(674) Loss of a functional CFTR leads to a calpain-dependent degradation of ezrin in activated CF macrophages

Background: We have previously shown that cystic fibrosis (CF) macrophages (MΦs) and lung tissues of CF-affected mice have lower levels of ezrin, a filamentous-actin binding protein and component of the ezrin-radixin-moesin (ERM) protein family. In response to Pseudomonas aeruginosa (PA)-LPS, ezrin fails to be activated (phosphorylation at Thr567; p-ezrin) and to localize to the plasma membrane (PM) of MΦs, thus altering their immune activation. The mechanism/s by which lack of CFTR leads to the abnormal ezrin activation in CF MΦs is unknown. In contrast to the protein levels, ezrin expression (mRNA) is not lower in CF-KO compared to WT MΦs. Based on these data, we hypothesize that ezrin protein is degraded in the absence of functional CFTR.

Aim: To assess that lack of a functional CFTR leads to a calpain-dependent degradation of ezrin.

Methods: MΦs were generated by culturing bone marrow cells from CF (B6-KOCftrtm1UNC), G551D mice (kindly provided by Dr. D. Bedwell, UAB), and wild-type (WT) littermate mice in MCSF. CFTR-G551D protein is at the PM of cells but does not transport chloride. WT-MΦs were pre-incubated overnight with 20 µM of CFTR inhibitor (CFTRinh172) before adding PA-LPS. After 6h, ezrin cellular localization was assessed by immunofluorescence. To test ezrin degradation, CF MΦs were pre-incubated for 30 min with 10 µM Mg132 before adding PA-LPS. To test the role of calpain in ezrin degradation, WT, CF-KO, and EZ-KO MΦs were challenged with PA-LPS and then treated with ALLN (125nM), a calpain inhibitor. Levels of ezrin and cytokines were assessed by WB and real-time PCR, respectively.

Results: 1) Treatment of WT MΦs with a CFTR inhibitor (CFTRinh172) that blocks chloride ion transport is sufficient to decrease ezrin levels and its localization at the PM of activated MΦs Similar to CF-KO MΦs, G551D-MΦs (n=3) had impaired p-ezrin, blunted PI3K/AKT signaling, and increased pro-inflammatory cytokines expression than WT cells. These data suggest that functional CFTR is needed for controlling ezrin activation during MΦs activation. 2) CF MΦs (n=3) pretreated with the proteasome inhibitor Mg132 preserved high ezrin levels in response to LPS, thus supporting the hypothesis that Ezrin is degraded in CF MΦs. Based on published studies showing that calpain, a Ca2+-dependent protease that cleaves ezrin, has higher activity in peripheral blood mononuclear cells from CF patients than healthy controls (REF), we tested the calpain-specific inhibitor ALLN. Treatment of WT MΦs, CF-KO, and G551D MΦs (n=4) with ALLN led to increased levels of p-ezrin, but not total ezrin, and significantly reduced expression of pro-inflammatory cytokines (e.g., IL-6, TNFalpha, and Cxcl1) compared to vehicle-treated cells in response to LPS. EZ-KO MΦs treated with ALLN also showed a reduced expression of pro-inflammatory cytokines in response to LPS but to a lesser extent than WT. These data suggest that the anti-inflammatory effect of ALLN is partially dependent on ezrin.

Conclusions: In summary, our results suggest that functional CFTR is required for the induction and phosphorylation of ezrin during MΦs activation, which is necessary for a controlled immune response. Reduced ezrin level in CF MΦs is caused by protein degradation. Blocking the protease calpain is sufficient to rescue ezrin phosphorylation and reduce inflammation in CF MΦs.

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