Joo Hong Woo

Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes

To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-gamma-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFalpha, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRbeta formed a trimer with PIAS1-pSTAT1, whereas LXRalpha formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRbeta and LXRalpha were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFalpha expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-gamma-stimulated brain astrocytes.

The 15-Deoxy-Δ12,14-Prostaglandin J2 Suppresses Monocyte Chemoattractant Protein-1 Expression in IFN-γ-Stimulated Astrocytes through Induction of MAPK Phosphatase-1

The 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a cyclopentene PG generated from PGD2. It is an endogenous ligand of the peroxisome proliferator-activated receptor-γ that is primarily involved in adipocyte differentiation and lipid metabolism. Its anti-inflammatory actions have recently attracted considerable research attention, although the precise role and underlying mechanisms of these actions are largely unknown. In the present study, we focused on the inhibitory action of 15d-PGJ2 on the chemokine MCP-1, which plays a key role in the initiation and progression of inflammation by recruiting inflammatory cells to lesion sites. We found that 15d-PGJ2 suppressed MCP-1 transcription and protein secretion in IFN-γ-stimulated brain astrocytes. The inhibitory effects of 15d-PGJ2 on MCP-1 resulted from its actions on the transcription factors, AP-1 and specificity protein-1, which play key roles in IFN-γ-induced MCP-1 expression in astrocytes. Of interest, the negative effects of 15d-PGJ2 on AP-1/specificity protein-1 signaling and the resulting inhibition of MCP-1 expression were mediated by MAPK phosphatase (MKP)-1 activity, which was induced by 15d-PGJ2 in a peroxisome proliferator-activated receptor-independent manner. Thus, our data demonstrate a novel anti-inflammatory mechanism of 15d-PGJ2 involving MKP-1. Considering the importance of MCP-1 in inflammatory processes, our results suggest that 15d-PGJ2 analogues may have therapeutic potential to attenuate inflammatory brain diseases by inducing MKP-1 expression.

PINK1 expression increases during brain development and stem cell differentiation, and affects the development of GFAP-positive astrocytes

BackgroundMutation of PTEN-induced putative kinase 1 (PINK1) causes autosomal recessive early-onset Parkinson’s disease (PD). Despite of its ubiquitous expression in brain, its roles in non-neuronal cells such as neural stem cells (NSCs) and astrocytes were poorly unknown.ResultsWe show that PINK1 expression increases from embryonic day 12 to postnatal day 1 in mice, which represents the main period of brain development. PINK1 expression also increases during neural stem cell (NSC) differentiation. Interestingly, expression of GFAP (a marker of astrocytes) was lower in PINK1 knockout (KO) mouse brain lysates compared to wild-type (WT) lysates at postnatal days 1-8, whereas there was little difference in the expression of markers for other brain cell types (e.g., neurons and oligodendrocytes). Further experiments showed that PINK1-KO NSCs were defective in their differentiation to astrocytes, producing fewer GFAP-positive cells compared to WT NSCs. However, the KO and WT NSCs did not differ in their self-renewal capabilities or ability to differentiate to neurons and oligodendrocytes. Interestingly, during differentiation of KO NSCs there were no defects in mitochondrial function, and there were not changes in signaling molecules such as SMAD1/5/8, STAT3, and HES1 involved in differentiation of NSCs into astrocytes. In brain sections, GFAP-positive astrocytes were more sparsely distributed in the corpus callosum and substantia nigra of KO animals compared with WT.ConclusionOur study suggests that PINK1 deficiency causes defects in GFAP-positive astrogliogenesis during brain development and NSC differentiation, which may be a factor to increase risk for PD.

5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

BackgroundThe peroxisome proliferator-activated receptor (PPAR)-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA), is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation.MethodsTo determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1) were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR) was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment.ResultsWe found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK) phosphorylation and activator protein 1 (AP1) activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery.ConclusionETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests eicosanoids as potential therapeutic modulators of inflammation that act through a novel target.

Phosphatidylinositol 4-phosphate 5-kinase α is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kα is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kα mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kα knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1β and TNFα. PIP5Kα knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-κB and the degradation of IκB-α, indicating that PIP5Kα knockdown interfered with the ganglioside-activated NF-κB signaling. Together, these results suggest that PIP5Kα is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-κB activation in ganglioside-stimulated astrocytes.

Small heterodimer partner SHP mediates liver X receptor (LXR)–dependent suppression of inflammatory signaling by promoting LXR SUMOylation specifically in astrocytes

The transcriptional regulator SHP promotes SUMOylation of the LXR transcriptional factors specifically in astrocytes to limit inflammatory signaling. SHP delivers SUMO to suppress inflammation Inflammation contributes to various neurodegenerative diseases and occurs in response to brain injury. LXR transcriptional repressors suppress the expression of inflammatory genes. Lee et al. found that an orphan nuclear receptor called SHP (also known as NR0B2) facilitated the attachment of SUMO to LXRs specifically in astrocytes, either by acting as a bridge between LXRα and its SUMO-conjugating enzyme HDAC4 or by preventing the degradation of PIAS1, the SUMO-conjugating enzyme for LXRβ. The findings present opportunities for future drug development to limit neuroinflammation. Liver X receptors (LXRs) suppress the expression of inflammatory genes in a context-specific manner. In astrocytes, SUMOylation of LXRs promotes their anti-inflammatory effects. We found that small heterodimer partner (SHP), also known as NR0B2 (nuclear receptor subfamily 0, group B, member 2), facilitates the anti-inflammatory actions of LXRs by promoting their SUMOylation. Knockdown of SHP abrogated SUMOylation of LXRs, preventing their anti-inflammatory effects, in primary rat astrocytes but not macrophages. The underlying mechanisms differed according to LXR isoform. SHP promoted SUMO2 and SUMO3 attachment to LXRα by interacting directly with the histone deacetylase and E3 SUMO ligase HDAC4. In contrast, SHP promoted SUMO1 attachment to LXRβ by stabilizing the E3 SUMO ligase PIAS1. SHP bound PIAS1 and disrupted its interaction with the E3 ubiquitin ligase SIAH1. Knocking down SIAH1 rescued LXRβ SUMOylation in SHP-deficient astrocytes. Our data collectively suggested that SHP mediates the anti-inflammatory actions of LXRs through differential regulation of receptor SUMOylation specifically in astrocytes, thereby revealing potential avenues for therapeutic development in diseases associated with brain inflammation.

22(R)-hydroxycholesterol induces HuR-dependent MAP kinase phosphatase-1 expression via mGluR5-mediated Ca(2+)/PKCα signaling.

MAP kinase phosphatase (MKP)-1 plays a pivotal role in controlling MAP kinase (MAPK)-dependent (patho) physiological processes. Although MKP-1 gene expression is tightly regulated at multiple levels, the underlying mechanistic details remain largely unknown. In this study, we demonstrate that MKP-1 expression is regulated at the post-transcriptional level by 22(R)-hydroxycholesterol [22(R)-HC] through a novel mechanism. 22(R)-HC induces Hu antigen R (HuR) phosphorylation, cytoplasmic translocation and binding to MKP-1 mRNA, resulting in stabilization of MKP-1 mRNA. The resulting increase in MKP-1 leads to suppression of JNK-mediated inflammatory responses in brain astrocytes. We further demonstrate that 22(R)-HC-induced phosphorylation of nuclear HuR is mediated by PKCα, which is activated in the cytosol by increases in intracellular Ca(2+) levels mediated by the phospholipase C/inositol 1,4,5-triphosphate receptor (PLC/IP3R) pathway and translocates from cytoplasm to nucleus. In addition, pharmacological interventions reveal that metabotropic glutamate receptor5 (mGluR5) is responsible for the increases in intracellular Ca(2+) that underlie these actions of 22(R)-HC. Collectively, our findings identify a novel anti-inflammatory mechanism of 22(R)-HC, which acts through PKCα-mediated cytoplasmic shuttling of HuR to post-transcriptionally regulate MKP-1 expression. These findings provide an experimental basis for the development of a RNA-targeted therapeutic agent to control MAPK-dependent inflammatory responses.

Erratum: PINK1 expression increases during brain development and stem cell differentiation, and affects the development of GFAPpositive astrocytes (Molecular Brain (2016) 9:5 DOI 10.1186/s13041-016-0186-6)

Author details Neuroscience Graduate Program Department of Biomedical Sciences, Ajou University School of Medicine, Suwon, Korea. Chronic Inflammatory Disease Research Center, Ajou University School of Medicine, Suwon, Korea. Department of Pharmacology, Ajou University School of Medicine san-5, Woncheon-dong, Youngtong-gu, SuwonKyunggi-do 442–721Korea. Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea. Department of Anatomy and Division of Brain Korea 21 Plus Biomedical Science, Korea University College of Medicine, Seoul 136-705, Korea. Department of Brain Science, Ajou University School of Medicine, Suwon, Korea. Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea.

Identification of hexapeptides that render C2 myoblasts the resistant to menadione‐induced cell death

Abstract Menadione induced cell death in cultured C2 myoblasts. By screening synthetic peptide libraries composed of random sequence of hexapeptides, we identified the hexa‐peptides pool of (Ala/Ile)‐(Ile/Met)‐Val‐Ile‐Asp‐(Met/ Ser)‐NH2 that protected the myoblasts against menadione‐induced cell death. Pre‐incubation with the hexapeptide pool reduced the number of cells detached from culture dish substrate and increased the ratio of relative viability against menadione. In addition, the peptides strongly increased the expression of Bcl‐2, an anti‐apoptotic protein. These results suggest that the hexapeptides might enhance the resistance to cell death against menadione by increasing the expression of Bcl‐2.