PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

Syntenin negatively regulates TRAF6-mediated IL-1R/TLR4 signaling

Toll-like receptors are involved in host defense against invading pathogens. The two members of this superfamily, IL-1R and TLR4, activate overlapping NF-kappaB activate signaling pathway mediated by TRAF6. In this study, we identified syntenin as a negative regulator of IL-1R and TLR4 mediated NF-kappaB activation. Overexpressed syntenin inhibited IL-1- or LPS-, but not TNF- induced NF-kappaB activation and IL-8 mRNA expression in a dose dependent manner. Syntenin specifically interacted with TRAF6 in human 293 cells, and inhibited TRAF6 induced NF-kappaB and AP-1 activation. Syntenin also associated with TRAF6 under physiological condition, and dissociated from TRAF6 upon IL-1 stimulation. This might be due to a competition between syntenin and IRAK1, as overexpression of IRAK1 disrupted the interaction of syntenin with TRAF6, and rescued syntenin induced reduction of TRAF6 ubiquitination. Moreover, knockdown of syntenin potentiated IL-1- or LPS- triggered NF-kappaB activation and IL-8 mRNA expression. These findings suggest that syntenin is a physiological suppressor of TRAF6 and plays an inhibitory role in IL-1R- and TLR4- mediated NF-kappaB activation pathways.     

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages

Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development and progression of many chronic diseases.     

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.     

Human Fas-associated factor 1 interacts with heat shock protein 70 and negatively regulates chaperone activity

We examined the cell death-inducing property of human Fas-associated factor 1 (hFAF1) in the heat shock signaling pathway. By employing co-immunoprecipitation and peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that hFAF1 binds to the 70-kDa heat shock protein family (Hsc70/Hsp70). Interaction mapping indicated that the 82-180 sequence of hFAF1 directly binds to the N-terminal region containing sequence 1-120 of Hsc70/Hsp70. This binding is very tight regardless of ATP and heat shock treatment. Hsc70/Hsp70 and hFAF1 co-localized in the cytosol and nucleus and concentrated to the perinuclear region by heat shock treatment. We examined how hFAF1 regulates Hsp70 function, and found that hFAF1 inhibited the Hsp70 chaperone activity of refolding denatured protein substrates, accelerated heat shock-induced SAPK/JNK activation, and raised heat shock-induced cell death in a binding dependent manner. These results suggest that hFAF1 prevents cells from recovery after stress by binding to and inhibiting the chaperone activity of Hsp70.     

Decursin negatively regulates LPS-induced upregulation of the TLR4 and JNK signaling stimulated by the expression of PRP4 in vitro

ABSTRACT

The current investigation was carried out to analyze the correlation of bacterial lipopolysaccharide (LPS) and pre-mRNA processing factor 4B (PRP4) in inducing inflammatory response and cell actin cytoskeleton rearrangement in macrophages (Raw 264.7) and colorectal (HCT116) as well as skin cancer (B16-F10) cells. Cell lines were stimulated with LPS, and the expression of PRP4 as well as pro-inflammatory cytokines and proteins like IL-6, IL-1β, TLR4, and NF-κB were assayed. The results demonstrated that LPS markedly increased the expression of PRP4, IL-6, IL-1β, TLR4, and NF-κB in the cells. LPS and PRP4 concomitantly altered the morphology of cells from an aggregated, flattened shape to a round shape. Decursin, a pyranocoumarin from Angelica gigas, inhibited the LPS and PRP4-induced inflammatory response, and reversed the induction of morphological changes. Finally, we established a possible link of LPS with TLR4 and JNK signaling, through which it activated PRP4. Our study provides molecular insights for LPS and PRP4-related pathogenesis and a basis for developing new strategies against metastasis in colorectal cancer and skin melanoma. Our study emphasizes that decursin may be an effective treatment strategy for various cancers in which LPS and PRP4 perform a critical role in inducing inflammatory response and morphological changes leading to cell survival and protection against anti-cancer drugs.     

MCPIP1 negatively regulates toll-like receptor 4 signaling and protects mice from LPS-induced septic shock

Septic shock is one of leading causes of morbidity and mortality in hospital patients. However, genetic factors predisposing to septic shock are not fully understood. Our previous work showed that MCP-induced protein 1 (MCPIP1) was induced by lipopolysaccharides (LPSs), which then negatively regulates LPS-induced inflammatory signaling in vitro. Here we report that although MCPIP1 was induced by various toll-like receptor (TLR) ligands in macrophages, MCPIP1-deficient mice are extremely susceptible to TLR4 ligand (LPS)-induced septic shock and death, but not to the TLR2, 3, 5 and 9 ligands-induced septic shock. Consistently, LPS induced tumor necrosis factor α (TNFα) production in MCPIP1-deficient mice was 20-fold greater than that in their wild-type littermates. Further analysis revealed that MCPIP1-deficient mice developed severe acute lung injury after LPS injection and JNK signaling was highly activated in MCPIP1-deficient lungs after LPS stimulation. Finally, macrophage-specific MCPIP1 transgenic mice were partially protected from LPS-induced septic shock, suggesting that inflammatory cytokines from sources other than macrophages may significantly contribute to the pathogenesis of LPS-induced septic shock. Taken together, these results suggest that MCPIP1 selectively suppresses TLR4 signaling pathway and protects mice from LPS-induced septic shock.     

Zebrafish heat shock protein a4 genes in the intestinal epithelium are up-regulated during inflammation

A number of heat shock proteins (HSPs), including Hsp70 and Hsp110, function as molecular chaperones within intestinal epithelial cells that line the mammalian digestive system. HSPs confer cellular protection against environmental stress induced by chemical toxins or pathogens. There is interest in how members of this protein family might influence the progression of inflammatory bowel disease. Using the zebrafish model system, we report the expression of the duplicated hspa4 genes within the intestinal epithelium. The hspa4 genes belong to the Hsp110 family. We show that under inflammatory stress conditions within the gut, expression of these genes is up-regulated in a similar manner to that previously observed for mammalian Hsp70. Because of the amenability of the zebrafish to whole-animal screening protocols, the hspa4 genes could be used as effective read-outs for genetic, chemical and environmental factors that might influence intestinal inflammation.     

Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening

Cigarette smoke is associated with increased carotid intimal thickening or stroke. Preliminary work showed that exposure to smoke resulted in a 4.5-fold reduction of heat shock protein-70 (HSP70) expression in spleens of mice using gene microarray analysis. In the current study, we investigated the role of extracellular HSP70 in carotid intimal thickening of mice exposed to cigarette smoke. Intimal thickening was induced by placement of a cuff around the right carotid artery of mice. Cuff injury resulted in increased HSP70 mRNA expression in carotid arteries that persisted for 21 days. Cigarette smoke exposure decreased arterial HSP70 expression and significantly increased intimal thickening compared with mice exposed to air. Treatment of mice exposed to cigarette smoke with intravenous recombinant HSP70 attenuated intimal thickening through reduced phosphorylated extracellular signal-regulated kinase (pERK) expression in the arterial wall. In vitro experiments with rat aortic smooth muscle cells confirmed that recombinant HSP70 decreases pERK and proliferating cell nuclear antigen (PCNA) expression in cells exposed to cigarette smoke extract and H(2)O(2). Our study suggests that decreased expression of arterial HSP70 is an important mechanism by which exposure to cigarette smoke augments intimal thickening. The effects of recombinant HSP70 suggest a role for extracellular HSP70.     

The heat shock response and cytoprotection of the intestinal epithelium

Following heat stress, the mammalian intestinal epithelial cells respond by producing heat shock proteins that confer protection under stressful conditions, which would otherwise lead to cell damage or death. Some of the noxious processes against which the heat shock response protects cells include heat stress, infection, and inflammation. The mechanisms of heat shock response-induced cytoprotection involve inhibition of proinflammatory cytokine production and induction of cellular proliferation for restitution of the damaged epithelium. This can mean selective interference of pathways, such as nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinase (MAPK), that mediate cytokine production and growth responses. Insight into elucidating the exact protective mechanisms could have therapeutic significance in treating intestinal inflammations and in aiding maintenance of intestinal integrity. Herein we review findings on heat shock response-induced intestinal epithelial protection involving regulation of NF-kappaB and MAPK cytokine production.     

Stress management - heat shock protein-70 and the regulation of apoptosis

Apoptosis, molecularly regulated cell death, can be induced by a range of environmental, physical or chemical stresses, and is characterized by a sequence of precisely regulated events that culminate in the self-destruction of a cell. Fascinating biochemical and genetic parallels exist between the cell death pathways of different animal species. However, an even more highly conserved and evolutionarily ancient cellular response can be engaged as a consequence of stress, which functions to maintain cellular survival. This response is mediated by the heat-shock or stress proteins. This article discusses the functional interactions between the stress response and the apoptotic cell death pathway and how these might impact on determining cellular survival.     

Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells

Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-kappaB activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 microg/ml HSP-70) for 18 h, followed by LPS stimulation (1 microg/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-kappaB-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP-70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100 degrees C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor kappaB-alpha and inhibited activation of inhibitor kappaB kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-kappaB following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.     

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.     

Leptin downregulates heat shock protein-70 (HSP-70) gene expression in chicken liver and hypothalamus

Heat shock protein (HSP)-70 is expressed in normal and stressed cells but is highly stress-inducible. Although leptin has long been suggested to be involved in the regulation of stress response, its interaction with the HSP-70 gene is still unknown, under both unstressed and stressed conditions. The present study has aimed to investigate the effect of leptin on HSP-70 gene expression in normal chicken liver, hypothalamus, and muscle. Continuous infusion of recombinant chicken leptin (8 μg/kg per hour) at a constant rate of 3 ml/h for 6 h in 3-week-old broiler chickens significantly (P < 0.05) decreased food intake and HSP-70 mRNA levels in liver and hypothalamus, but not in muscle. In an attempt to discriminate between the effect of leptin and of leptin-reduced food intake on HSP-70 gene expression, we also evaluated the effect of food deprivation on the same cellular responses in two broiler chicken lines genetically selected for low (LL) or high (FL) abdominal fat pad size. Food deprivation for 16 h did not affect HSP-70 gene expression in any of the studied tissues indicating that the effect of leptin was independent of the inhibition of food intake. Regardless of the nutritional status, HSP-70 mRNA levels were significantly (P < 0.05) higher in the hypothalamus of FL compared with LL chickens consistent with higher mRNA levels for hypothalamic corticotropin-releasing factor. To assess, whether the effects of leptin were direct or indirect, we carried out in vitro studies. Leptin treatments did not affect HSP-70 mRNA levels in a leghorn male hepatoma cell line or quail myoblast cell line suggesting that the effect of leptin on HSP-70 gene expression is mediated through the central nervous system. Furthermore, HSP-70 gene expression was gender-dependent with significantly (P < 0.05) higher levels in male than in female chickens. This work was supported by a research grant (G.0402.05) from the FWO-Flanders (Belgium). No conflicts of interest would prejudice impartiality.     

Intracellular localization of constitutive and inducible heat shock protein 70 in rat liver after in vivo heat stress

The level and intracellular redistribution of the two nucleo-cytoplasmic members of 70 kDa heat shock protein family (constitutive, Hsc70 or Hsp73, and inducible, Hsp72) were studied in rat liver during a 24-h period after exposure of the animals to 41 degrees C whole body hyperthermic stress. The examined proteins were detected in the liver cytosol and nuclei by Western blotting and immunohistochemical staining of paraffin sections, as well as by immnocytochemical staining of isolated nuclear smears. All three techniques applied were based on the use of monoclonal antibodies recognizing both constitutive and inducible Hsp70 isoforms or only the inducible isoform, and gave consistent results. The exposure of the animals to in vivo heat stress was shown to induce the synthesis of otherwise non-existing Hsp72, rendering Hsc70 level unchanged in comparison to unstressed controls. However, immediately after the stress the intracellular redistribution of Hsc70, i.e. its nuclear accumulation, was observed. The maximal level of Hsp70 both in the cytoplasm and in the nuclei was registered 5 h after the stress, which coincided with the maximal level of Hsp72 induction. The alterations in the level and intracellular distribution of examined proteins were still noticeable 24 h after the stress. The results of this study could shed some more light on, as yet uncertain, differences between cellular functions of these two proteins, as well as on the role of the constitutive form under normal and stress conditions.