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.

Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of STAT1 inflammatory signaling in IFN-gamma-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.

MAP kinase phosphatase-1 expression is regulated by 15-deoxy-Δ12,14-prostaglandin J2 via a HuR-dependent post-transcriptional mechanism.

In the present study, we demonstrate a mechanism through which 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) induces MKP-1 expression in rat primary astrocytes, leading to the regulation of inflammatory responses. We show that 15d-PGJ2 enhances the efficiency of MKP-1 pre-mRNA processing (constitutive splicing and 3'-end processing) and increases the stability of the mature mRNA. We further report that this occurs via the RNA-binding protein, Hu antigen R (HuR). Our experiments show that HuR knockdown abrogates the 15d-PGJ2-induced increases in the pre-mRNA processing and mature mRNA stability of MKP-1, whereas HuR overexpression further enhances the 15d-PGJ2-induced increases in these parameters. Using cysteine (Cys)-mutated HuR proteins, we show that the Cys-245 residue of HuR (but not Cys-13 or Cys-284) is critical for the direct binding of HuR with 15d-PGJ2 and the effects downstream of this interaction. Collectively, our data show that HuR is a novel target of 15d-PGJ2 and reveal HuR-mediated pre-mRNA processing and mature mRNA stabilization as important regulatory steps in the 15d-PGJ2-induced expression of MKP-1. The potential to use a small molecule such as 15d-PGJ2 to regulate the induction of MKP-1 at multiple levels of gene expression could be exploited as a novel therapeutic strategy aimed at combating a diverse range of MKP-1-associated pathologies.

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

BACKGROUND: The 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. METHODS: To 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. RESULTS: We 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. CONCLUSION: ETYA 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.

25-Hydroxycholesterol suppress IFN-γ-induced inflammation in microglia by disrupting lipid raft formation and caveolin-mediated signaling endosomes.

Acute microglial activation plays an important role in neuroprotection. However, dysregulated, prolonged microgliosis exacerbates neurodegeneration through excessive release of pro-inflammatory cytokines and cytotoxic factors. Interferon-gamma (IFN-γ), an inflammatory cytokine, exacerbates the detrimental microglial response. Although various anti-inflammatory drugs have been evaluated as interventions for microglia-mediated neuroinflammation, no anti-inflammatories are in clinical use for microgliosis. The present study evaluated the anti-inflammatory mechanisms of oxysterols, blood brain barrier (BBB) penetrable bioactive lipids, revealing that this intervention suppresses neuroinflammation by disrupting membrane lipid raft formation and caveolae-mediated endosomal IFN-γ signaling. We find that 25-hydroxycholesterol (25-HC) rapidly repressed IFN-γ receptor trafficking to lipid rafts in microglia by disrupting raft formation, thereby suppressing microglial inflammatory response. IFN-γ treatment upregulated expression of Cav-1, a major component of caveolae, and IFN-γ signaling was sustained through Cav-1+ signaling endosomes. 25-HC repressed IFN-γ induction of Cav-1 expression in microglia, and subsequently suppressed the chronic inflammatory response. Taken together, these findings demonstrated that 25-HC effectively regulate the inflammatory status of microglia by mediating the formation of rafts and caveolae-dependent signaling endosomes. Given the important roles of IFN-γ and microglia in the pathology of neurodegenerative brain diseases, a novel anti-inflammatory mechanism of 25-HC that is not receptor-dependent, but rather is related to the regulation of membrane rafts and caveolae, suggests a new therapeutic target for inflammatory neurodegenerations.

Interleukin-6 signaling requires EHD1-mediated alteration of membrane rafts.

Interleukin-6 (IL-6) is involved in many inflammatory diseases. IL-6 binds to membrane-bound IL-6 receptor α (IL-6Rα) (classic signaling) or soluble IL-6Rα (trans-signaling); this complex then associates with the signal-transducing membrane protein gp130. IL-6Rα and gp130 float on membrane (i.e., lipid) rafts; however, how membrane rafts regulate IL-6 signaling remains unclear. Here, we demonstrate that both IL-6 classic signaling and trans-signaling depend on membrane cholesterol, an essential raft component. Super-resolution fluorescence imaging using perfringolysin O D4 fragments that selectively bind to high cholesterol concentrations revealed that IL-6 and hyper-IL-6, a fusion protein of IL-6 and soluble IL-6Rα, induce the alteration of membrane rafts. IL-6 and hyper-IL-6 induced D4-positive raft (D4 raft) formation without affecting cholera toxin subunit B (CTB)-positive rafts (CTB rafts). Receptor clustering of IL-6Rα and gp130 and STAT3 phosphorylation occurred in D4 rafts. These results indicate that D4 rafts serve as platforms for the assembly of functional IL-6 receptor complexes. We found that Eps15 homology domain-containing protein 1 (EHD1) mediates the formation of functional IL-6 receptor complexes through D4 rafts. Overall, we uncover a novel regulatory mechanism of the EHD1-mediated alteration of membrane raft in IL-6 signaling.

The 15-deoxy-delta 12,14-prostaglandin J2 suppresses monocyte chemoattractant protein-1 expression in IFN-gamma-stimulated astrocytes through induction of MAPK phosphatase-1.

The 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a cyclopentene PG generated from PGD(2). It is an endogenous ligand of the peroxisome proliferator-activated receptor-gamma 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-PGJ(2) 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-PGJ(2) suppressed MCP-1 transcription and protein secretion in IFN-gamma-stimulated brain astrocytes. The inhibitory effects of 15d-PGJ(2) on MCP-1 resulted from its actions on the transcription factors, AP-1 and specificity protein-1, which play key roles in IFN-gamma-induced MCP-1 expression in astrocytes. Of interest, the negative effects of 15d-PGJ(2) 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-PGJ(2) in a peroxisome proliferator-activated receptor-independent manner. Thus, our data demonstrate a novel anti-inflammatory mechanism of 15d-PGJ(2) involving MKP-1. Considering the importance of MCP-1 in inflammatory processes, our results suggest that 15d-PGJ(2) analogues may have therapeutic potential to attenuate inflammatory brain diseases by inducing MKP-1 expression.

Phosphorylation of eukaryotic elongation factor 2 can be regulated by phosphoinositide 3-kinase in the early stages of myoblast differentiation.

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in G2/M and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in G0/G1 while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.

Lipid products of phosphoinositide 3-kinase abrogate genistein-induced fusion inhibition in myoblasts.

Genistein (4',5,7-trihydroxyisoflavone) is a tyrosine kinase inhibitor. Although the agent has shown to inhibit myoblast differentiation, neither intracellular target(s) as a tyrosine kinase inhibitor nor action mechanism of the agent is well known. Here we studied the effect of genistein on the differentiation of myoblasts. Genistein strongly but reversibly blocked both myoblast fusion and synthesis of the muscle-specific proteins. The agent also reversibly reduced the phosphorylation level of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and its interaction with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3-kinase). In addition, genistein indirectly inhibited PI3-kinase activity and blocked calcium influx which is required for myoblast fusion. However, both genistein-induced inhibition of cell fusion and calcium influx were abrogated by the lipid products of PI3-kinase. These results demonstrate that genistein can exert their effect on the signaling pathway from FAK to calcium influx via PI3-kinase in the differentiation of myoblasts.

PINK1 Deficiency Decreases Expression Levels of mir-326, mir-330, and mir-3099 during Brain Development and Neural Stem Cell Differentiation.

PTEN-induced putative kinase 1 (PINK1) is a Parkinson's disease (PD) gene. We examined miRNAs regulated by PINK1 during brain development and neural stem cell (NSC) differentiation, and found that lvels of miRNAs related to tumors and inflammation were different between 1-day-old-wild type (WT) and PINK1-knockout (KO) mouse brains. Notably, levels of miR-326, miR-330 and miR-3099, which are related to astroglioma, increased during brain development and NSC differentiation, and were significantly reduced in the absence of PINK1. Interestingly, in the presence of ciliary neurotrophic factor (CNTF), which pushes differentiation of NSCs into astrocytes, miR-326, miR-330, and miR-3099 levels in KO NSCs were also lower than those in WT NSCs. Furthermore, mimics of all three miRNAs increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) during differentiation of KO NSCs, but inhibitors of these miRNAs decreased GFAP expression in WT NSCs. Moreover, these miRNAs increased the translational efficacy of GFAP through the 3'-UTR of GFAP mRNA. Taken together, these results suggest that PINK1 deficiency reduce expression levels of miR-326, miR-330 and miR-3099, which may regulate GFAP expression during NSC differentiation and brain development.

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

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 LXRß by stabilizing the E3 SUMO ligase PIAS1. SHP bound PIAS1 and disrupted its interaction with the E3 ubiquitin ligase SIAH1. Knocking down SIAH1 rescued LXRß 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.

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

BACKGROUND: Mutation 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. RESULTS: We 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. CONCLUSION: Our 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.

Control of Inflammatory Responses: a New Paradigm for the Treatment of Chronic Neuronal Diseases.

The term 'inflammation' was first introduced by Celsus almost 2000 years ago. Biological and medical researchers have shown increasing interest in inflammation over the past few decades, in part due to the emerging burden of chronic and degenerative diseases resulting from the increased longevity that has arisen thanks to modern medicine. Inflammation is believed to play critical roles in the pathogenesis of degenerative brain diseases, including Alzheimer's disease and Parkinson's disease. Accordingly, researchers have sought to combat such diseases by controlling inflammatory responses. In this review, we describe the endogenous inflammatory stimulators and signaling pathways in the brain. In particular, our group has focused on the JAK-STAT pathway, identifying anti-inflammatory targets and testing the effects of various anti-inflammatory drugs. This work has shown that the JAK-STAT pathway and its downstream are negatively regulated by phosphatases (SHP2 and MKP-1), inhibitory proteins (SOCS1 and SOCS3) and a nuclear receptor (LXR). These negative regulators are controlled at various levels (e.g. transcriptional, post-transcriptional and post-translational). Future study of these proteins could facilitate the manipulation of the inflammatory response, which plays ubiquitous, diverse and ambivalent roles under physiological and pathological conditions.

Phosphatidylinositol 4-phosphate 5-kinase alpha 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 alpha (PIP5K alpha) 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 PIP5K alpha 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 PIP5K alpha 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 PIP5K alpha knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1 beta and TNFalpha. PIP5K alpha knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-kappaB and the degradation of I kappaB-alpha, indicating that PIP5K alpha knockdown interfered with the ganglioside-activated NF-kappaB signaling. Together, these results suggest that PIP5K alpha is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-kappaB activation in ganglioside-stimulated astrocytes.

Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.