IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG‐63 cells

Insulin has been proposed to be an anabolic agent in bone, but the mechanisms underlying insulin effects on osteoblast differentiation are still not clear. To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line‐MG‐63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3‐K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG‐63 cells in a time‐ and dose‐dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3‐K inhibitor LY294002 significantly reversed the down‐regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up‐regulation of Osx and IGF‐1 expression after insulin treatment. Therefore, the data obtained from this study suggested that insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG‐63 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Guanosine inhibits CD40 receptor expression and function induced by cytokines and beta amyloid in mouse microglia cells

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-gamma (10 ng/ml) plus TNF-alpha (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta(1-42); 500 nM). Culture pretreatment with guanosine (10-300 microM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-gamma/TNF-alpha challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-gamma-induced phosphorylation on Ser(727) and translocation of STAT-1alpha to the nucleus as well as TNF-alpha-/Abeta-induced IkappaBalpha and NF-kappaB p65/RelA subunit phosphorylation, thus inhibiting NF-kappaB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-gamma/TNF-alpha-induced CD40 expression and function as well as on STAT-1alpha or NF-kappaB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer's disease.     

Pertussis toxin activates tyrosine kinase signaling cascade in myelomonocytic cells: a mechanism for cell adhesion

Pertussis toxin (PTX) has recently been shown to specifically bind to CD14 to promote myelomonocytic cell adhesion to serum. The present study investigated the signaling mechanisms responsible for PTX-induced differentiated U937 cell adhesion. PTX-induced myelomonocytic cell adhesion was blocked by genistein or tyrphostin-47 (two protein tyrosine kinase inhibitors), LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), or PD098059 (a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor). PTX induced a rapid tyrosine phosphorylation of several discrete cytoplasmic proteins, which could be inhibited by genistein or tyrphostin 47. In addition, PTX induced phosphorylation of Akt and of ERK2, which could be completely blocked by LY294002 and PD098059, respectively, and by genistein or tyrphostin 47 as well. All of these PTX-induced signaling events could be reproduced using purified PTX B-oligomer (PTX-B) alone. Our data show that PTX can activate tyrosine kinase signaling cascade, including the downstream PI3K and ERK/MAPK pathways, in myelomonocytic cells to induce cell adhesion to serum.     

Tryptase activates PKB in inflammatory reaction in ECV304 cells

Tryptase is involved in proteinase-activated receptor-2 (PAR-2) mediated up-regulation of IL-8 expression. The present report showed the effects of tryptase on gene expression and activation, including up-regulation IL-8 expression. The expression of mRNA for NF-kappaB first increased at 1 h after tryptase-treatment (1 ng/ml) and reached the plateau after 4 h. The NF-kappaB mRNA increased by 3-fold (n = 3, P < 0.05), AP-1 by 2-fold (n = 3, P < 0.05), and PKB by 10-fold (n = 3, P < 0.05). However, tryptase-treatment did not affect the expression of JNK and p38 MAPK when compared with control cells at mRNA level. Furthermore, in addition to increasing phosphorylation of p38 MAPK, tryptase-treatment also increased phosphorylation of PKB by 2-fold at 15 min following the treatment. The up-regulation and phosphorylation of PKB by tryptase could be abolished by either phosphoinositol-3-kinase (PI3K) inhibitor (LY294002) at 10 microM or antisense PKB cDNA transfection. The up-regulation of NF-kappaB expression could be inhibited by LY294002 and antisense PKB cDNA. These results indicate that tryptase can activate PI3K-PKB pathway and enhance IL-8 expression.     

Insulin-Stimulated Glycogen Synthesis in Cultured Hepatoma Cells: Differential Effects of Inhibitors of Insulin Signaling Molecules

Abstract

In rat HTC hepatoma cells overexpressing human insulin receptors, insulin stimulated glycogen synthesis by 55–70%. To study postreceptor signaling events leading to insulin-stimulated glycogen synthesis in these cells, we have employed pathway-specific chemical inhibitors such as LY294002, rapamycin and PD98059 to inhibit phosphatidylinositol-3-kinase (PI3K), p70 ribosomal S6 kinase and mitogen-activated protein kinase (MAPK) kinase/MAPK, respectively. LY294002 (50 μM) completely abolished insulin-stimulated glycogen synthesis whereas rapamycin (2–20 nM) partially inhibited it. Neither LY294002 nor rapamycin significantly affected the basal glycogen synthesis. However, PD98059 (100 μM) significantly inhibited the basal glycogen synthesis without affecting insulin-stimulated glycogen synthesis. In these cells, insulin at 100 nM decreased glycogen synthase kinase 3α (GSK3α) activity by 30–35%. LY294002, but neither rapamycin nor PD98059, abolished insulin-induced inactivation of GSK3α. These data suggest that insulin-stimulated glycogen synthesis in rat HTC hepatoma cells is mediated mainly by PI3K-dependent mechanism. In these cells, inactivation of GSK3α, downstream of PI3K, may play a role in insulin-stimulated glycogen synthesis.     

Supernatant from bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells

Background

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, the signaling pathways engaged by probiotics are poorly understood. We have previously reported that a fermentation product from Bifidobacterium breve C50 (BbC50sn) could induce maturation, high IL-10 production and prolonged survival of DCs via a TLR2 pathway. We therefore studied the roles of mitogen-activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K) pathways on biological functions of human monocyte-derived DCs treated with BbC50sn.

Methodology/Principal Findings

DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50sn, lipopolysaccharide (LPS) or Zymosan, with or without specific inhibitors of p38MAPK (SB203580), ERK (PD98059), PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and GSK3 (SB216763). We found that 1) the PI3K pathway was positively involved in the prolonged DC survival induced by BbC50sn, LPS and Zymosan in contrast to p38MAPK and GSK3 which negatively regulated DC survival; 2) p38MAPK and PI3K were positively involved in DC maturation, in contrast to ERK and GSK3 which negatively regulated DC maturation; 3) ERK and PI3K were positively involved in DC-IL-10 production, in contrast to GSK3 that was positively involved in DC-IL-12 production whereas p38MAPK was positively involved in both; 4) BbC50sn induced a PI3K/Akt phosphorylation similar to Zymosan and a p38MAPK phosphorylation similar to LPS.

Conclusion/Significance

We report for the first time that a fermentation product of a bifidobacteria can differentially activate MAPK, GSK3 and PI3K in order to modulate DC biological functions. These results give new insights on the fine-tuned balance between the maintenance of normal mucosal homeostasis to commensal and probiotic bacteria and the specific inflammatory immune responses to pathogen bacteria.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma

Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen-activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up-regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF-induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF-induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF-induced B16F10 melanogenesis by up-regulating tyrosinase expression.