Regulation of gene expression in pulmonary inflammation and differentiation : a role for C/EBP transcription factors

CCAAT/enhancer-binding protein (C/EBP) transcription factors play essential roles in gene regulation. The lung-enriched isoform C/EBPα is known to inhibit proliferation, promote differentiation and stimulate gene expression characteristic of the mature differentiated pulmonary epithelium. C/EBPβ, also enriched in the lung, plays a role in cell differentiation and the regulation of inflammatory and host defense genes in several organs. The activity of C/EBPβ is decreased in smokers with chronic obstructive pulmonary disease (COPD), indicating a role in COPD pathogenesis. The objective of this thesis was to investigate the unique or overlapping roles of C/EBPα and C/EBPβ in lung epithelial differentiation, and to assess the contribution of C/EBPβ in regulating pulmonary inflammation. To investigate unique vs. overlapping roles of C/EBPα and C/EBPβ in the lung, the pulmonary phenotype of mice lacking C/EBPα (Cebpa mice), C/EBPβ (Cebpb mice) or both C/EBPα and C/EBPβ (Cebpa; Cebpb mice) specifically in the lung epithelium, all generated by SFTPC-Cre mediated excision, was investigated. Cell culture experiments suggested that C/EBP and C/EBP bind the same elements within a lungspecific promoter, and that their functions are partially overlapping. Pre-natal Cebpa mice and Cebpa; Cebpb mice displayed immature lungs similar to the lungs of premature infants, and Cebpa; Cebpb mice exhibited even more impaired airway epithelial cell differentiation than the Cebpa mice. The proportion of Cebpa mice that survived and reached adulthood spontaneously developed a majority of the histopathological hallmarks of COPD, possibly caused by infiltrating inflammatory cells – similar to what is observed in COPD and what is mechanistically proposed to drive COPD pathogenesis. These findings are indicative of a relationship between immature lungs at birth, C/EBPs and the development of inflammatory lung disease. Considering the previous documentation of decreased airway epithelial C/EBPβ activity in smokers with COPD, C/EBPβ could have a role in COPD pathogenesis. The role of C/EBPβ in regulating inflammatory and innate immune responses in the lung was on this account investigated by employing a translational approach encompassing clinical samples as well as in vitro and in vivo experiments. CEBPB was significantly down-regulated in the airway epithelium of both current and former smokers compared to never-smokers, and in cigarette smoke extract-treated primary human airway epithelial cells in vitro, suggesting that C/EBPβ plays a role in smoking-induced disease. Supporting this, inhibition of CEBPB in human airway cells in vitro resulted in a compromised inflammatory response to smoke. Moreover, cigarette smoke-exposed Cebpb mice displayed reduced respiratory neutrophilia and induction of inflammatory mediators, including the neutrophil chemoattractant Groa, compared to smoke-exposed controls. LPS-challenged Cebpb mice also exhibited blunted respiratory neutrophilia and lower pulmonary expression of Groa, compared to LPS-challenged control littermates. In addition, suppression of LPSinduced neutrophilia and inflammatory gene expression by formoterol, a long acting β2adrenoceptor agonist used in treatment of COPD, was impaired in Cebpb mice. C/EBP transactivation was increased by treatment with formoterol in vitro, possibly through a β2adrenoceptor and cAMP-dependent mechanism. This demonstrates that both inflammatory as well as anti-inflammatory stimuli involve regulation of gene transcription by C/EBPβ. Taken together, these findings demonstrate that C/EBPα and C/EBPβ play pivotal and partly overlapping roles in airway epithelial differentiation, and that C/EBP and the lung epithelium orchestrates inflammatory responses as well as anti-inflammatory signaling by β2-adrenoceptor agonists in the lung. Thus, C/EBPs may influence tissue regeneration in lung homeostasis and disease as well as inflammatory and anti-inflammatory signaling, and are potential contributors to COPD pathogenesis. ISBN 978-91-7457-775-4