L-selectin activation regulates Rho GTPase activity via Ca⁺² influx in Sertoli cell line, ASC-17D cells

In seminiferous epithelium, tight junctions (TJs) between adjacent Sertoli cells constitute the blood-testis barrier and must change synchronically for germ cells to translocate from the basal to the adluminal compartment during the spermatogenic cycle. Rho GTPase activation through stimulation with specific L-selectin ligands has been proposed to modulate tight junctional dynamics. However, little is known regarding the role of Ca⁺² dynamics in Sertoli cell and how Ca⁺² relays L-selectin signals to modulate Rho GTPase activity in Sertoli cells, thus prompting us to investigate the Ca⁺² flux induced by L-selectin ligand in ASC-17D cells. Using fluorescent real-time image, we first demonstrated the increase of intracellular Ca⁺² level following L-selectin ligand stimulation. This Ca⁺² increase was inhibited in ASC-17D cells pretreated with nifedipine, the L-type voltage-operated Ca⁺² channel (VOCC) blocker, but not mibefradil, the T-type VOCC blocker. We then demonstrated the up-regulation of Rho and Rac1 in ASC-17D cells following the administration of L-selectin ligand, and the pre-treatment with nifedipine, but not mibefradil, prior to L-selectin ligand-binding abolished the activation of both Rho and Rac1. Together, we conclude that the activation of L-selectin induces Ca⁺² influx through the L-type VOCC, which up-regulates Rho and Rac1 proteins, in ASC-17D cells.