Hydroethanolic extract of Cirsium setidens ameliorates doxorubicin-induced cardiotoxicity by AMPK-PGC-1α-SOD-mediated mitochondrial protection.

BACKGROUND: Doxorubicin (DOX) is an effective anticancer agent. However, the clinical outcomes of DOX-based therapies are severely hampered by their significant cardiotoxicity. PURPOSE: We investigated the beneficial effects of an ethanol extract of Cirsium setidens (CSE) on DOX-induced cardiomyotoxicity (DICT). METHODS: UPLC-TQ/MS analysis was used to identify CSE metabolite profiles. H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells were used to evaluate the effects of CSE on DICT-induced cell death. To elucidate the mechanism underlying it, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma co-activator l-alpha (PGC1-α), nuclear respiratory factor 1 (NRF1), NRF2, superoxide dismutase (SOD1), and SOD2 expression was detected using western blot analysis. The oxygen consumption rate (OCR), cellular ROS, and mitochondrial membrane potential were measured. Finally, we confirmed the cardioprotective effect of CSE against DICT in both C57BL/6 mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) by observing various parameters, such as electrophysiological changes, cardiac fibrosis, and cardiac cell death. RESULTS: Chlorogenic acid and nicotiflorin were the major compounds in CSE. Our data demonstrated that CSE blocked DOX-induced cell death of H9c2 cells without hindrance of its apoptotic effects on MDA-MB-231 cells. DOX-induced defects of OCR and mitochondrial membrane potential were recovered in a CSE through upregulation of the AMPK-PGC1-α-NRF1 signaling pathway. CSE accelerated NRF1 translocation to the nucleus, increased SOD activity, and consequently blocked apoptosis in H9c2 cells. In mice treated with 400 mg/kg CSE for 4 weeks, electrocardiogram data, creatine kinase and lactate dehydrogenase levels in the serum, and cardiac fibrosis, were improved. Moreover, various electrophysiological features indicative of cardiac function were significantly enhanced following the CSE treatment of hiPSCCMs. CONCLUSION: Our findings demonstrate CSE that ameliorates DICT by protecting mitochondrial dysfunction via the AMP- PGC1α-NRF1 axis, underscoring the therapeutic potential of CSE and its underlying molecular pathways, setting the stage for future investigations into its clinical applications.

Human induced pluripotent stem cell line YCMi007-A generated from a dilated cardiomyopathy patient with a heterozygous dominant c.613C > T (p. Arg205Trp) variant of the TNNT2 gene.

Cardiac muscle troponin T protein binds to tropomyosin and regulates the calcium-dependent actin-myosin interaction on thin filaments in cardiomyocytes. Recent genetic studies have revealed that TNNT2 mutations are strongly linked to dilated cardiomyopathy (DCM). In this study, we generated YCMi007-A, a human induced pluripotent stem cell (hiPSC) line from a DCM patient with a p. Arg205Trp mutation in the TNNT2 gene. The YCMi007-A cells show high expression of pluripotent markers, normal karyotype, and differentiation into three germ layers. Thus, YCMi007-A-an established iPSC-could be useful for the investigation of DCM.

Dnmt1/Tet2-mediated changes in Cmip methylation regulate the development of nonalcoholic fatty liver disease by controlling the Gbp2-Pparγ-CD36 axis.

Dynamic alteration of DNA methylation leads to various human diseases, including nonalcoholic fatty liver disease (NAFLD). Although C-Maf-inducing protein (Cmip) has been reported to be associated with NAFLD, its exact underlying mechanism remains unclear. Here, we aimed to elucidate this mechanism in NAFLD in vitro and in vivo. We first identified alterations in the methylation status of the Cmip intron 1 region in mouse liver tissues with high-fat high-sucrose diet-induced NAFLD. Knockdown of DNA methyltransferase (Dnmt) 1 significantly increased Cmip expression. Chromatin immunoprecipitation assays of AML12 cells treated with oleic and palmitic acid (OPA) revealed that Dnmt1 was dissociated and that methylation of H3K27me3 was significantly decreased in the Cmip intron 1 region. Conversely, the knockdown of Tet methylcytosine dioxygenase 2 (Tet2) decreased Cmip expression. Following OPA treatment, the CCCTC-binding factor (Ctcf) was recruited, and H3K4me3 was significantly hypermethylated. Intravenous Cmip siRNA injection ameliorated NAFLD pathogenic features in ob/ob mice. Additionally, Pparγ and Cd36 expression levels were dramatically decreased in the livers of ob/ob mice administered siCmip, and RNA sequencing revealed that Gbp2 was involved. Gbp2 knockdown also induced a decrease in Pparγ and Cd36 expression, resulting in the abrogation of fatty acid uptake into cells. Our data demonstrate that Cmip and Gbp2 expression levels are enhanced in human liver tissues bearing NAFLD features. We also show that Dnmt1-Trt2/Ctcf-mediated reversible modulation of Cmip methylation regulates the Gbp2-Pparγ-Cd36 signaling pathway, indicating the potential of Cmip as a novel therapeutic target for NAFLD.

Derivation of YCMi005-A, a human-induced pluripotent stem cell line, from a patient with dilated cardiomyopathy carrying missense variant in TPM1 (p. Glu192Lys).

Dilated cardiomyopathy (DCM) is one of the leading causes of heart transplantation. The clinical feature of DCM is characterized by enlarged heart and impaired function of the left or both ventricles, while its etiology is varied. In this study, we generated YCMi005-A, a human-induced pluripotent stem cell (hiPSC) line from a patient with DCM carrying the missense mutation of p.Glu192Lys in the TPM1 genes. YCMi005-A, an established hiPSC, showed the normal karyotype (46, XX) and high expression of pluripotency markers. In addition, it was confirmed that YCMi005-A has the differentiation potential assessed by staining of three germ layer markers.

Glial TLR2-driven innate immune responses and CD8+ T cell activation against brain tumor.

Growing interest has been focused on the roles of microglia as sentinels and effector cells that guard diverse pathological milieu in the brain. Here, it has been reported that microglial TLR2 is a crucial molecule that confers innate and adaptive immunity against brain tumor. TLR2 is preferentially expressed on microglia, brain-resident immune cells, in the tumor-bearing cerebral hemisphere of mouse and rat intracranial tumor models. Microglial TLR2 rapidly responds to brain tumor and modulates the inflammation-associated immune responses including phagocytosis, which are markedly decreased in TLR2-deficient mice. We further reveal that TLR2, but not TLR4, is essential for the tumor-triggered increase of MHC I in microglia. in vitro co-culture and in vivo experiments show that the glial TLR2-MHC I axis contributes to the proliferation and activation of CD8+ T cells by brain tumor. In addition, brain tumor-bearing ß2m-/- , Tlr2-/- , or Rag2 -/- γc -/- mice exhibit higher tumor volumes compared with WT mice with tumor. Survival analysis of GL26-bearing MHC I-defective mice also support the contribution of glial TLR2-MHC I axis to brain tumor immunity. Moreover, using publicly available data sets of human brain tumor patients, we find that glioblastoma (GBM) tissues with high TLR2 level have similar co-occurrence patterns with MHC I molecules, and the amounts and activity of infiltrating CD8+ T cells are correlated with TLR2 level in tissues from GBM patients. Collectively, our findings provide the importance of glial TLR2-driven innate and adaptive immune responses in the brain tumor microenvironment.

Glatiramer acetate attenuates the activation of CD4+ T cells by modulating STAT1 and -3 signaling in glia.

Interactions between immune effector cells of the central nervous system appear to directly or indirectly influence the progress/regression of multiple sclerosis (MS). Here, we report that glial STAT1 and -3 are distinctively phosphorylated following the interaction of activated lymphocytes and glia, and this effect is significantly inhibited by glatiramer acetate (GA), a disease-modifying drug for MS. GA also reduces the activations of STAT1 and -3 by MS-associated stimuli such as IFNγ or LPS in primary glia, but not neurons. Experiments in IFNγ- and IFNγ receptor-deficient mice revealed that GA-induced inhibitions of STAT signaling are independent of IFNγ and its receptor. Interestingly, GA induces the expression levels of suppressor of cytokine signaling-1 and -3, representative negative regulators of STAT signaling in glia. We further found that GA attenuates the LPS-triggered enhancement of IL-2, a highly produced cytokine in patients with active MS, in CD4+ T cells co-cultured with glia, but not in CD4+ T cells alone. Collectively, these results provide that activation of glial STATs is an essential event in the interaction between glia and T cells, which is a possible underlying mechanism of GA action in MS. These findings provide an insight for the development of targeted therapies against MS.

The HIF-1/glial TIM-3 axis controls inflammation-associated brain damage under hypoxia.

Inflammation is closely related to the extent of damage following cerebral ischaemia, and the targeting of this inflammation has emerged as a promising therapeutic strategy. Here, we present that hypoxia-induced glial T-cell immunoglobulin and mucin domain protein (TIM)-3 can function as a modulator that links inflammation and subsequent brain damage after ischaemia. We find that TIM-3 is highly expressed in hypoxic brain regions of a mouse cerebral hypoxia-ischaemia (H/I) model. TIM-3 is distinctively upregulated in activated microglia and astrocytes, brain resident immune cells, in a hypoxia-inducible factor (HIF)-1-dependent manner. Notably, blockade of TIM-3 markedly reduces infarct size, neuronal cell death, oedema formation and neutrophil infiltration in H/I mice. Hypoxia-triggered neutrophil migration and infarction are also decreased in HIF-1α-deficient mice. Moreover, functional neurological deficits after H/I are significantly improved in both anti-TIM-3-treated mice and myeloid-specific HIF-1α-deficient mice. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against hypoxia-associated brain diseases.

Anti-neuroinflammatory effects of DPTP, a novel synthetic clovamide derivative in in vitro and in vivo model of neuroinflammation.

Neuroinflammation is one of the critical pathological mechanisms influencing various neurodegenerative disorders. Most of the neurodegenerative diseases involve over-activation of microglial cells contributing to the demise of neurons. The objective of the current study is to evaluate the anti-inflammatory effect of novel synthetic clovamide derivative on the suppression of microglial activation in an in vitro and in vivo model of neuroinflammation. We have used lipopolysaccharide (LPS) to induce an inflammatory response in murine BV-2 microglial cells. Molecular tools like immunocytochemistry and immunoblotting were used to study the activity of novel synthetic clovamide derivative to inhibit inflammation induced by LPS in microglial cells. In in vivo experiments, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mouse model of neuroinflammation was developed to investigate the anti-neuroinflammatory effects of DPTP [3-(3,4-Dihydroxy-phenyl)-2-[4-(3-trifluoromethylphenyl)-but-2-enoylamino]-propionic acid methyl ester]. DPTP was observed to reduce the proinflammatory response in BV-2 cells induced by LPS. Further investigation revealed that DPTP attenuated phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), which was accompanied by a decrease in nuclear translocation of nuclear factor-κB (NF-κB) in LPS-treated BV2 microglia. Moreover, prophylactic treatment with DPTP (20mg/kg) for 7 days suppressed MPTP induced glial activation and behavioral impairment. Overall, our findings suggested that, DPTP exerts anti-neuroinflammatory effects against activated microglia in an in vitro and in vivo model and hence might be a promising therapeutic agent for alleviating the evolvement of neurodegenerative diseases associated with microglial activation.

Distinctive responses of brain tumor cells to TLR2 ligands.

Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll-like receptors (TLR)-2-mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2-deficient, interferon (IFN)-γ-deficient, and IFNγ-receptor-1-deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2-dependent pathway, not IFNγ-receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2-dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2-associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors.

EOP, a newly synthesized ethyl pyruvate derivative, attenuates the production of inflammatory mediators via p38, ERK and NF-κB pathways in lipopolysaccharide-activated BV-2 microglial cells.

Microglia-induced neuroinflammation is an important pathological mechanism influencing various neurodegenerative disorders. Excess activation of microglia produces a myriad of proinflammatory mediators that decimate neurons. Hence, therapeutic strategies aimed to suppress the activation of microglia might lead to advancements in the treatment of neurodegenerative diseases. In this study, we synthesized a novel ethyl pyruvate derivative, named EOP (S-ethyl 2-oxopropanethioate) and studied its effects on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in rat primary microglia and mouse BV-2 microglia. EOP significantly decreased the production of NO, inducible nitric oxide synthase, cyclooxygenase and other proinflammatory cytokines, such as interleukin (IL)-6, IL-1ß and tumor necrosis factor-α, in LPS-stimulated BV-2 microglia. The phosphorylation levels of extracellular regulated kinase, p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB were also inhibited by EOP in LPS-activated BV-2 microglial cells. Overall, our observations indicate that EOP might be a promising therapeutic agent to diminish the development of neurodegenerative diseases associated with microglia activation.

Regulation of microglia activity by glaucocalyxin-A: attenuation of lipopolysaccharide-stimulated neuroinflammation through NF-κB and p38 MAPK signaling pathways.

Microglial cells are the resident macrophages and intrinsic arm of the central nervous system innate immune defense. Microglial cells become activated in response to injury, infection, environmental toxins, and other stimuli that threaten neuronal survival. Therefore, regulating microglial activation may have therapeutic benefits that lead to alleviating the progression of inflammatory-mediated neurodegeneration. In the present study, we investigated the effect of glaucocalyxin A (GLA) isolated from Rabdosia japonica on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated primary microglia and BV-2 cells. GLA significantly inhibited LPS-induced production of nitric oxide and reversed the morphological changes in primary microglia. Further, GLA suppressed expression of inducible nitric oxide synthase and cyclooxygenase-2 dose-dependently at the mRNA and protein levels. The production of proinflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß (IL)-1ß, and IL-6 were inhibited by suppressing their transcriptional activity. Furthermore, GLA suppressed nuclear factor-κB activation by blocking degradation of IκB-α and inhibited the induction of lipocalin-2 expression in LPS-stimulated BV-2 cells. Mechanistic study revealed that the inhibitory effects of GLA were accompanied by blocking the p38 mitogen activated protein kinase signaling pathway in activated microglia. In conclusion, given that microglial activation contributes to the pathogenesis of neurodegenerative diseases, GLA could be developed as a potential therapeutic agent for treating microglia-mediated neuroinflammatory diseases.

Dual functionality of myeloperoxidase in rotenone-exposed brain-resident immune cells.

Rotenone exposure has emerged as an environmental risk factor for inflammation-associated neurodegenerative diseases. However, the underlying mechanisms responsible for the harmful effects of rotenone in the brain remain poorly understood. Herein, we report that myeloperoxidase (MPO) may have a potential regulatory role in rotenone-exposed brain-resident immune cells. We show that microglia, unlike neurons, do not undergo death; instead, they exhibit distinctive activated properties under rotenone-exposed conditions. Once activated by rotenone, microglia show increased production of reactive oxygen species, particularly HOCl. Notably, MPO, an HOCl-producing enzyme that is undetectable under normal conditions, is significantly increased after exposure to rotenone. MPO-exposed glial cells also display characteristics of activated cells, producing proinflammatory cytokines and increasing their phagocytic activity. Interestingly, our studies with MPO inhibitors and MPO-knockout mice reveal that MPO deficiency potentiates, rather than inhibits, the rotenone-induced activated state of glia and promotes glial cell death. Furthermore, rotenone-triggered neuronal injury was more apparent in co-cultures with glial cells from Mpo(-/-) mice than in those from wild-type mice. Collectively, our data provide evidence that MPO has dual functionality under rotenone-exposed conditions, playing a critical regulatory role in modulating pathological and protective events in the brain.

Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.

Galectin-3, a ß-galactoside-binding lectin, has been proposed to have multifaceted functions in various pathophysiological conditions. However, the characteristics of galectin-3 and its molecular mechanisms of action are still largely unknown. In this study, we show that galectin-3 exerts cytokine-like regulatory actions in rat and mouse brain-resident immune cells. Both the expression of galectin-3 and its secretion into the extracellular compartment were significantly enhanced in glia under IFN-γ-stimulated, inflamed conditions. After exposure to galectin-3, glial cells produced high levels of proinflammatory mediators and exhibited activated properties. Notably, within minutes after exposure to galectin-3, JAK2 and STAT1, STAT3, and STAT5 showed considerable enhancement of tyrosine phosphorylation; thereafter, downstream events of STAT signaling were also significantly enhanced. Treatment of the cells with pharmacological inhibitors of JAK2 reduced the galectin-3-stimulated increases of inflammatory mediators. Using IFN-γ receptor 1-deficient mice, we further found that IFN-γR 1 might be required for galectin-3-dependent activation of the JAK-STAT cascade. However, galectin-3 significantly induced phosphorylation of STATs in glial cells from IFN-γ-deficient mice, suggesting that IFN-γ does not mediate activation of STATs. Collectively, our findings suggest that galectin-3 acts as an endogenous danger signaling molecule under pathological conditions in the brain, providing a potential explanation for the molecular basis of galectin-3-associated pathological events.

Sulfatide, a major lipid component of myelin sheath, activates inflammatory responses as an endogenous stimulator in brain-resident immune cells.

Sulfatide, a major lipid component of myelin sheath, participates in diverse cellular events of the CNS, and its cellular level has recently been implicated in many inflammation-associated neuronal diseases. Herein, we report that sulfatide alone can trigger pathological inflammatory responses in glia, brain-resident immune cells. We show that sulfatide changed the morphology of primary microglia to their activated form, and it significantly induced the production of various inflammatory mediators in primary microglia and astrocytes. Moreover, sulfatide rapidly triggered the phosphorylation of p38, ERK, and JNK within 30 min, and it markedly enhanced the NF binding activity to NF-kappaB and AP-1 binding elements. However, nonsulfated galactocerebroside, another major lipid component of myelin, had no effect on activation of glia. We further reveal that CD1d did not contribute to sulfatide-stimulated activation of MAPKs, although its expression was enhanced by sulfatide and sulfatide-treated microglial cells actually stimulated type II NKT cells. Sulfatide significantly stimulated the phosphorylation of MAPKs in glia from CD1d-deficient mice, and the phosphorylation levels were similar to those in wild-type littermates. Sulfatide-triggered inflammatory events appear to occur at least in part through an L-selectin-dependent mechanism. L-selectin was dramatically down-regulated upon exposure to sulfatide, and inhibition of L-selectin resulted in suppression of sulfatide-triggered responses. Collectively, these results show that abnormally released sulfatide at demyelinated regions may act as an endogenous stimulator in the brain immune system, thus causing and further exacerbating pathological conditions in the brain.

Regulation of TLR2 expression by prostaglandins in brain glia.

TLR have emerged as important primary sensors for diverse stimuli and are increasingly implicated in various diseases. However, the molecular mechanisms underlying the regulation of the TLR system remain poorly understood. In this study, we report that some PGs may control TLR-mediated inflammatory events through modulation of TLR2 expression in brain immune cells. We first found that 15-deoxy-Delta12,14-PG J(2) (15d-PGJ(2)) markedly altered the expression of TLR2 but not TLR4, TLR1, and TLR9 at the message and protein levels in activated glia. Down-regulation of TLR2 expression and downstream events of TLR2 activation, including phagocytosis by 15d-PGJ(2), were also observed in cells treated with representative TLR2 ligands such as lipoteichoic acid and Pam(3)CSK(4). We further revealed that certain 15d-PGJ(2)-related PGs such as 15d-PGD(2) and PGD(2) also suppressed the ligand-stimulated increase of TLR2 expression, whereas PGE(2) and arachidonic acids did not. Interestingly, TLR2 expression was down-regulated even when such PGs were added at several hours after stimulator treatment. These findings appear to be independent of peroxisome proliferator-activated receptor gamma and D prostanoid receptors (DPs) because potent synthetic peroxisome proliferator-activated receptor gamma agonists, selective DP1 agonist, or DP2 agonist did not mimic the effects of such PGs on TLR2 expression. Taken together, our results suggest that 15d-PGJ(2), 15d-PGD(2), and PGD(2) may play notable roles as modulators of the TLR2-mediated inflammatory events, and provide new insight into the resolution of inflammation in the brain.

Contribution of TLR2 to the initiation of ganglioside-triggered inflammatory signaling.

Gangliosides, sialic acid-containing glycosphingolipids, are implicated in many neuronal diseases, but the precise molecular mechanisms underlying their pathological activities are poorly understood. Here we report that TLR2 participates in the initiation of ganglioside-triggered inflammatory signaling responses. Using FACS analysis and immunofluorescence microscopy, we found that gangliosides rapidly enhanced the cell surface expression of TLR2 in microglia, while reducing that of TLR4. The ganglioside-dependent increase of TLR2 expression was also observed at the messenger and protein levels. We also showed that gangliosides stimulate the interaction of TLR2 with Myd88, an adaptor for TLRs, and obtained evidence that lipid raft formation is closely associated with the ganglioside-induced activation of TLR2 and subsequent inflammatory signaling. These results collectively suggest that TLR2 contributes to the ability of gangliosides to cause inflammatory conditions in the brain.

Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: implications for death of activated microglia.

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.

Anti-inflammatory roles of retinoic acid in rat brain astrocytes: Suppression of interferon-gamma-induced JAK/STAT phosphorylation.

The anti-inflammatory effect of retinoic acid (RA) has been investigated for several decades. However, the underlying mechanisms responsible for this effect are largely unknown. In this study, we demonstrate that 9-cis-RA (cRA) and all-trans-RA (tRA) inhibit interferon-gamma (IFN-gamma)-induced inflammatory responses in astrocytes. In primary cultured rat brain astrocytes and C6 astroglioma cells, both cRA and tRA decreased IFN-gamma-induced expression of interferon regulatory factor-1. Both RA isoforms also reduced IFN-gamma-induced activation of signal transducers and activators of transcription (STAT)1, STAT3, Janus kinase (JAK)1, and JAK2. This inhibitory effect was significant when cells were pre-treated with RA prior to IFN-gamma. Furthermore, the effect of pre-treated RA was abolished in the presence of cycloheximide, indicating a requirement for de novo protein synthesis. Suppressors of cytokine signaling (SOCS), which are negative regulators of the JAK/STAT pathway, may be candidate mediators of the anti-inflammatory function of RA. Both cRA and tRA induced SOCS3 mRNA expression. These results suggest that RA induces an anti-inflammatory effect by suppressing the activation of the JAK/STAT pathway in IFN-gamma-treated astrocytes. SOCS3 may be at least one of the mechanisms that mediate the anti-inflammatory roles of RA.

Nordihydroguaiaretic acid inhibits IFN-gamma-induced STAT tyrosine phosphorylation in rat brain astrocytes.

The Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal cascades are major pathways that mediate the inflammatory functions of interferon-gamma (IFN-gamma), an important pro-inflammatory cytokine. Therefore, regulation of JAK/STAT signaling should modulate IFN-gamma-mediated inflammation. In this study, we found that nordihydroguaiaretic acid (NDGA), a well-known lipoxygenase (LO) inhibitor, suppressed IFN-gamma-induced inflammatory responses in brain astrocytes. In the presence of NDGA, interferon regulatory factor-1 expression was significantly reduced. Expression of monocyte chemotactic protein-1 and interferon-gamma inducible protein-10 mRNA in response to IFN-gamma was significantly suppressed in the presence of NDGA, as was tyrosine-phosphorylation of JAK and STAT. However, the 5-LO products, leukotriene B(4) (LTB(4)) and leukotriene C(4), were not detected in cells treated with IFN-gamma, indicating that the effect of NDGA seemed to be independent of 5-LO inhibition. In addition, two other 5-LO inhibitors (Rev5901 and AA861) did not mimic the effect of NDGA, and the 5-LO metabolites, 5-hydroxyeicosatetraenoic acid and LTB(4), were unable to reverse NDGA-driven suppression of STAT activation or affect basal STAT phosphorylation. Taken together, these results suggest that NDGA regulates IFN-gamma-mediated inflammation through mechanisms unrelated to the inhibition of 5-LO.

Rac1 contributes to maximal activation of STAT1 and STAT3 in IFN-gamma-stimulated rat astrocytes.

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-gamma-induced inflammatory responses in rat astrocytes. We revealed that IFN-gamma rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-gamma-responsive transcriptional responses. Compared with levels in control cells, IFN-gamma-induced IFN-gamma-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-gamma-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-gamma-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-gammaRalpha subunit and IFN-gamma-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-gammaRalpha and augment the interaction of IFN-gammaR with either STAT1 or STAT3 in response to IFN-gamma. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-gamma-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-gamma-responsive inflammatory signaling in rat astrocytes.