Molecular and cellular changes induced by the activation of CB2 cannabinoid receptors in trabecular meshwork cells.
OBJECTIVE
To study the role of CB2 cannabinoid receptors in cellular functions of trabecular meshwork (TM) cells including cytoskeleton changes and migration and to investigate the possible signaling pathways utilized by CB2 receptor for these cellular functions.


METHODS
JWH015, a selective CB2 receptor agonist, SR144528, a selective CB2 receptor antagonist, and SR141716A, a selective CB1 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 coverslips. 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 activity. Western-blot analysis with an anti-phospho-cofilin antibody was used to measure the levels of active cofilin.


RESULTS
JWH015 (100 nM) significantly inhibited the formation of actin stress fibers and focal adhesions in cultured TM cells. The effect of 100 nM of JWH015 on the cytoskeleton was completely blocked by 1 microM of SR144528 but not by SR141716A. The addition of 100 nM JWH015 decreased the migration of TM cells in wound-healing assays and this effect of JWH015 was blocked by 1 microM of SR144528. In contrast, SR141716A had no effect on the inhibitory effect of JWH015 on TM cell migration. In Rac1-GTP pull-down assays, treatment of TM cells with 100 nM of JWH015 decreased the activity of Rac1 GTPase activity in a time-dependent manner. Pretreatment with 1 microM SR144528, but not with SR141716A, blocked the effect of JWH015 on Rac1 activity. Western blot analysis revealed that JWH015 also diminished the level of phosphorylated cofilin in TM cells and this effect of JWH015 was antagonized by SR144528 but not by SR141716A, indicating a CB2 receptor-mediated activation of cofilin.


CONCLUSIONS
This study demonstrates that by acting through CB2 receptors, the CB2-selective cannabinoid agonist JWH015 modulates the TM cell actin's cytoskeleton and migration. This study also shows that JWH015 modulates the activities of Rac1-GTPase and cofilin, which are important signaling molecules for the cytoskeletal and migratory properties of TM cells.
Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, USA.
Fang
F
He
Zhao-Hui
ZH
Song
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects; D022001:Focal Adhesions / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism*; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D051339:Actin Depolymerizing Factors; D000199:Actins; D007211:Indoles; D043885:Receptor, Cannabinoid, CB2; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; C402944:JHW 015