Abstract
Purpose::
To study the roles of CB2 cannabinoid receptors in cellular functions of trabecular meshwork (TM) cells, including cytoskeleton changes and cell migration, and to investigate the signaling pathways utilized by CB2 receptor for these cellular functions.
Methods::
JWH015, a selective CB2 receptor agonist, and SR144528, a selective CB2 receptor antagonist, were used on cultured porcine TM cells. In cytoskeleton studies, Alexafluor 488-labeled phalloidin staining was used to examine actin filaments, and immunocytochemistry using an anti-paxillin antibody was used to detect focal adhesions on fibronectin-coated glass cover-slips. Standard wound healing assays were used to study cell migration. Rac1-GTP pull-down assays were conducted to examine the changes in the Rac1-GTPase activities. Western-Blot analysis with an anti-phospho-cofilin antibody was used to measure the levels of active cofilin.
Results::
JWH015 (100nM) significantly inhibited the formation of actin stress fibers and focal adhesions. The effect of 100 nM of JWH015 on the cytoskeleton was completely blocked by 1 µM of SR144528. The addition of 100 nM JWH015 decreased the migration of TM cells in wound healing assays. At 24 h after scratching, the distance between the edges of the wound region was presented as a percentage of the distance at 0 h. These percentages were calculated to be12.9±1.2, 48.5±5.3, 28.6±2.7, and 15.0±2.9% (mean ±SE), respectively, for vehicle control, 100 nM JWH015 alone, 100 nM JWH015 + 1 µM SR144528, and 1 µM SR144528 alone. In pull down assays, treatment of TM cells with 100 nM of JWH015 decreased the activity of Rac1 in a time-dependent manner. Pretreatment with 1 µM SR144528 blocked the effect of JWH015 on Rac1 activity. Western-Blots analysis revealed that JWH015 also diminished the phosphorylation level of cofilin in TM cells in a time-dependent manner.
Conclusions::
This study demonstrates that by acting through CB2 receptors, the CB2-selective cannabinoid agonist JWH015 modulates the TM cell actin cytoskeleton and migration. This study also indicates that JWH015 modulates the activities Rac1 and cofilin, which are important signaling molecules for the cellular function of TM cells.
Keywords: receptors • trabecular meshwork • signal transduction