Hypoxia-mediated mechanism of MUC5AC production in human nasal epithelia and its implication in rhinosinusitis

PLoS One. 2014 May 19;9(5):e98136. doi: 10.1371/journal.pone.0098136. eCollection 2014.

Abstract

Background: Excessive mucus production is typical in various upper airway diseases. In sinusitis, the expression of MUC5AC, a major respiratory mucin gene, increases. However, the mechanisms leading to mucus hypersecretion in sinusitis have not been characterized. Hypoxia due to occlusion of the sinus ostium is one of the major pathologic mechanisms of sinusitis, but there have been no reports regarding the mechanism of hypoxia-induced mucus hypersecretion.

Methods and findings: This study aims to identify whether hypoxia may induce mucus hypersecretion and elucidate its mechanism. Normal human nasal epithelial (NHNE) cells and human lung mucoepidermoid carcinoma cell line (NCI-H292) were used. Sinus mucosa from patients was also tested. Anoxic condition was in an anaerobic chamber with a 95% N2/5% CO2 atmosphere. The regulatory mechanism of MUC5AC by anoxia was investigated using RT-PCR, real-time PCR, western blot, ChIP, electrophoretic mobility shift, and luciferase assay. We show that levels of MUC5AC mRNA and the corresponding secreted protein increase in anoxic cultured NHNE cells. The major transcription factor for hypoxia-related signaling, HIF-1α, is induced during hypoxia, and transfection of a mammalian expression vector encoding HIF-1α results in increased MUC5AC mRNA levels under normoxic conditions. Moreover, hypoxia-induced expression of MUC5AC mRNA is down-regulated by transfected HIF-1α siRNA. We found increased MUC5AC promoter activity under anoxic conditions, as indicated by a luciferase reporter assay, and mutation of the putative hypoxia-response element in MUC5AC promoter attenuated this activity. Binding of over-expressed HIF-1α to the hypoxia-response element in the MUC5AC promoter was confirmed. In human sinusitis mucosa, which is supposed to be hypoxic, expression of MUC5AC and HIF-1α is higher than in control mucosa.

Conclusion: The results indicate that anoxia up-regulates MUC5AC by the HIF-1α signaling pathway in human nasal epithelia and suggest that hypoxia might be a pathogenic mechanism of mucus hypersecretion in sinusitis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Line, Tumor
  • Chromatin Immunoprecipitation
  • Electrophoretic Mobility Shift Assay
  • Humans
  • Hypoxia / etiology
  • Hypoxia / metabolism*
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
  • Luciferases
  • Mucin 5AC / metabolism*
  • Mucus / metabolism*
  • Nasal Mucosa / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / genetics
  • Signal Transduction / physiology*
  • Sinusitis / complications
  • Sinusitis / metabolism*

Substances

  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • MUC5AC protein, human
  • Mucin 5AC
  • Luciferases

Grants and funding

This research was supported by grants 2010-0008442 and 2012R1A1A2042476 to C.H. Kim, and 2012-0000803 to J.H. Yoon, from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology. This study was also supported by a faculty research grant from the Yonsei University College of Medicine for 2011 (6-2011-0140) to C.H. Kim. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.