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

Area of Science:

• Cellular and Molecular Biology
• Respiratory Medicine
• Pharmacology

Background:

• Asthma involves bronchial inflammation and hyperresponsiveness.
• Mast cell tryptase and protease-activated receptor 2 (PAR-2) play a role in asthma.
• Tryptase increases intracellular calcium concentration ([Ca2+]i), activating human airway smooth muscle cells (HASMC).

Purpose of the Study:

• To investigate the effect of PAR-2 gene silencing on HASMC calcium response.
• To assess the utility of siRNA for studying PAR-2 function in HASMC.
• To address the lack of available PAR-2 antagonists.

Main Methods:

• Synthesized five small interfering RNAs (siRNA) targeting PAR-2.
• Transfected HASMC with siRNA using lipid agents.
• Validated PAR-2 inhibition via Western blot, flow cytometry, immunocytochemistry, and RT-PCR.
• Measured [Ca2+]i using microspectrofluorimetry in response to tryptase, SLIGKV peptide, trypsin, and caffeine.

Main Results:

• Two siRNA significantly reduced PAR-2 expression at both protein and mRNA levels.
• Tryptase- and SLIGKV-induced [Ca2+]i increases were significantly inhibited by the most effective siRNA.
• Responses to trypsin and caffeine remained unaltered.
• Control siRNA had no effect on PAR-2 expression or calcium response.
• Maximal transfection efficiency was observed at 24 hours, diminishing by 48 hours.

Conclusions:

• Gene silencing using siRNA is a viable in vitro method to assess PAR-2 function in HASMC.
• PAR-2 activation by tryptase is a key pathway for increasing [Ca2+]i in HASMC.
• This approach provides a tool for functional studies in the absence of specific antagonists.