Monocyte Chemotactic Protein-induced Protein Research Articles

The role of MCPIP‐1 in maintaining lipid homeostasis (LB156)

Background:Increased inflammation is associated with alterations of lipid and lipoprotein metabolism. Recent studies suggest that MCPIP1 (Monocyte Chemotactic Protein induced Protein‐1) may have a role in maintaining normal metabolic balance by negatively regulating chronic inflammation. Our preliminary data revealed that the MCPIP1 deficient (MCPIP1‐/‐) mice have reduced body lipid deposits compared to control mice. The current project was designed to test if MCPIP1 is involved in maintaining lipid homeostasis by affecting hepatic lipid production and distribution. Methods: MCPIP1‐/‐ mice (C57/BL6 background, 6 weeks old) were identified by PCR. The serum and liver from control and MCPIP1‐/‐ mice were collected after fasting. Serum lipids (LDL/VLDL‐cholesterol and total triglyceride) and liver lipids (total cholesterol and triglyceride) were measured using commercially available kits. Results: The data collected from sixteen samples (eight control and eight MCPIP1‐/‐) showed that in MCPIP1 ‐/‐ mice the mean serum HDL‐cholesterol and total triglyceride concentrations were about 49% and 31% lower respectively, compared to control mice. In contrast, serum concentration of LDL/VLDL‐cholesterol in MCPIP1‐/‐ mice increased about 66% when compared to control. Triglyceride in the liver tissue of MCPIP‐/‐ mice was also drastically decreased (72% lower than control). No changes were observed in total cholesterol in liver tissue. Conclusion: The lack of MCPIP1 is associated with alterations of lipid homeostasis.Grant Funding Source: Supported by KCOM Graduate Program in Biomedical Sciences

Monocyte chemotactic protein induced protein 1 (MCPIP1) fails to affect adipogenesis in immortalized mouse embryonic fibroblasts (iMEF) (LB149)

Background: Increased adipogenesis is associated with chronic inflammation. MCPIP1 has been shown to suppress inflammation through regulating pro‐inflammatory cytokines in the immune system. Recent studies found that MCPIP1 is involved in the process of adipogenesis in 3T3‐L1 cells but the results are controversial. Our preliminary data indicates that MCPIP1 knockout impaired hepatic insulin signaling. The current study was designed to deterimine if MCPIP1 affects adipogenesis by regulating the insulin signal transduction pathway. Methods: MEFs were isolated from both MCPIP1 heterozygous (no phenotypic changes) and knockout C57/BL6 mice and immortalized. At 2 days post‐confluency, the cells were treated with or without a differentiation cocktail containing dexamethasone, IBMX, trogliterazone, and different concentrations of insulin. After 48 hours, cells were switched to medium containing 10% FBS with or without the different concentrations of insulin for the remainder of the differentiation period. Adipocyte differentiation was measured using Oil Red‐O staining by visualizing the intracellular lipid droplets. Lipids were extracted with isopropanol. The concentration of lipid was measured, calculated, and expressed by normalizing to the protein concentration. Results: Although a slight increase in adipogenesis was seen in the MCPIP1 KO model compared to the heterozygous model, overall there was no significant difference between these groups (p>0.05). Also, no difference in adipogenesis was observed among the different concentrations of insulin groups in cells from either heterozygous or MCPIP1 KO mice (p>0.05). Conclusion: From the data we collected thus far, we conclude that MCPIP1 might not play a key role in adipogenesis in iMEFs. Insulin may not be required to stimulate adipogenesis in iMEFs isolated from MCPIP1 knockout and heterozygous mice.Grant Funding Source: Supported by ATSU‐KCOM Biomedical Sciences Program

Expression of the monocyte chemotactic protein-1-induced protein 1 decreases human neuroblastoma cell survival

The importance of monocyte chemotactic protein-1-induced protein 1 (MCPIP1) in the negative regulation of inflammatory reactions has already been extensively studied. However, its role in cancer is not yet established. We studied MCPIP1 gene expression in primary human neuroblastomas and several neuroblastoma cell lines. Our results showed a lack of MCPIP1 expression in primary neuroblastoma tumors. Moreover, it was found that the low expression of the protein measured in human neuroblastoma cell lines might be important for neuroblastoma survival, since enforced MCPIP1 gene expression in human neuroblastoma BE(2)-C cells caused a significant decrease in neuroblastoma cell viability and proliferation.

Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.

MCPIP1 restricts HIV infection and is rapidly degraded in activated CD4+ T cells

HIV-1 primarily infects activated CD4+ T cells and macrophages. Quiescent CD4+ T cells, however, possess cellular factors that limit HIV-1 infection at different postentry steps of the viral life cycle. Here, we show that the previously reported immune regulator monocyte chemotactic protein-induced protein 1 (MCPIP1) restricts HIV-1 production in CD4+ T cells. While the ectopic expression of MCPIP1 in cell lines abolished the production of HIV-1, silencing of MCPIP1 enhanced HIV-1 production. Subsequent analysis indicated that MCPIP1 imposes its restriction by decreasing the steady levels of viral mRNA species through its RNase domain. Remarkably, common T-cell stimuli induced the rapid degradation of MCPIP1 in both T-cell lines and quiescent human CD4+ T cells. Lastly, blocking the proteosomal degradation of MCPIP1 by MG132 abrogated HIV-1 production in phorbol 12-myristate 13-acetate/ionomycin-stimulated human CD4+ T cells isolated from healthy donors. Overall, MCPIP1 poses a potent barrier against HIV-1 infection at a posttranscriptional stage. Although the observed HIV restriction conferred by MCPIP1 does not seem to be overcome by any viral protein, it is removed during cellular stimulation. These findings provide insights into the mechanisms of cellular activation-mediated HIV-1 production in CD4+ T cells.

Antidicer RNAse activity of monocyte chemotactic protein-induced protein-1 is critical for inducing angiogenesis

Inflammatory angiogenesis involves the induction of a novel gene ZC3H12A encoding monocyte chemoattractant protein-1 (MCP-1)-induced protein-1 (MCPIP1) that has deubiquitinase and antidicer RNAse activities. If and how these enzymatic activities of MCPIP1 mediate the biological functions of MCPIP1 are unknown. Present studies with human umbilical vein endothelial cells suggest that MCPIP-induced angiogenesis is mediated via hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF), and silent information regulator (SIRT-1) induction that results in the inhibition of angiogenesis inhibitor thrombospondin-1. MCPIP1 expression inhibited the production of the antiangiogenic microRNA (miR)-20b and -34a that repress the translation of HIF-1α and SIRT-1, respectively. The RNase-dead MCPIP mutant D141N not only did not induce angiogenesis but also failed to inhibit the production of miR-20b and -34a suggesting that the antidicer RNase activity of MCPIP1 is involved in MCPIP-mediated angiogenesis. Mimetics of miR-20b and -34a inhibited MCPIP1-induced angiogenesis confirming that MCPIP1 suppresses the biogenesis of miR-20b and -34a. Furthermore, our results indicate that MCPIP expression induces nuclear translocation of HIF-1α. We show that under hypoxia angiogenesis is mediated via induction of MCPIP1 and under normoxia, in vitro, MCPIP deubiquitinates ubiquitinated HIF-1α and the stabilized HIF-1α enters the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and VEGF, suggesting that the deubiquitinase activity of MCPIP may also promote angiogenesis. The present results show for the first time that the antidicer RNase activity of MCPIP1 is critical in mediating a biological function of MCPIP, namely angiogenesis.

Delayed brain ischemia tolerance induced by electroacupuncture pretreatment is mediated via MCP-induced protein 1

BackgroundEmerging studies have demonstrated that pretreatment with electroacupuncture (EA) induces significant tolerance to focal cerebral ischemia. The present study seeks to determine the involvement of monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified novel modulator of inflammatory reactions, in the cerebral neuroprotection conferred by EA pretreatment in the animal model of focal cerebral ischemia and to elucidate the mechanisms of EA pretreatment-induced ischemic brain tolerance.MethodsTwenty-four hours after the end of the last EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 90 minutes in male C57BL/6 mice and MCPIP1 knockout mice. Transcription and expression of MCPIP1 gene was monitored by qRT-PCR, Western blot and immunohistochemistry. The neurobehavioral scores, infarction volumes, proinflammatory cytokines and leukocyte infiltration in brain and NF-κB signaling were evaluated after ischemia/reperfusion.ResultsMCPIP1 protein and mRNA levels significantly increased specifically in mouse brain undergoing EA pretreatment. EA pretreatment significantly attenuated the infarct volume, neurological deficits, upregulation of proinflammatory cytokines and leukocyte infiltration in the brain of wild-type mice after MCAO compared with that of the non-EA group. MCPIP1-deficient mice failed to evoke EA pretreatment-induced tolerance compared with that of the control MCPIP1 knockout group without EA treatment. Furthermore, the activation of NF-κB signaling was significantly reduced in EA-pretreated wild-type mice after MCAO compared to that of the non-EA control group and MCPIP1-deficient mice failed to confer the EA pretreatment-induced inhibition of NF-κB signaling after MCAO.ConclusionsOur data demonstrated that MCPIP1 deficiency caused significant lack of EA pretreatment-induced cerebral protective effects after MCAO compared with the control group and that MCPIP1 is involved in EA pretreatment-induced delayed brain ischemia tolerance.

Proteasome inhibitor MG‐132 induces MCPIP1 expression

The proteasome is a protein complex responsible for the degradation of polyubiquitin-tagged proteins. Besides the removal of target proteins, the proteasome also participates in the regulation of gene transcription in both proteolytic and non-proteolytic fashion. In this study the effect of proteasome inhibition on the basal expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1) was examined. Treatment of HepG2 or HeLa cells with proteasome inhibitor MG-132 resulted in a significant increase of MCPIP1 expression, both at mRNA and protein level. Interestingly, MG-132 did not alter MCPIP1 stability. Instead, the observed protein increase was blocked by actinomycin D, suggesting the involvement of de novo mRNA synthesis in the increase of MCPIP1 protein following MG-132 treatment. Using several inhibitors we determined the participation of extracellular-signal-regulated kinase 1/2 and p38 kinases in MCPIP1 upregulation by MG-132. Our findings show for the first time the impact of proteasome inhibition on MCPIP1 protein expression by modulation of the activity of intracellular signaling pathways. Overexpression of MCPIP1-myc protein decreased the viability of HeLa cells but not HepG2 cells, which correlates with the increased susceptibility of HeLa cells to MG-132 toxicity. Notably, both MG-132 treatment and MCPIP1-myc overexpression led to the activation of apoptosis, as revealed by the induction of caspases 3/7 in both types of cell lines. This suggests the involvement of MCPIP1 upregulation in toxic properties of proteasome inhibition, which is an acknowledged approach to the treatment of several cancer types.

Resveratrol Inhibits IgE-Mediated Basophilic Mast Cell Degranulation and Passive Cutaneous Anaphylaxis in Mice

AbstractResveratrol is a phytoalexin abundantly found in red grape skin and is effective in antitumor and antiinflammation associated with immune responses. This study investigated whether resveratrol suppressed immunoglobulin (Ig)E-mediated allergic responses and passive cutaneous anaphylaxis (PCA) in rat RBL-2H3 mast cells and in BALB/c mice. The release of β-hexosaminidase and histamine was enhanced in mast cells sensitized with anti-dinitrophenyl (DNP)-IgE and subsequently stimulated by DNP-human serum albumin (HSA), indicative of mast cell degranulation. When mast cells were pretreated with nontoxic resveratrol at 1–25 μmol/L, such induction was dose dependently diminished. Spleen tyrosine kinase (Syk) and phospholipase Cγ (PLCγ) of sensitized mast cells were activated by stimulation with DNP-HSA antigen, which was dampened by ≥5 μmol/L resveratrol. The phosphorylation of protein kinase C (PKC)μ and PKCθ was attenuated by administering resveratrol to DNP-HSA–exposed mast cells, whereas quiescent PKCζ/λ in sensitized cells was dose-dependently activated by resveratrol. Male BALB/c mice were sensitized for 24 h with DNP-IgE and orally administered with resveratrol 1 h before the DNP-HSA challenge. The histamine concentration was enhanced in sensitized mice challenged to DNP-HSA, which was reversed by administration of 10 mg/kg resveratrol. Additionally, it encumbered the tissue activation of Syk, PLCγ, and PKCμ in antigen-exposed mice. Resveratrol decreased IgE-mediated PCA and alleviated allergic edema of mouse ear and dorsal skin. Mast cell degranulation and allergic inflammation, accompanying the induction of monocyte chemotactic protein-1 and macrophage inflammatory protein-2, were inhibited by supplementing resveratrol to antigen-challenged mice. Resveratrol inhibited mast cell-derived, immediate-type allergic reactions, and these responses of resveratrol suggest possible therapeutic strategies in preventing allergic inflammatory diseases.

Quantum dots induced monocyte chemotactic protein-1 expression via MyD88-dependent Toll-like receptor signaling pathways in macrophages

Quantum dots (QDs) are nano-sized semiconductors. Previously, intratracheal instillation of QD705s induces persistent inflammation in mouse lungs. In our present study, QD705-COOH and QD705-PEG activated NF-κB and increased monocyte chemotactic protein-1 (MCP-1) expression in macrophages RAW264.7 via MyD88 dependent Toll-like receptor (TLR) signaling pathways. MyD88 is an adapter protein for most TLRs to activate NF-κB. Silencing expression of MyD88 or p65 with siRNA or co-treatment with a NF-κB inhibitor tremendously abolished QD705s-induced NF-κB activity and MCP-1 expression. The involved TLRs might locate either on the cell surface or inside of cells. Co-treatment with a TLR4 inhibitor completely prevented MCP-1 induction by QD705-PEG. Nevertheless, QD705-COOH readily entered cells, and co-treatment with either inhibitors of endocytosis or intracellular TLRs prevented MCP-1 induction. These findings indicate that, depending on their surface modification, OD705s activate MyD88 dependent-TLRs at the surface or inside of the cells, which is an important mechanism for nanoparticles-induced inflammatory responses. But other MyD88-independent pathways may also involve in these responses.

HMGB1-mediated activation of TLR4 signaling in hepatic stellate cells

To determine the potential of the high mobility group box-1 protein 1 (HMGB1) to activate Toll-like receptor 4 (TLR4) signaling in hepatic stellate cells (HSCs) and investigate the subsequent transition of HSC towards the inflammatory phenotype. Three immortalized mouse HSC cell lines, wild-type (JS1), TLR4-/- (JS2) and MyD88-/- (JS3), were subcultured in plates and divided into groups of normal control (untreated), postive control (lipopolysaccaride, LPS treatment), and experimental (HMGB1 treatment). All groups were transfected with luciferase reporter plasmids carrying responsive elements for either the nuclear factor-kappa B (NF-kB) or activator protein-1 AP-1 transcription factors. Following stimulation with normal saline, LPS (100 ng/mL) or HMGB1 (100 ng/mL), the activation of NF-kB or AP-1 was detected by a dual-luciferase reporter assay system. The induction of monocyte chemotactic protein-1 (MCP-1) transcription was determined by measuring the mRNA levels using real time quantitative reverse transcription PCR (qRT-PCR). The secreted protein levels of MCP-1 were determined by enzyme-linked immunosorbent assay (ELISA) of the culture supernatants. Activation of NF-kB- and AP-1-responsive reporters was significantly up-regulated in JS1 cells treated with HMGB1 or LPS, and the activation was coincident with markedly up-regulated transcription and secretion of MCP-1. However, HMGB1 and LPS treatment produced no responsive of the NF-kB and AP-1 reporters, and no increase in expression or secretion of MCP-1, in JS2 or JS3 cells. As an endogeous ligand of TLR4, HMGB1 may activate TLR4 signaling and the TLR4-mediated inflammatory response of HSC.

Monocyte chemotactic protein-induced protein (MCPIP) promotes inflammatory angiogenesis via sequential induction of oxidative stress, endoplasmic reticulum stress and autophagy

Major diseases such as cardiovascular diseases, rheumatoid arthritis, diabetes, obesity and tumor growth are known to involve inflammation. Inflammatory molecules such as MCP-1, TNF-α, IL-1β and IL-8 are known to promote angiogenesis. MCP-induced protein (MCPIP), originally discovered as a novel zinc finger protein induced by MCP-1, is also induced by other inflammatory agents. MCPIP was shown to mediate MCP-1-induced angiogenesis. Whether angiogenesis induced by other inflammatory agents is mediated via MCPIP is unknown and the molecular mechanisms involved in angiogenesis induced by MCPIP have not been elucidated. The aim of this study was to bridge this gap and delineate the sequential processes involved in angiogenesis mediated via MCPIP. siRNA knockdown of MCPIP was used to determine whether different inflammatory agents, MCP-1, TNF-α, IL-1β and IL-8, mediate angiogenesis via MCPIP in human umbilical vein endothelial cells (HUVECs). Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3′methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as a tool to study angiogenic differentiation induced by inflammatory agents or MCPIP overexpression in HUVECs. Tube formation induced by inflammatory agents, TNF-α, IL-1β, IL-8 and MCP-1 was inhibited by knockdown of MCPIP. Forced MCPIP-expression induced oxidative stress, ER stress, autophagy and angiogenic differentiation in HUVECs. Inhibition of each step caused inhibition of each subsequent step postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via induction of MCPIP that causes oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. The sequence of events suggested to be involved in inflammatory angiogenesis by MCPIP could serve as possible targets for therapeutic intervention of angiogenesis-related disorders.

혈관내피세포에서 라이코펜이 염증유전자 발현에 미치는 영향

Lycopene, found in tomatoes and tomato products, has antioxidant, anticancer, and anti-inflammatory effects. High-mobility-group box 1 (HMGB1) mediates the pro-inflammatory responses in several inflammatory diseases. In this study, the potential roles of lycopene in the HMGB1-mediated pro-inflammatory gene expressions in the primary human-umbilical-vein endothelial cells (HUVECs) were investigated. The data showed that HMGB1 upregulated the expressions of monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6), secretory phospholipase A2 (sPLA2)-IIA, and prostaglandin E2 (PGE2). Lycopene pre-incubation for 6 h decreased the HMGB1-mediated induction of MCP-1, IL-6, sPLA2-IIA, and PGE2. Further study revealed that the inhibitory effects of lycopene on the HMGB-1 induced expression of pro-inflammatory genes were mediated by the inhibition of two important inflammatory cytokines: tumor necrosis factor (TNF)- and nuclear factor (NF)-. These results suggest that HMGB1 upregulated the expression of pro-inflammatory genes and lycopene inhibited HMGB-1-induced pro-inflammatory genes by inhibiting TNF- and NF-. This finding will serve as an important evidence in the development of a new medicine for the treatment of inflammatory diseases.

CD4+ T cells Regulate Cardiac Ischemia/Reperfusion Injury

The mechanisms of healing following myocardial infarction are multifaceted. We tested the hypothesis that chronic, post‐ischemic, cardiac inflammation is regulated by CD4+ T cells through cytokine release and metabolism, and this leads to impaired cardiac function. For these studies, a mouse model of myocardial I/R (30 minutes/24 hours) was employed using wild type (WT) C57Bl/6J mice and CD4+ −/− mice. Based on several hemodynamic measures (EF, SW, dP/dtmax), the cardiac function of CD4+ −/− mice was impaired. Significant induction of monocyte chemotactic protein‐1 (1.9x; MCP‐1) and interferon gamma (3.4x; IFNγ) protein expression occurred following I/R in WT but was absent in CD4+ −/− (n=4; p<0.05). These observations suggested that CD4+ T cells play a pivotal role in the recruitment of innate immune responses following I/R. CD4+ −/− mice had increased cardiac TNFα (3x), ADAM10 (3x), ADAM17 (1.5x), and TLR‐2 (2.3x) mRNA expression relative to WT (n=5; p<0.05). Collectively, these results suggest that CD4+ T cells mediate cardiac dysfunction via TNFα metabolism and inflammatory cascades linked to MCP‐1, IFNγ, and TLR‐2. We propose that CD4+ T cells may bridge the innate immune response to the adaptive immune response in cardiac I/R. This may provide novel mechanistic strategies to target CD4+ T cells and treat chronic reperfusion injury syndromes. Supported by HL077566, HL085119, AHA SDG and AHA 10POST3870022.

Monocyte Chemotactic Protein-induced Protein 1 (MCPIP1) Suppresses Stress Granule Formation and Determines Apoptosis under Stress

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.

Induction of brain natriuretic peptide and monocyte chemotactic protein-1 gene expression by oxidized low-density lipoprotein: relevance to ischemic heart failure

Brain natriuretic peptide (BNP) and monocyte chemotactic protein-1 (MCP-1) are biomarkers of heart failure (HF). The aim of the present study was to determine the role of oxidized low-density lipoprotein (Ox-LDL) in the induction of these biomarkers and the signaling pathways involved in vitro. Incubation of HL-1 cardiomyocytes and human myocytes with Ox-LDL induced the expression of BNP and MCP-1 genes, while native LDL had no effect. When peroxides associated with Ox-LDL were reduced to hydroxides, the ability to induce BNP and MCP-1 gene expression was abolished. Furthermore, exposure of HL-1 cells to ischemic conditions alone had no effect on BNP gene expression, while ischemia followed by reperfusion resulted in increased expression of BNP gene. Inhibitors of ERK and JNK inhibited the induction of BNP. Signaling array results suggested that the induction of both MAPK and NF-κB pathways is involved in the induction of BNP by Ox-LDL. These results suggest that Ox-LDL or peroxidized lipids formed in oxidatively stressed myocytes during ischemia-reperfusion injury may play a role in the induction of BNP and MCP-1.

Extracellular HIV-1 Tat upregulates TNF-α dependent MCP-1/CCL2 production via activation of ERK1/2 pathway in rat hippocampal slice cultures: Inhibition by resveratrol, a polyphenolic phytostilbene

Human immunodeficiency virus-1 (HIV-1) associated dementia (HAD) has been attributed to an encephalitis resulting from intense infiltration of monocytes. Evidence suggests that the viral protein Tat, which is released actively from HIV-1 infected cells, can contribute significantly to this process. Therefore, the principal objective of this study was to evaluate the potential molecular basis for the role of extracellular HIV-1 Tat in the induction of monocyte chemotactic protein-1 (MCP-1/CCL2) in the hippocampus, which is primarily linked to cognitive function and most commonly damaged in HAD. We also attempted to identify the mechanism by which resveratrol (trans-3,5,4′-trihydroxystilbene) modulates MCP-1 release in hippocampal tissues exposed to Tat. An ex vivo study using rat hippocampal slices demonstrated a time- and dose-dependent increase in MCP-1 production from Tat-treated hippocampal tissues. This increase was accompanied by the activation of the MEK/ERK pathway and TNF-α production. Tat-induced MCP-1 release was abrogated by inhibitors of tyrosine kinases (TK), herbimycin A or genistein, a finding that supports the MAPK signaling mechanism. The inhibition of the ERK1/2 pathway with SL327 induced a near-complete abolition of the observed Tat-induced effects. Furthermore, anti-TNF-α antibodies suppressed Tat-induced MCP-1 release. Resveratrol, to a level similar to that of SL327, downregulated Tat-induced proinflammatory responses via the inactivation of ERK1/2. These results indicate that the activation of the ERK1/2 pathway and TK are critical factors in the production of TNF-α and MCP-1 in the Tat-exposed hippocampus. Additionally, the inhibition of Tat-induced production of MCP-1 and TNF-α via the inactivation of the ERK1/2 pathway may represent the anti-inflammatory mechanism of resveratrol in the hippocampus.

Early Inflammatory Reactions in Atherosclerosis Are Induced by Proline-Rich Tyrosine Kinase/Reactive Oxygen Species–Mediated Release of Tumor Necrosis Factor-α and Subsequent Activation of the p21 Cip1 /Ets-1/p300 System

Reactive oxygen species (ROS) are involved in the initial process of atherosclerosis, whereas it remains to be determined how atherogenic stimulus causes ROS-mediated proinflammatory reactions. Here, we focused on proline-rich tyrosine kinase (PYK2)-mediated ROS generation and examined how atherogenic stimulus causes early proinflammatory reactions. PYK2-deficient (knockout [KO]) (PYK2-KO) mice were crossbred with apolipoprotein E (ApoE)-deficient (PYK2-KO/ApoE-KO) mice. PYK2-KO/ApoE-KO mice and endothelial cells (EC) were used for the study. Aortic atherogenic lesions in PYK2-KO/ApoE-KO mice were markedly decreased (55% versus ApoE-KO) after 8 weeks of a Western diet. Aortic PYK2 was activated as early as 7 days after the Western diet, when inflammatory cells were not yet activated. Addition of the proatherogenic oxidized phospholipid lysophosphatidylcholine caused activation of endothelial PYK2. Lysophosphatidylcholine-activated PYK2 induced NADPH oxidase-mediated ROS generation and ROS-mediated synthesis of tumor necrosis factor-α (TNFα), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and p21Cip1/Ets-1. Neutralizing anti-TNFα antibody or knockdown of p21Cip1/Ets-1 system blocked the induction of VCAM-1 and MCP-1. PYK2 deficiency abolished these ROS-mediated proinflammatory reactions. Further analysis revealed that PYK2/ROS-mediated p21Cip1/Ets-1 activation upregulated the transcription of the MCP-1 gene in collaboration with p300 transcription coactivator. PYK2 is a key tyrosine kinase activated by high cholesterol exposure, which causes ROS-mediated TNFα release and induces TNFα-dependent expression of proinflammatory molecules via the p21Cip1/Ets-1/p300 transcription system.

Participation of MCP-induced protein 1 in lipopolysaccharide preconditioning-induced ischemic stroke tolerance by regulating the expression of proinflammatory cytokines

BackgroundLipopolysaccharide (LPS) preconditioning-induced neuroprotection is known to be related to suppression of the inflammatory response in the ischemic area. This study seeks to determine if monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified CCCH Zn finger-containing protein, plays a role in focal brain ischemia and to elucidate the mechanisms of LPS-induced ischemic brain tolerance.MethodsTranscription and expression of MCPIP1 gene was monitored by qRT-PCR and Western blot. Mouse microglia was prepared from cortices of C57BL/6 mouse brain and primary human microglia was acquired from Clonexpress, Inc. Wild type and MCPIP1 knockout mice were treated with LPS (0.2 mg/kg) 24 hours before brain ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsMCPIP1 protein and mRNA levels significantly increased in both mouse and human microglia and mouse brain undergoing LPS preconditioning. MCPIP1 mRNA level significantly increased in mice ipsilateral brain than that of contralateral side after MCAO. The mortality of MCPIP1 knockout mice was significantly higher than that of wild-type after MCAO. MCPIP1 deficiency caused significant increase in the infarct volume compared with wild type mice undergoing LPS preconditioning. MCPIP1 deficiency caused significant upregulation of proinflammatory cytokines in mouse brain. Furthermore, MCPIP1 deficiency increased c-Jun N terminal kinase (JNK) activation substantially. Inhibition of JNK signaling decreased the production of proinflammatory cytokines in MCPIP1 knock out mice after MCAO.ConclusionsOur data indicate that absence of MCPIP1 exacerbates ischemic brain damage by upregulation of proinflammatory cytokines and that MCPIP1 participates in LPS-induced ischemic stroke tolerance.

MCP-induced protein 1 deubiquitinates TRAF proteins and negatively regulates JNK and NF-κB signaling

The intensity and duration of macrophage-mediated inflammatory responses are controlled by proteins that modulate inflammatory signaling pathways. MCPIP1 (monocyte chemotactic protein-induced protein 1), a recently identified CCCH Zn finger-containing protein, plays an essential role in controlling macrophage-mediated inflammatory responses. However, its mechanism of action is poorly understood. In this study, we show that MCPIP1 negatively regulates c-Jun N-terminal kinase (JNK) and NF-κB activity by removing ubiquitin moieties from proteins, including TRAF2, TRAF3, and TRAF6. MCPIP1-deficient mice spontaneously developed fatal inflammatory syndrome. Macrophages and splenocytes from MCPIP1(-/-) mice showed elevated expression of inflammatory gene expression, increased JNK and IκB kinase activation, and increased polyubiquitination of TNF receptor-associated factors. In vitro assays directly demonstrated the deubiquitinating activity of purified MCPIP1. Sequence analysis together with serial mutagenesis defined a deubiquitinating enzyme domain and a ubiquitin association domain in MCPIP1. Our results indicate that MCPIP1 is a critical modulator of inflammatory signaling.