Dyrk2 mediated the release of proinflammatory cytokines in LPS-induced BV2 cells
Introduction
The activation of glial cells in the central nervous system(CNS) is one of the major features of the occurrence of neurogenic inflammation, including the activation of microglia and astrocytes [1]. With the activation of inflammatory cells and infiltration of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), the receptors on the surface of neurons are activated, resulting in the activation of downstream signaling pathways and neuronal damage [2], [3]. Accumulating evidences suggest NF-κB and p38MAPK are main pathways in affecting the release of proinflammatory cytokines in microglial cells,while phosphorylation of upstream and downstream proteins is a key factor in activating these pathways [4], [5]. In the canonical NF-κB activation pathway, as the cytoplasmic inhibitors of NF-κB, IκBα will be phosphorylated and subsequently marked by ubiquitination and finally degraded by proteasome pathway [6]. The degradation of IκBα promotes the phosphorylation of NF-κB subunit p65 and leads to its translocation into the nucleus, resulting in transcription of various genes including encoding various inflammatory cytokines, such as TNF-α and IL-1β [7]. In addition, extracellular stimulation activates p38MAPK by promoting phosphorylation of p38MAPK, which further transfers into nucleus and enhance transcription of proinflammatory cytokines [8]. Although, phosphorylation of NF-κB and p38MAPK plays a key procedure in inducing their own nuclear transfer, the related mechanisms are complex and there is no definite conclusion. On the other hand, researches indiate that activating the phosphoinositide 3-kinase/Akt signaling pathway attenuates LPS-induced inflammatory responses, where phosphorylation of PI3k and Akt are the key conditions to activate Akt signaling pathway [9]. Therefore, protein phosphorylation modification plays an important role in regulating the release of proinflammatory cytokines in microglia.
As a novel phosphorylation-regulated kinase, Dyrk2 (Dual-specificity tyrosine- phosphorylation-regulated kinase 2) can prime phosphorylation of c-Jun and c-Myc in cancer cells and control p53 via Ser46 phosphorylation in reponse to DNA damage [10], [11]. Until now, researches on the functional role for Dyrk2 were not very adequate, and as a member of the Dyrks family that autophosphorylate a critical tyrosine in their activation loop but phosphorylate the substrates on their serine/threonine residues, few substrates for Dyrk2 by serine/threonine phosphorylation are reported, especially in the area of inflammatory [12]. However, recently findings revealed that Dyrk2 negatively regulates virus-induced type I interferon by promoting TBK1 degradation via Ser527 phosphorylation [13]. TBK1 is a downstream molecule of TLR4, which mediate the activation of NF-κB signaling pathway and the production of proinflammatory cytokines [14], [15]. Which suggests Dyrk2 potentially have an impact on the activation of microglia and the release of proinflammatory cytokines.
In this study, we investigated the role of Dyrk2 in regulating the release of proinflammatory cytokines in LPS-induced the activation of BV2 cells. We revealed that LPS-stimulation caused a different expression of Dyrk2 in the nucleus and cytoplasm of BV2 cells, which is probably one of the reasons that cause the different expression trends of phospho-p65 and phospho-p38MAPK in the nucleus and cytoplasm of BV2 cells. Moreover, we demonstrated that Dyrk2 interacted with p65, Akt and p38MAPK to regulate their phosphorylation levels, and Dyrk2 suppressed the release of proinflammatory cytokines in LPS-induce BV2 cells.
Section snippets
Cell Cultures and Stimulation
BV2 cells were cultured in PMI-1640 medium(HyClone, Logan, UT, United States) supplemented with 10% (v/v) fetal bovine serum and 100 mg/ml penicillin- streptomycin, and incubated at 37 ∘C under 5% CO2 in humidified air. The cells were passed every 2–3 days. BV2 cells were stimulated with 1 μg/ml LPS (Escherichia coli LPS 055:B5,Sigma Aldrich, L6529) for the required time.
DNA Transfection
Dyrk2 DNA plasmids were constructed by PCR, which was transiently transfected into BV2 cells via Lipofectamine2000 Reagent
Dyrk2 was expressed in the nucleus and cytoplasm of BV2 cells
To investigate the role of Dyrk2 in microglia, we first examined its expression in microglia. BV2 cells were cultured in normal conditions, immunofluorescence was used to detect its expression in microglia. Single immunofluorescence staining indicated that Dyrk2 was present in the nucleus and cytoplasm of BV2 cells (Fig. 1A). In order to further confirm the existence of Dyrk2 in the nucleus and cytoplasm, we respectively extracted the proteins from the nucleus and cytoplasm of BV2 cells without
Discussion
LPS-caused M1-polarization of microglia and release of proinflammatory cytokines are detrimental to the repair of damaged neurons [24], [25]. However, several lines of evidence suggested phosphorylation of p65(S536) and p38MAPK(T180/Y182) is important for the release of proinflammatory cytokines [26], [27]. In this study, we focused on a novel phosphokinase Dyrk2 and its role in affecting the activation of microglia by regulating the phosphorylation of Akt, p65 and p38MAPK. Investigations
Declaration of interest
The author reports no conflicts of interest in this work.
Acknowledgment
We thank New Cell & Molecular Biotech Co, Ltd (www.ncmbio.com) for its products (Antibody Diluent, Fast PAGE Plus) are a great help to this experiment.
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Yuxiang Sun, Li Xu contributed equally to this work.