Suppression of the maturation and activation of the dendritic cell line DC2.4 by melanoma-derived factors

Dendritic cells play critical roles in both innate and adaptive immunity, and their numerous functions are tightly linked to their maturation and activation status. Here, we characterize the murine dendritic cell line DC2.4 as a model for studying dendritic cell maturation and activation, and we evaluate the influence of melanoma tumor cells on these processes. Exposure of DC2.4 cells to the Toll-like receptor ligand lipopolysaccharide induces both maturation and activation of these cells, characterized by upregulation of costimulatory molecule expression and proinflammatory cytokine/chemokine production. This maturation and activation is suppressed by soluble factors derived from both the highly tumorigenic B16-F1 and the poorly tumorigenic D5.1G4 murine melanoma cell lines. Interestingly, the extent of DC2.4 immunosuppression by these melanomas correlates with their tumorigenicity, suggesting a potentially vital role for dendritic cell/tumor cell interactions in the regulation of anti-tumor immunity and tumor outgrowth.     

生物肽-蛋氨酸脑啡肽增强树突状细胞抗肿瘤活性研究

采用反复冻融法制备Rac-1淋巴瘤细胞总抗原,与树突状细胞（DC2.4）共培养制备DC瘤苗;设置不同实验组,M组及RM组加入蛋氨酸脑啡肽（MENK）,研究MENK对DC瘤苗抗肿瘤细胞的杀伤活性影响。结果表明：MENK作用于DC瘤苗后,DC2.4形态上更趋向于成熟,且CD86、MHC-Ⅱ类分子表达水平与对照组相比较明显升高;DC2.4酸性磷酸酶（ACP）含量与对照组相比较明显减少而IL-12的分泌水平升高;同时,DC2.4诱导淋巴细胞增殖能力增强,且诱导活化的淋巴细胞对Rac-1淋巴瘤细胞的杀伤活性与对照组相比较明显增强。结果可见,MENK可明显促进特异性负载Rac-1淋巴瘤抗原的DC2.4的成熟,并增强特异性诱导活化的淋巴细胞对Rac-1淋巴瘤细胞的杀伤活性。     

Role of oxidative stress in ERK and p38 MAPK activation induced by the chemical sensitizer DNFB in a fetal skin dendritic cell line

The intracellular mechanisms involved in the early phase of dendritic cell (DC) activation upon contact with chemical sensitizers are not well known. The strong skin sensitizer 2,4-dinitrofluorobenzene (DNFB) was shown to induce the activation of mitogen-activated protein kinases (MAPK) in DC. In the present study, we investigated a putative role for oxidative stress in DNFB-induced MAPK activation and upregulation of the costimulatory molecule CD40. In a DC line generated from fetal mouse skin, DNFB induced a significant increase in protein oxidation, measured by the formation of carbonyl groups, while it had almost no effect on lipid peroxidation. The antioxidants glutathione and vitamin E, which inhibit protein and lipid oxidation, respectively, were used to assess the role of oxidative stress in DNFB-induced MAPK activation. Glutathione, but not vitamin E, inhibited DNFB-induced p38 MAPK and ERK1/2 phosphorylation, whereas none of the antioxidants interfered significantly with the DNFB-induced upregulation of CD40 protein levels. Taken together, these results indicate that DNFB activates p38 MAPK and ERK1/2 via production of reactive oxygen species, and that protein oxidation plays an important role in MAPK activation.     

Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1

Integrin LFA-1 is a receptor that is able to transmit multiple intracellular signals in leukocytes. Herein we show that LFA-1 induces a potent and transient increase in the activity of the small GTPase Rac-1 in T cells. Maximal Rac-1 activity peaked 10-15 min after LFA-1 stimulation and rapidly declined to basal levels at longer times. We have identified Vav, a guanine nucleotide exchange factor for Rac-1, and PI3K/Akt, as regulators of the activation and inactivation phases of the activity of Rac-1, respectively, in the context of LFA-1 signaling based on the following experimental evidence: (i) LFA-1 induced activation of Vav and PI3K/Akt with kinetics consistent with a regulatory role for these molecules on Rac-1, (ii) overexpression of a constitutively active Vav mutant induces activation of Rac independently of LFA-1 stimulation whereas overexpression of a dominant-negative Vav mutant blocks LFA-1-mediated Rac activation, (iii) pharmacological inhibition of PI3K/Akt prevented the fall in the activity of Rac-1 after its initial activation but had no effect on Vav activity, and (iv) overexpression of a dominant-negative or a constitutively active Akt-1 induced or inhibited, respectively, Rac-1 activity. Finally, we show that T cells with a sustained Rac activity have impaired capacity to elongate onto ICAM-1. These results demonstrate that down-regulation of the activity of this GTPase is a requirement for the regulation of T cell morphology and motility and highlight the importance of temporal regulation of the signaling triggered from this integrin.     

Rac-1 Superactivation Triggers Insulin-independent Glucose Transporter 4 (GLUT4) Translocation That Bypasses Signaling Defects Exerted by c-Jun N-terminal kinase (JNK)- and Ceramide-induced Insulin Resistance

Insulin activates a cascade of signaling molecules, including Rac-1, Akt, and AS160, to promote the net gain of glucose transporter 4 (GLUT4) at the plasma membrane of muscle cells. Interestingly, constitutively active Rac-1 expression results in a hormone-independent increase in surface GLUT4; however, the molecular mechanism and significance behind this effect remain unresolved. Using L6 myoblasts stably expressing myc-tagged GLUT4, we found that overexpression of constitutively active but not wild-type Rac-1 sufficed to drive GLUT4 translocation to the membrane of comparable magnitude with that elicited by insulin. Stimulation of endogenous Rac-1 by Tiam1 overexpression elicited a similar hormone-independent gain in surface GLUT4. This effect on GLUT4 traffic could also be reproduced by acutely activating a Rac-1 construct via rapamycin-mediated heterodimerization. Strategies triggering Rac-1 “superactivation” (i.e. to levels above those attained by insulin alone) produced a modest gain in plasma membrane phosphatidylinositol 3,4,5-trisphosphate, moderate Akt activation, and substantial AS160 phosphorylation, which translated into GLUT4 translocation and negated the requirement for IRS-1. This unique signaling capacity exerted by Rac-1 superactivation bypassed the defects imposed by JNK- and ceramide-induced insulin resistance and allowed full and partial restoration of the GLUT4 translocation response, respectively. We propose that potent elevation of Rac-1 activation alone suffices to drive insulin-independent GLUT4 translocation in muscle cells, and such a strategy might be exploited to bypass signaling defects during insulin resistance.     

p38alpha MAPK can positively or negatively regulate Rac-1 activity depending on the presence of serum

The small GTP-ase Rac-1 can trigger p38 MAPK activation and, in turn, p38alpha can regulate signalling pathways that potentially impinge on Rac-1 activity. We have investigated the cross-talk between p38alpha and Rac-1 and found that p38alpha regulates the association between Rac-1 and caveolin-1 in serum-deprived cardiomyocytes. This interaction depends on cell attachment and correlates with higher levels of active Rac-1. Actin organization might regulate the formation of Rac-1-caveolin-1 complexes. In contrast, the Rac-1-caveolin-1 interaction is almost undetectable in the presence of serum, where Rac-1 activity is negatively regulated by p38alpha. Our results indicate that p38alpha can differentially contribute to Rac-1 activation depending on the presence of serum.     

Induction of the Galpha(q) signaling cascade by the human immunodeficiency virus envelope is required for virus entry

Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of Galpha(q) and its downstream targets. Fusion and Rac-1 activation are mediated by Galpha(q) and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting Galpha(q) or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca2+ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for Galpha(i) or Galpha(s) signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the Galpha(q) pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance.     

Pigment epithelium-derived factor (PEDF) blocks the interleukin-6 signaling to C-reactive protein expression in Hep3B cells by suppressing Rac-1 activation

There is a growing body of evidence to show that that C-reactive protein (CRP), an acute phase reactant, is one of the most valuable predictors of future cardiovascular events. Since CRP proteins directly contribute to the development and progression of atherosclerosis as well, reduction of CRP levels may be a novel therapeutic target for the treatment of cardiovascular disease. In this study, we examined whether pigment epithelium-derived factor (PEDF) could block the interleukin-6-induced CRP expression in cultured human hepatoma cells and the way that it might achieve this effect. PEDF inhibited the IL-6-induced CRP expression in Hep3B cells at both mRNA and proteins levels. PEDF suppressed the intracellular reactive oxygen species generation in IL-6-exposed Hep3B cells. Anti-oxidants mimicked the effects of PEDF. PEDF was also found to inhibit the IL-6-elicited Rac-1 activation, whereas dominant-negative Rac-1 dose-dependently decreased the CRP mRNA levels. PEDF blocked the IL-6-induced STAT3 phosphorylations and NF-kappaB p65 activity in Hep3B cells. Our present study suggests that PEDF could be one of the potent suppressors of CRP production by the liver and may play a protective role against atherosclerosis.     

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.     

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation, whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development.     

Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N -formyl- l -Methionyl- l -Leucyl- l -P henylalanine (f MLP) with a rise in the intracellular calcium concentration ([Ca2]i), NADPH oxidase activation, and increased superoxide anion (O2-) production. [Ca2+]i mobilization and superoxide production were largely dependent on extracellular calcium (Ca2+]e) and a capacitative calcium entry. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We tested the effect of removal of Ca2+]e on Rac-1 plasma membrane sequestration and activation of NADPH oxidase using immunodetection and a double labelling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and that Rac-1 membrane sequestration was preceded by [Ca2+]i mobilization following entry of Ca2+ e. Therefore, we propose that O2- production is dependent on activation of PTX-sensitive G-proteins and sequestration of Rac-1 in the plasma membrane, following entry of Ca2+ e.     

Myosin IIB deficiency in embryonic fibroblasts affects regulators and core members of the par polarity complex

Wild-type (WT) and myosin heavy chain IIB null [MHCIIB (−/−)] embryonic fibroblasts were used as an experimental model to assess the role of the isoform B of myosin II (MII) in the regulation of the cell shape and intrinsic polarity. Genetic ablation of MHCIIB causes a persistent albeit, unstable protrusive activity in embryonic fibroblasts (Lo et al. in Nonmuscle myosin IIB is involved in the guidance of fibroblast migration. Mol Biol Cell 15:982–989, 2004). Here, we show that MHCIIB-deficient fibroblasts are characterized by a sustained guanine nucleotide exchange factor (GEF)-dependent activation of the small GTPase Rac-1 that is responsible for the continual lamellipodium formation. Moreover, we observed a sustained PKC-ζ activation and an increased association of cortactin with the plasma membrane in the MHCIIB (−/−) cells that were also dependent on GEF-mediated Rac-1 activation. Rac-1 activation and its downstream effects were induced in WT fibroblasts by inhibiting MII ATPase and crosslinking activities, suggesting that an altered actin-MII interaction favours Rac-1 activation, regardless of the MII isoform implicated. In addition, we found MIIB isoform-specific effects that were independent of Rac-1 activation. MHCIIA interacts with cortactin whereas MHCIIB does not. By contrast, MHCIIB interacts with Lgl1, a member of the Scribble/Dlg/Lgl polarity complex, whereas MHCIIA does not. MHCIIB (−/−) fibroblasts exhibited deregulated endogenous levels of the Par polarity complex members, Par3 and Par6. Together, the data show that MHCIIB deficiency causes imbalances in signalling pathways that are responsible for cell polarity determination. The results suggest that these pathways are targets of MIIB in the regulation of the cell’s shape and polarity.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of T cell stimulation

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.     

Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.     

Light-mediated activation of Rac-1 in photoreceptor outer segments

Small GTP binding proteins regulate diverse biological processes including gene expression, cytoskeleton reorganization, and protein and vesicular transport. While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin. To increase their sensitivity to light, photoreceptors accumulate remarkably high concentrations of rhodopsin and transducin in specialized cellular compartments, the outer segments (OS). Transport of these proteins from the inner segments is regulated by the small GTPases Rab6 and Rab8, which do not enter OS. Here, we asked if small G proteins have other functions in photoreceptors. We show that OS contain the small GTPase Rac-1, a member of the Rho family. In contrast to other cells, Rac-1 in OS is exclusively associated with the membranes and resides in lipid rafts. Most importantly, Rac-1 is activated by light. This activation is specifically blocked by a synthetic peptide corresponding to the Asn-Pro-X-X-Tyr motif found in rhodopsin, and Rac-1 coprecipitates with rhodopsin on Concanavalin A Sepharose. These data provide the first direct evidence for the existence of a novel pathway activated by rhodopsin.     

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.     

Endothelial ICAM-1 protein induction is regulated by cytosolic phospholipase A2α via both NF-κB and CREB transcription factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.     

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.     

Oxidative stress-mediated mesangial cell proliferation requires RAC-1/reactive oxygen species production and beta4 integrin expression

Lipid abnormalities and oxidative stress, by stimulating mesangial cell (MC) proliferation, can contribute to the development of diabetes-associated renal disease. In this study we investigated the molecular events elicited by oxidized low density lipoproteins (ox-LDL) in MC. We demonstrate that in MC cultured in the presence of ox-LDL, survival and mitogenic signals on Akt and Erk1/2 MAPK pathways are induced, respectively. Moreover, as shown by the expression of the dominant negative Rac-1 construct, we first report that ox-LDL-mediated cell survival and cell cycle progression depend on Rac-1 GTPase-mediated reactive oxygen species production and on epidermal growth factor receptor transactivation. By silencing Akt and blocking Erk1/2 MAPK pathways, we also demonstrate that these signals are downstream to Rac-1/reactive oxygen species production and epidermal growth factor receptor activation. Finally, by endogenous depletion of beta4 integrin, expressed in MC, we provide evidence that the expression of this adhesion molecule is essential for ox-LDL-mediated MC dysfunction. Our data identify a novel signaling pathway involved in oxidative stress-induced diabetes-associated renal disease and provide the rationale for therapeutically targeting beta4 integrin.