Mucus overproduction and airway obstruction are important factors in the pathology of inflammatory airway diseases such as asthma and cystic fibrosis, two major childhood diseases for which there are no cures. Mucus is composed of mucin glycoproteins, and mucin overproduction is attributed to increase in inflammatory cytokines in airways. Levels of the pro-inflammatory cytokine tumor necrosis factor-a (TNF) is elevated early during the pathogenesis of airway diseases, and is hypothesized to regulate expression of the mucin gene MUC5AC, at the transcriptional and post-transcriptional levels through cis sequences.
AIM I will characterize TNF mediated regulation of MUC5AC expression at the transcriptional level by transiently transfecting airway epithelial cells (A549 and NHBE) with 1.4 kb of the MUC5AC promoter, followed by exposure to TNF (0.001-1.0 nM) for various times (0.5, 1, 2, 4, 8, or 24 hrs). Preliminary data demonstrate that TNF decreases transcriptional activity at the MUC5AC promoter in a concentration-and time-dependent fashion. Deletion constructs of the promoter (1, 0.5, 0.2, and 0.1 kb) followed by exposure to TNF will determine the region(s) of the promoter involved in transcriptional regulation.
Aim II will identify by the electromobility shift assay (EMSA) the cis sequences and putative trans-acting factors that participate in TNF-mediated regulation of MUC5AC transcription. Post-transcriptional regulation of MUC5AC gene expression by TNF will be investigated in AIM III by identifying putative cis sequences in the 3? untranslated region expression (UTR) of the MUC5AC gene using the EMSA. Regulation of gene expression by the regions of the 3?-UTR identified by EMSA will be verified by transfecting the 3?-UTR vectors into airway epithelial cellar followed by exposure to TNF. A better understanding of how TNF regulates MUC5AC gene expression is paramount to controlling and or preventing the processes that greatly contribute to the morbidity and mortality of airway diseases.
