Involvement of RSK1 activation in malformin-enhanced cellular fibrinolytic activity

Abstract

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.

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation

“…Similar to the effects on c-Ski, MEK inhibition did not completely reduce the levels of p-ERK, p-CREB or cell proliferation to that of the control group. This phenomenon may be caused by failure of the MEK inhibitor to completely suppress ERK activity, as found in previous studies [22][23][24]. These data indicate that TGF-β1 is an important factor mediating increases in c-Ski through the ERK/CREB pathway.…”

Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1

“…Our recent report showed that fluorescence‐labeled MA1 was enriched in ER‐like intracellular compartments, so, out of the candidate genes, we focused on SQLE localized in ER membrane (Figure G) and validated the effects of depletion by individual sgRNA transduction on MA1 cytotoxicity in U937 cells. Protein expression levels of SQLE were depleted by about 70 % or more after sgRNA transduction (Figure A).…”

“…MA1 acts on urokinase‐producing monocytes and increases cellular plasmin activity . A recent study also showed that the activation of RSK1 by way of the MAP kinase pathway leads to an increase in urokinase expression for fibrinolytic activity enhancement of MA1 . Although MA1 exhibits undesirable cytotoxicity together with the enhancement of cellular fibrinolytic activity, our previous structure–activity relationship study for MA1 suggested that the cytotoxic effects of MA1 could be split from its fibrinolysis‐enhancing effect .…”

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina

“…However, from a pharmaceutical angle, malpicyclins A and C are structurally identical to the cyclopentapeptide plactin B and D, respectively, which were previously isolated from an uncharacterized fungus (38,74) and exhibit fibrinolytic activities by elevating the activity of cellular urokinase-type plasminogen activator (39). The anticoagulating effects of plactins and derived cyclopentapeptides such as malformin A1 are currently under investigation in treatment of thrombic disorders (75). Hence, M. alpina is not only of nutritional benefit by production of polyunsaturated fatty acids, but also of pharmaceutical interest as producer of bioactive natural products.…”