is not clear. The goal of this project was to analyse the localization of CFTR in membrane micro-domains of airway epithelial cells, depending on the presence or absence of mutations in its gene and on the pro-inflammatory conditions. In all cases, the protein and protein complex composition of CFTR-containing domains was determined, in order to identify proteins functionally associated with CFTR. The obtained results could be summarised as follows:
1. in basal conditions CFTR was mainly localised to detergent soluble microdomains (DSM), while a significative fraction localised to detergent insoluble microdomains (DIM or rafts), in Calu-3 and IB3/S9 cells
2. in Calu-3 cells, cholesterol depletion led to a decrease in the total amount of CFTR and annexin-1 as well as to a delocalisation of these two proteins from DIM
3. proinflammatory treatments with either IL1ß or TNFa led to a time-dependent redistribution of CFTR, annexin-1 (Anx-A1) and cytosolic phospholipase A2 (cPLA2) between DIM and DSM
4. specific inhibition of CFTR prevented relocalization of CFTR, Anx-A1 and cPLA2 to DIM in proinflammatory conditions. This result strongly suggested that a functional CFTR might be necessary in the relocalization of these two proteins to DIM
5. specific inhibition of CFTR also induced an increase in LTB4 production, suggesting that CFTR might participate in the regulation of this inflammatory pathway
5. Anx-A1 and cPLA2 interacted with each other both in DIM and DSM
6. the integrity of membrane microdomains could be an important factor in the correct regulation of inflammatory processes in CF
7. the double sodium dodecyl sulfate polyacrylamide gel electrophoresis (dSDS-PAGE) was revealed as a valuable technique to proteomic analysis of membrane microdomains
8. proteomic analysis revealed two differentially overxpressed proteins upon long TNFa stimulation in Calu-3 cells with highly significant scores, namely cytokeratin 18 and disulfide isomerise
9. three proteins were differentially expressed in cystic fibrosisbronchial epithelial (CFBE) cells containing the F508del mutation with respect to wild-type CFTR. Cytokeratin 18 was found upregulated in mutated cells, while a-actin was downregulated.
In case cytoskeletal proteins were confirmed to be differentially expressed as a result of inflammation and CFTR mutation, these results would reinforce the hypothesis of cytoskeleton as a therapeutic target in cystic fibrosis. In the current case, our results not only pointed to the possibility of F508del CFTR addressing to the apical membrane, and to DIM in particular, but also to the role of cytoskeleton regulation in the management of the inflammatory response.
A remarkable result was the effect of CFTR inhibition on the microdomain distribution of CFTR, Anx-A1 and cPLA2, suggesting that the integrity of membrane microdomains and membrane lipid composition might play a role in the regulation of inflammation in CF. Concerning the interaction of CFTR, annexin-1 and cPLA2 within a potential functional complex and the involvement of this interaction in the regulation of inflammation, our results, if confirmed, would lead to two immediate consequences. Firstly, this would explain, at least partially, the mechanistic link between CFTR function and inflammation. Secondly, this would suggest the cPLA2 inflammatory pathway and the microdomain dynamics as potential targets for therapies in cystic fibrosis.
1. in basal conditions CFTR was mainly localised to detergent soluble microdomains (DSM), while a significative fraction localised to detergent insoluble microdomains (DIM or rafts), in Calu-3 and IB3/S9 cells
2. in Calu-3 cells, cholesterol depletion led to a decrease in the total amount of CFTR and annexin-1 as well as to a delocalisation of these two proteins from DIM
3. proinflammatory treatments with either IL1ß or TNFa led to a time-dependent redistribution of CFTR, annexin-1 (Anx-A1) and cytosolic phospholipase A2 (cPLA2) between DIM and DSM
4. specific inhibition of CFTR prevented relocalization of CFTR, Anx-A1 and cPLA2 to DIM in proinflammatory conditions. This result strongly suggested that a functional CFTR might be necessary in the relocalization of these two proteins to DIM
5. specific inhibition of CFTR also induced an increase in LTB4 production, suggesting that CFTR might participate in the regulation of this inflammatory pathway
5. Anx-A1 and cPLA2 interacted with each other both in DIM and DSM
6. the integrity of membrane microdomains could be an important factor in the correct regulation of inflammatory processes in CF
7. the double sodium dodecyl sulfate polyacrylamide gel electrophoresis (dSDS-PAGE) was revealed as a valuable technique to proteomic analysis of membrane microdomains
8. proteomic analysis revealed two differentially overxpressed proteins upon long TNFa stimulation in Calu-3 cells with highly significant scores, namely cytokeratin 18 and disulfide isomerise
9. three proteins were differentially expressed in cystic fibrosisbronchial epithelial (CFBE) cells containing the F508del mutation with respect to wild-type CFTR. Cytokeratin 18 was found upregulated in mutated cells, while a-actin was downregulated.
In case cytoskeletal proteins were confirmed to be differentially expressed as a result of inflammation and CFTR mutation, these results would reinforce the hypothesis of cytoskeleton as a therapeutic target in cystic fibrosis. In the current case, our results not only pointed to the possibility of F508del CFTR addressing to the apical membrane, and to DIM in particular, but also to the role of cytoskeleton regulation in the management of the inflammatory response.
A remarkable result was the effect of CFTR inhibition on the microdomain distribution of CFTR, Anx-A1 and cPLA2, suggesting that the integrity of membrane microdomains and membrane lipid composition might play a role in the regulation of inflammation in CF. Concerning the interaction of CFTR, annexin-1 and cPLA2 within a potential functional complex and the involvement of this interaction in the regulation of inflammation, our results, if confirmed, would lead to two immediate consequences. Firstly, this would explain, at least partially, the mechanistic link between CFTR function and inflammation. Secondly, this would suggest the cPLA2 inflammatory pathway and the microdomain dynamics as potential targets for therapies in cystic fibrosis.