P2X7 receptor-Pannexin1 interaction mediates stress-induced interleukin-1 beta expression in human periodontal ligament cells.
OBJECTIVE
Pannexin 1 (Panx1) has been found to form nonjunctional hemichannels. It is also proposed to combine with the P2X7 receptor, forming a complex involved in adenosine triphosphate (ATP)-induced interleukin-1beta (IL-1β) release in macrophages. Previously, we reported that mechanical stress induced IL-1β expression via the ATP/P2X7 receptor-dependent pathway in human periodontal ligament (HPDL) cells and that ATP was released through the connexin 43 (Cx43) hemichannel. In the present work, we examined the role of Panx1 in stress-induced IL-1β induction in HPDL cells.


METHODS
Cultured HPDL cells were treated with compressive loading or ATP to stimulate IL-1β expression. Inhibitors, antagonists and the small interfering RNA technique were used to investigate the involvement of Panx1 in IL-1β induction. Co-immunoprecipitation (Co-IP) and immunostaining were used to determine the association of Panx1 with the P2X7 receptor. The IL-1β release mechanism was analyzed using inhibitors.


RESULTS
Blocking Panx1 significantly decreased ATP release, as well as IL-1β up-regulation, upon stimulation with stress or ATP. Co-IP revealed the association of Panx1 and the P2X7 receptor in HPDL cells, which was increased in response to mechanical loading. Pretreatment with vesicular trafficking inhibitors significantly reduced the amount of IL-1β released from stimulated cells, suggesting that IL-1β might be released through vesicles.


CONCLUSIONS
We clearly illustrated the contribution of Panx1 in ATP release, as well as in IL-1β induction in HPDL cells. The association of Panx1 and the P2X7 receptor might be required for IL-1β induction, and their possible novel role in IL-1β vesicular release was indicated.
Graduate School of Oral Biology, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand; Research Unit of Mineralized Tissues, Faculty of Dentistry, Chulalongkorn University, Pathumwan, Bangkok, Thailand.
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Kanjanamekanant
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Luckprom
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Pavasant
D000255:Adenosine Triphosphate / Q000032:analysis / Q000494:pharmacology; D001696:Biomechanical Phenomena; D002229:Carbenoxolone / Q000494:pharmacology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018031:Connexin 43 / Q000037:antagonists & inhibitors / Q000276:immunology; D017630:Connexins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D006801:Humans; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors / Q000276:immunology*; D008469:Meclofenamic Acid / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D010513:Periodontal Ligament / Q000166:cytology* / Q000276:immunology; D011339:Probenecid / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D058486:Receptors, Purinergic P2X7 / Q000276:immunology*; D013096:Spermine / Q000494:pharmacology; D013314:Stress, Mechanical; D000099264:Connexin 50
D000255:Adenosine Triphosphate; D002229:Carbenoxolone; D018031:Connexin 43; D017630:Connexins; D053583:Interleukin-1beta; D008469:Meclofenamic Acid; D009419:Nerve Tissue Proteins; D011339:Probenecid; D011803:Quinine; D034741:RNA, Small Interfering; D058486:Receptors, Purinergic P2X7; D013096:Spermine; D000099264:Connexin 50; C484032:GJD4 protein, human; C492764:PANX1 protein, human
P2X7 receptor; Pannexin1; adenosine triphosphate; human periodontal ligament cells; interleukin-1beta; mechanical stress