Leukamenin E Induces K8/18 Phosphorylation and Blocks the Assembly of Keratin Filament Networks Through ERK Activation.

Bo Xia,Hui Zhang,Shilong Du,Minghui Yang,Lan Ding,Jingxin Wei

doi:10.3390/ijms21093164

Bo Xia, Hui Zhang + Show 4 more

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https://doi.org/10.3390/ijms21093164

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Abstract

Leukamenin E is a natural ent-kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.

Highlights

Keratins are the largest subfamily of intermediate filaments (IFs) and the most abundant cellular proteins in simple epithelial cells, and they form cytoskeletal networks that contribute to cell-type-specific functions such as adhesion, migration, and metabolism [1]
To exclude apoptotic cells induced by leukamenin E for subsequent experiments, we examined the effects of leukamenin E at different concentrations on cell viability and apoptosis by MTT and acridine orange/ethidium bromide (AO/EB) staining, respectively
The leukamenin E showed obvious proliferation inhibition at 2.0–4.0 μM against primary liver carcinoma cells (PLC), human umbilical vein endothelial cells (HUVECs) and Panc-1 cells when compared with the control group (Figure 1B), but the apoptosis rate and necrosis rate of leukamenin E-treated cells were less than 3.2% and 1.2%, respectively, in all groups (Figure 1C–E)

Summary

Introduction

Keratins are the largest subfamily of intermediate filaments (IFs) and the most abundant cellular proteins in simple epithelial cells, and they form cytoskeletal networks that contribute to cell-type-specific functions such as adhesion, migration, and metabolism [1]. The molecular mechanisms governing keratin assembly–disassembly and their non-mechanical functions are largely unknown, in part due to the lack of useful chemical inhibitors as well as the unique properties of keratin filaments (KFs) [1,3,4]. Cytochalasin and colchicine analogs as specific small-molecule inhibitors of microfilament (MFs) and microtubules (MTs) have become successful models for research on MFs and MTs assembly and their cellular function [5]. Small-molecule compounds that can phosphorylate keratin to inhibit keratin assembly have similar effects as keratin inhibitors. Recent studies have shown that some small molecular compounds can phosphate keratin at specific sites, such as sphingosylphosphorylcholine (SPC) at K8-Ser431 and K18-Ser in Panc-1 cells, 12-Otetradecanoylphorbol-13-acetate (TPA) at K8-Ser431 in Panc-1 cells, and prostaglandins at K20-Ser in HT29-MTX cells [6,9,10,11]

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