Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21(ras) protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IkappaB-alpha. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-alpha production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-alpha production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism.
"Cells that encounter MSU crystals express a broad array of inflammatory mediators that contribute to acute gouty inflammation . The role of SHIP-1 is essential for the suppressor activity of macrophages and is a potent inhibitor of many inflammatory pathways [24,25]. Recently, it has been shown that monocytes and macrophages overexpressing miR-155 exhibit decreased SHIP-1 expression that may lead to increased production of pro-inflammatory cytokines . "
[Show abstract][Hide abstract] ABSTRACT: Gout is characterized by episodes of intense joint inflammation in response to intra-articular monosodium urate monohydrate (MSU) crystals. miR-155 is crucial for the proinflammatory activation of human myeloid cells and antigen-driven inflammatory arthritis. The functional role of miR-155 in acute gouty arthritis has not been defined. Therefore, the aim of this study was to examine the role of miR-155 in pathogenesis of acute gouty arthritis.
Samples from 14 patients with acute gouty arthritis and 10 healthy controls (HCs) were obtained. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were cultured in vitro with MSU crystals, and gene expression (human miR-155 and SHIP-1) were assessed by real-time PCR. THP-1 cells were stimulated by MSU crystals and/or miR-155 transfection and then subjected to Western blot analysis. Levels of human tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in cell culture supernatants were measured by Luminex. Immunohistochemistry was performed on formalin-fixed gout tissues with anti-SHIP-1 antibody. A C57BL/6J male mouse model of gout was used to analyze the expressions of miR-155, SHIP-1, and inflammatory cytokines.
The samples from gouty arthritis were highly enriched in miR-155, with levels of expression being higher than those found in PBMC from HC. Treatment of the cells with MSU crystals strongly induced miR-155. In addition, overexpression of miR-155 in the cells decreased levels of SHIP-1 and promoted production of MSU-induced proinflammatory cytokines, such as TNF-alpha and IL-1beta. Consistent with in vitro observations, miR-155 expression was elevated in the mouse model of gout. The production of inflammatory cytokines was markedly increased in MSU crystal induced peritonitis mice.
Overexpression of miR-155 in the gouty SFMC leads to suppress SHIP-1 levels and enhance proinflammatory cytokines.
"MAPKs can be activated by Toll-like receptor 4 (TLR4) leading to the activation of nuclear translocation of NF-κB and finally initiates pro-inflammatory responses . NF-κB is activated by phosphorylation of IκBα via activation of MAPKs such as ERK1/2, JNK and p38 subfamilies, and then migrates into the nucleus and activates the expression of inflammatory cytokines and mediators [12,19,22]. The activation of NF-κB in response to pro-inflammatory stimuli such as TNF-α through phosphorylation of IκBα results in the NF-κB p65–p50 heterodimer to migrate into the nucleus and up-regulating the expression of pro-inflammatory and anti-apoptotic genes . "
[Show abstract][Hide abstract] ABSTRACT: Wedelolactone (WEL), a major coumestan ingredient in Wedelia chinensis, has been used to treat septic shock, hepatitis and venom poisoning in traditional Chinese medicines. The objective of the study was to elucidate the anti-inflammatory effects and mechanism of WEL with a cellular model of lipopolysaccharide (LPS)-induced RAW264.7 cells.
To study the role of WEL in pro-inflammation, we measured key inflammation mediators and end products including nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) by using the Griess method, enzyme linked immunosorbent assay (ELISA) and Western blotting. Nuclear factor-kappaB (NF-kappaB) transcription activity was detected by luciferase reporter assay. The important pro-inflammatory transcription factors, NF-kappaB p65 and inhibitory kappaB alpha (IkappaB-alpha); and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) were analyzed by Western blotting. Our study showed that WEL (0.1, 1, 10 muM) significantly inhibited the protein expression levels of iNOS and COX-2 in LPS-stimulated cells, as well as the downstream products, including NO, PGE2 and TNF-alpha. Moreover, WEL also inhibited LPS-induced NF-kappaB p65 activation via the degradation and phosphorylation of IkappaB-alpha and subsequent translocation of the NF-kappaB p65 subunit to the nucleus.
Our results revealed that WEL has a potential to be a novel anti-inflammatory agent targeting on the NF-kappaB signaling pathway.
"Although innate inflammatory responses induced by ligands for TLR9 and other TLRs are essential for the eradication of infectious microorganisms, excessive and prolonged activation of innate immunity is detrimental to the host. As a part of negative regulatory mechanisms to prevent exaggerated inflammatory reactions, TLR ligands induce several negative regulators, such as IL-10, Src homology 2 domain-containing inositol polyphosphate phosphatase 1, suppressor of cytokine signaling proteins, and IRAK-monocyte (IRAK-M) , , , , , . Among these, IRAK-M has been demonstrated to down-regulate the inflammatory response by directly blocking TLR/MyD88 signal transduction . "
[Show abstract][Hide abstract] ABSTRACT: As a part of the negative feedback mechanism, CpG DNA induces IRAK-M expression in monocytic cells. In the present study we investigated a biochemical signaling pathway and the transcription factors responsible for CpG DNA-mediated Irak-m gene expression. CpG DNA-induced Irak-m expression did not require new protein synthesis and was regulated at the transcriptional level through an endosomal pH-sensitive TLR9/MyD88 signaling pathway. Over-expression of the dominant negative (DN) form of or gene-specific knockdown of signaling modulators in the TLR9 pathway demonstrated that IRAK4, IRAK1, IRAK2, and PKD1 are required for Irak-m transcription induced by CpG DNA. Over-expression of DN-IRAK1 only partially, but significantly, inhibited CpG DNA-induced Irak-m promoter activity. While IRAK1 was critical for the initial phase, IRAK2 was required for the late phase of TLR9 signaling by sustaining activation of PKD1 that leads to activation of NF-κB and MAPKs. Irak-m promoter-luciferase reporters with alterations in the predicted cis-acting transcriptional regulatory elements revealed that the NF-κB consensus site in the Irak-m promoter region is absolutely required for Irak-m gene expression. AP-1 and CREB binding sites also contributed to the optimal Irak-m expression by CpG DNA. Collectively, our results demonstrate that IRAK2 plays a key role in the TLR9-mediated transcriptional regulation of Irak-m expression by sustaining activation of PKD1 and NF-κB.
PLoS ONE 08/2012; 7(8):e43970. DOI:10.1371/journal.pone.0043970 · 3.23 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.