PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms.
Asthma and chronic obstructive pulmonary disease are highly prevalent and economically important inflammatory airway diseases associated with mucus hypersecretion. Considerable additional morbidity and mortality are related to acute exacerbations, which are associated with further mucus hypersecretion. MUC5AC is a prominent airway mucin; however, the signalling pathways regulating MUC5AC hypersecretion are not fully characterised. We investigated the signalling pathway regulating phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC gene and protein expression in human respiratory epithelial cells. Using NCI-H292 cells, we demonstrated that treatment with PMA increased production of total and MUC5AC-specific mucin proteins. This increase was dependent on de novo MUC5AC gene transcription. We identified a short, proximal region of the MUC5AC promoter essential for this activity containing three specificity protein (Sp) 1 transcription factor-binding sites and a single CACCC site. By chemical inhibition, site-directed promoter mutagenesis and electrophoretic mobility-shift assay (EMSA), we demonstrated that PMA induced proteins binding to all three Sp1 sites and that they were all required for full induction of MUC5AC promoter activity. We then demonstrated a Ras-Raf-MEK/ERK signalling pathway was exclusively activated upstream of Sp1 activating the promoter and confirmed the requirement for matrix metalloproteinase activation leading to a ligand-dependent activation of the epidermal growth factor receptor. Finally, we demonstrated that activation of the novel protein kinase C isoforms delta and theta; was required upstream of the metalloproteinase activation. We have characterised a signalling pathway regulating PMA induction of MUC5AC. Studies such as this identify key signalling intermediates as targets for pharmacological intervention to treat mucus hypersecretion.
Hewson
Christopher A
CA
Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, UK.
Edbrooke
Mark R
MR
Johnston
Sebastian L
SL
D001483:Base Sequence; D001980:Bronchi / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D055271:Mucin 5AC; D009077:Mucins / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic; D016211:Transforming Growth Factor alpha / Q000378:metabolism*
D017931:DNA Primers; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D013755:Tetradecanoylphorbol Acetate