Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

Pharmacological interventions to enhance fibrinolysis are effective for treating thrombotic disorders. Utilizing the in vitro U937 cell line-based fibrin degradation assay, we had previously found a cyclic pentapeptide malformin A 1 (MA 1 ) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA 1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA 1 -induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA 1 showed that MA 1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA 1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA 1 -induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA 1 -enhanced fibrinolysis in U937 cells. Synthetic active MA 1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA 1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA 1 -induced phosphorylation of RSK1 and ERK1/2, indicating that MA 1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA 1 upregulated the expression of urokinase-type plasminogen activator (uPA) and increased uPA secretion. These inductions were abrogated in RSK1 knockout cells. These results indicate that RSK1 is a key regulator of MA 1 -induced extracellular fibrinolytic activity.