Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair.

Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect.

Cx26 regulates proliferation of repairing basal airway epithelial cells.

The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation. Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs. We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation.