Molecular and cellular changes induced by the activation of CB2cannabinoid receptors in trabecular meshwork cells

[摘要] Purpose: To study the role of CB2 cannabinoid receptors in cellularfunctions of trabecular meshwork (TM) cells including cytoskeletonchanges and migration and to investigate the possible signaling pathwaysutilized by CB2 receptor for these cellular functions.Methods: JWH015, a selective CB2 receptor agonist, SR144528, aselective CB2 receptor antagonist, and SR141716A, a selective CB1receptor antagonist were used on cultured porcine TM cells. Incytoskeleton studies, Alexafluor 488-labeled phalloidin staining wasused to examine actin filaments and immunocytochemistry using ananti-paxillin antibody was used to detect focal adhesions onfibronectin-coated glass coverslips. Standard wound-healing assays wereused to study cell migration. Rac1-GTP pull-down assays were conductedto examine the changes in the Rac1-GTPase activity. Western-blotanalysis with an anti-phospho-cofilin antibody was used to measure thelevels of active cofilin.Results: JWH015 (100 nM) significantly inhibited the formation ofactin stress fibers and focal adhesions in cultured TM cells. The effectof 100 nM of JWH015 on the cytoskeleton was completely blocked by 1 μM of SR144528 but not by SR141716A. The addition of 100 nM JWH015decreased the migration of TM cells in wound-healing assays and thiseffect of JWH015 was blocked by 1 μM of SR144528. In contrast,SR141716A had no effect on the inhibitory effect of JWH015 on TM cellmigration. In Rac1-GTP pull-down assays, treatment of TM cells with 100nM of JWH015 decreased the activity of Rac1 GTPase activity in atime-dependent manner. Pretreatment with 1 μM SR144528, but not withSR141716A, blocked the effect of JWH015 on Rac1 activity. Western blotanalysis revealed that JWH015 also diminished the level ofphosphorylated cofilin in TM cells and this effect of JWH015 wasantagonized by SR144528 but not by SR141716A, indicating a CB2receptor-mediated activation of cofilin.Conclusions: This study demonstrates that by acting through CB2receptors, the CB2-selective cannabinoid agonist JWH015 modulates the TMcell actin's cytoskeleton and migration. This study also shows thatJWH015 modulates the activities of Rac1-GTPase and cofilin, which areimportant signaling molecules for the cytoskeletal and migratoryproperties of TM cells.

[学科分类] 生物化学/生物物理