Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule

The ability of the CD44 adhesion molecule to interact with its ligand hyalu ronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-my ristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-bindi ng as well as the increase in CD44 expression are prevented by cycloheximid e, suggesting a requirement for new additional CD44 molecules on the cell s urface and/or cooperating proteins. In the present study, we have investiga ted which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capaci ty. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived f rom LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data sh ow that the PMA-induced HA-binding could not be blocked by agents inhibitin g protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, querceti n) or genestein, an inhibitor of tyrosine protein kinases. However, this PM A response was strongly inhibited by calmodulin antagonists (chlorpromazine , trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin a ntagonists inhibited the PMA-induced increase in CD44 expression on LB cell s, but had no influence on the ability of the constitutive HA-binder HA9 ce ll line to interact with HA, indicating an effect on the PMA induction phas e rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduc ed the ability of the HA9 cells to bind HA without affecting their CD44 exp ression level, In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.