RNA interference decreases PAR-2 expression and function in human airway smooth muscle cells

Am J Respir Cell Mol Biol. 2006 Jan;34(1):49-55. doi: 10.1165/rcmb.2005-0187OC. Epub 2005 Sep 29.


Asthma is characterized by bronchial inflammation and hyperresponsiveness that involves mast cell tryptase and potentially its specific receptor protease activated receptor 2 (PAR-2). Tryptase increases free intracellular calcium concentration ([Ca2+]i), a key step in activation of human airway smooth muscle cells (HASMC). The aim of this study was to analyze the effect of PAR-2 gene silencing on HASMC, in terms of calcium response, since no antagonist is available for this receptor. Five siRNA against PAR-2 were synthesized and transfected in HASMC using lipid agents, and PAR-2 expression was examined using Western blot, fluorescence-activated cell sorter, immunocytochemistry and RT-PCR. [Ca2+]i was measured using microspectrofluorimetry in response to tryptase, the activating peptide SLIGKV, trypsin, or caffeine. Two siRNA significantly inhibited PAR-2 expression in terms of both total and surface protein expression, as well as mRNA levels. Tryptase- and SLIGKV-induced transient increase in [Ca2+]i was significantly inhibited after transfection with the most appropriate siRNA, whereas neither trypsin nor caffeine response was altered. Two control siRNA had no effect in terms of both PAR-2 expression and calcium response. Transfection efficiency was maximal after 24 h and disappeared after 48 h. Gene silencing using siRNA can thus be used in vitro to assess the function of PAR-2 in HASMC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Asthma / metabolism
  • Calcium / metabolism
  • Calcium Signaling / physiology
  • Cells, Cultured
  • Female
  • Gene Silencing
  • Humans
  • Lung / anatomy & histology*
  • Male
  • Microspectrophotometry
  • Middle Aged
  • Myocytes, Smooth Muscle / cytology
  • Myocytes, Smooth Muscle / metabolism*
  • RNA Interference*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Receptor, PAR-2* / genetics
  • Receptor, PAR-2* / metabolism


  • RNA, Messenger
  • RNA, Small Interfering
  • Receptor, PAR-2
  • Calcium