title: PMA stimulates MUC5B gene expression through an Sp1-based mechanism in airway epithelial cells.
abstract: We previously showed that the MUC5B gene expression was elevated by phorbol 12-myristate 13-acetate (PMA) through an epidermal growth factor receptor-independent Ras/MEKK1/JNK and P38 signaling-based transcriptional mechanism. In the current study, we elucidated the molecular basis of this transcriptional regulation using promoter-reporter gene expression and chromatin immunoprecipitation (ChIP) assays with primary human bronchial epithelial cells that are cultured at the air-liquid interface. We have observed that PMA-induced MUC5B promoter activity is blocked by the Sp1-binding inhibitor, mithramycin A, in a dose-dependent manner. Deletion analysis with the MUC5B promoter construct demonstrated that both basal and PMA-induced promoter-reporter activities reside within the -222/-78 bp region relative to the transcriptional start site. NoShift transcriptional factor assays demonstrated that PMA stimulated Sp1 binding, but not STAT1 and c-Myc binding. Immunoprecipitation studies also verified the enhanced phosphorylation of Sp1 after PMA treatment. Site-directed mutagenesis and transfection studies demonstrated the involvement of Sp1-1 (-122/-114) and the Sp1-2 (-197/-186) cis elements in the basal and PMA-induced MUC5B promoter activity. The ChIP assay with anti-RNA polymerase II reconfirmed the PMA-induced MUC5B promoter activity by showing enhanced RNA polymerase II-DNA complex containing putative MUC5B Sp1-1, Sp1-2, or Sp1-3 sites. However, the ChIP assay using anti-Sp1 antibody demonstrated that the PMA-stimulated binding is only at Sp1-2. These results suggested an Sp1-based transcriptional mechanism with Sp1-1 as the regulator of basal MUC5B promoter activity and Sp1-2 as the regulator of PMA-induced MUC5B gene expression in the human airway epithelial cells.
authors.affiliation: Center for Comparative Respiratory Biology and Medicine, University of California at Davis, Davis, California 95616, USA.
authors.forename: Daphne Y C
authors.initials: DY
authors.lastname: Wu
authors.forename: Reen
authors.initials: R
authors.lastname: Wu
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: Natasha
authors.initials: N
authors.lastname: Tarasova
authors.forename: Mary M J
authors.initials: MM
authors.lastname: Chang
mesh_terms: D001483:Base Sequence; D002273:Carcinogens / Q000494:pharmacology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D055257:Mucin-5B; D009077:Mucins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D002273:Carcinogens; C524578:MUC5B protein, human; D055257:Mucin-5B; D009077:Mucins; D016329:Sp1 Transcription Factor; D013755:Tetradecanoylphorbol Acetate