RNA interference decreases PAR-2 expression and function in human airway smooth muscle cells.
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.
Trian
Thomas
T
Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E356, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France.
Girodet
Pierre-Olivier
PO
Ousova
Olga
O
Marthan
Roger
R
Tunon-de-Lara
J Manuel
JM
Berger
Patrick
P
D000328:Adult; D000368:Aged; D001249:Asthma / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008168:Lung / Q000033:anatomy & histology*; D008297:Male; D015591:Microspectrophotometry; D008875:Middle Aged; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D002118:Calcium