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PMA Stimulates MUC5B Gene Expression through an Sp1-Based Mechanism in Airway Epithelial Cells

¹ Center for Comparative Respiratory Biology and Medicine, University of California at Davis, Davis, California; and ² CIIT, Centers for Health Research, Research Triangle Park, North Carolina

Correspondence and requests for reprints should be addressed to Mary Mann-Jong Chang, Ph.D., Center for Comparative Respiratory Biology and Medicine, Genome and Biomedical Science Facility, Suite 6510, University of California, Davis, 451 East Health Sciences Drive, Davis, CA 95616. E-mail: mjchang{at}ucdavis.edu

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.

Key Words: MUC5B • Sp-1 • transcription • site-directed mutagenesis • ChIP

CLINICAL RELEVANCE Mucin overproduction is the hallmark of most airway diseases. It is important to understand its gene regulation.