TY - JOUR
T1 - Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity
AU - Koizumi, Yukio
AU - Nagai, Kenichiro
AU - Gao, Lina
AU - Koyota, Souichi
AU - Yamaguchi, Tomokazu
AU - Natsui, Miyuki
AU - Imai, Yumiko
AU - Hasumi, Keiji
AU - Sugiyama, Toshihiro
AU - Kuba, Keiji
N1 - Funding Information:
This work was supported in part by JSPS KAKENHI Grant Numbers 20200037 and 26350969 to Y.K.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - 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 A1 (MA1) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA1-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA1 showed that MA1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA1-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA1-enhanced fibrinolysis in U937 cells. Synthetic active MA1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA1-induced phosphorylation of RSK1 and ERK1/2, indicating that MA1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA1 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 MA1-induced extracellular fibrinolytic activity.
AB - 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 A1 (MA1) as a novel activating compound for cellular fibrinolytic activity. The mechanism by which MA1 enhances cellular fibrinolytic activity remains unknown. In the present study, we show that RSK1 is a crucial mediator of MA1-induced cellular fibrinolysis. Treatment with rhodamine-conjugated MA1 showed that MA1 localizes mainly in the cytoplasm of U937 cells. Screening with an antibody macroarray revealed that MA1 induces the phosphorylation of RSK1 at Ser380 in U937 cells. SL0101, an inhibitor of RSK, inhibited MA1-induced fibrinolytic activity, and CRISPR/Cas9-mediated knockout of RSK1 but not RSK2 suppressed MA1-enhanced fibrinolysis in U937 cells. Synthetic active MA1 derivatives also induced the phosphorylation of RSK1. Furthermore, MA1 treatment stimulated phosphorylation of ERK1/2 and MEK1/2. PD98059, an inhibitor of MEK1/2, inhibited MA1-induced phosphorylation of RSK1 and ERK1/2, indicating that MA1 induces the activation of the MEK-ERK-RSK pathway. Moreover, MA1 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 MA1-induced extracellular fibrinolytic activity.
UR - http://www.scopus.com/inward/record.url?scp=85044942502&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85044942502&partnerID=8YFLogxK
U2 - 10.1038/s41598-018-23745-0
DO - 10.1038/s41598-018-23745-0
M3 - Article
C2 - 29615689
AN - SCOPUS:85044942502
SN - 2045-2322
VL - 8
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 5472
ER -