PMA stimulates MUC5B gene expression through an Sp1-based mechanism in airway epithelial cells

Daphne Y.C. Wu, Reen Wu, Yin Chen, Natasha Tarasova, Mary Mann Jong Chang

Research output: Contribution to journalArticle

20 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)589-597
Number of pages9
JournalAmerican journal of respiratory cell and molecular biology
Volume37
Issue number5
DOIs
StatePublished - Nov 1 2007
Externally publishedYes

Keywords

  • ChIP
  • MUC5B
  • Site-directed mutagenesis
  • Sp-1
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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