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.