巨噬细胞免疫调变信号——PKA与PKC对MAPK信号通路的调节

以前的研究工作表明,细菌脂多糖（ＬＰＳ）可以调变抑制性巨噬细胞为增强Ｔ、Ｂ淋巴细胞及ＮＫ细胞活性,同时又能保持或增强其抗肿瘤效应。忆报道了在这一复杂的免疫调变过程中伴随有蛋白激酶Ｃ（ＰＫＣ）和促分裂原活化蛋白激酶（ＭＡＰＫ）信号转导通路的激活。为了探索免疫调变过程中其他信号对ＭＡＰＫ通路的影响,以ＬＰＳ调变小鼠腹腔抑制性巨噬细胞为模型,研究了ｃＡＭＰ／ＰＫＡ和佛波酯（ＰＭＡ）／ＰＫＣ信号对ＭＡＰＫ     

LPS介导细胞激活的信号转导：从CD14到p38MAPK通路的研究

近年来对脂多糖（ＬＰＳ）介导细胞激活的信号转导过程已取得实质性进展,ＬＰＳ与血浆ＬＰＳ结合蛋白（ＬＢＰ）结合被运输到单核巨噬细胞表面,与ｍＣＤ１４受体结合起起细胞激活。ＭＡＰＫ参与了ＬＰＳ激活细胞产生肿瘤坏死因子（ＴＮＦ）等活性物质的细胞内信号转导过程。ｐ３８ＭＡＰＫ对ＴＮＦ－α等细胞因子具有重要的调节作用。对ＬＰＳ激活细胞的信号转导研究呆能为治疗内毒素休克提供新的理论和思路。     

p38 MAPK信号传导通路

丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄｐｏｒｏｔｅｉｎｋｉｎａｓｅ,ＭＡＰＫ）介导了生长、发育,分裂,死亡,以及细胞间的功能同步等多种细胞生理功能,在哺乳动物细胞中已发现和克隆了ＥＲＫ、ＪＮＫ／ＳＡＰＫ,ＥＲＫ５／ＢＭＫ１和ｐ３８／ＲＫ四个ＭＡＰＫ亚族,这些新的ＭＡＰＫ介导了物理,化学反激,细菌产物,炎性细胞因子等多种刺激引起的细胞反应,ｐ３８亚族至少包括ｐ３８（α）,ｐ３８β,ｐ     

p38丝裂原活化蛋白激酶在不同细胞内定位的研究

用共聚焦显微镜对不同原代培养细胞的ｐ３８丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ,ＭＡＰＫ）进行定位,以探讨ｐ３８激活后入核在不同的细胞中是否具有普遍性。发现与单核细胞相似,未受刺激静止的心肌细胞、平滑肌细胞和内皮细胞的ｐ３８荧光强度呈弥散性分布;脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）刺激后,细胞核区荧光强度均明显增加,胞浆荧光强度均降低     

p38 MAPK参与LPS诱导RAW细胞TNF—α基因表达的调控

构建ＴＮＦ－α启动子驱动的荧光酶报告基因系统,研究ｐ３８ＭＡＰＫ信号转导系统对ＴＮＦ－α基因表达的影响,ＲＡＷ２６４．７细胞共转染实验发现,ＬＰＳ对ｐ３８的激活作用与其诱导ＴＮＦ－α转录活性的作用显著相关,虽然单纯转染ｐ３８未见明显诱导ＴＮＦ－α报告基因系统的转录活性。     

参与细胞凋亡的丝裂原活化蛋白激酶及其作用机制

丝裂原活化蛋白激酶家族（ＭＡＰＫｓ）参与细胞调亡的信号转导过程。在多数细胞中,ＪＮＫ／ＳＡＰＫｓ和ｐ３８诱导细胞调亡,ＥＲＫ促进细胞增殖;但在另一些细胞中则情况相反。ＭＡＰＫｓ途径与死亡受体途径之间存在一定的联系,它们间的交互作用尚待一步研究。     

p38MAPK信号通路

细胞表面受体到核的信号通路是现代生物学研究的主题之一。细胞外各种刺激通过和膜受体偶联的Ｇ蛋白和酪氨酸激酶介导了一系列丝氨酸／苏氨酸激酶介导的级联反应,即分裂原激活蛋白激酶（ＭＡＰＫ）级联反应。ＭＡＰＫ级联反应把细胞外信号传递到核,并汇总了各种信号通路来的传息,因此研究细胞内ＭＡＰＫ的信号通路是十分重要的．本文简要介绍了ＥＲＫ,ＪＮＫ和ｐ３８三种ＭＡＰＫ途径,着重叙述了ｐ３８ＭＡＰＫ信号途径的性质和功能以及在免疫细胞中的作用和某些疾病的临床关系。     

细胞信号转导分子在TNF—α诱导c—jun基因表达中的作用

前期研究表明ｐ３８丝裂原活化蛋白激酶（ＭＡＰＫ）通过磷酸化心肌细胞增强因子２（ｍｙｏｃｙｔｅ ｅｎｈａｎｃｅｒ ｆａｃｔｏｒ２,ＭＥＦ２）转录因子家族成员调节ｃ－Ｊｕｎ蛋白表达。ｃ－ｊｕｎ的启动子区存在ＭＥＦ２位点,ＭＥＦ２转录因子家族成员以同源或异源二聚体形式与其结合。研究了ｐ３８和ＢＭＫ１（ｂｉｇ ＭＡＰ ｋｉｎａｓｅ１）在ＴＮＦ－α诱导ｃ－ｊｕｎ基因表达中的调控作用。ｐ３８上调ＭＥＦ２Ａ的转     

LPS和PMA诱导小鼠腹腔巨噬细胞的PKC—α和PKC—ε的激活与转位

利用免疫印迹技术及内源性底物磷酸化方法,我们研究了在巨噬细胞的信号传递中起重要作用的ＰＫＣ同功酶的分布及其在免疫调变剂ＬＰＳ的刺激下产生的激活和转位。在未激活的巨噬细胞中,ＰＫＣ－β捻量高于ＰＫＣ－α和ε,它和ＰＫＣ－α的分布均是胞质中大于胞膜。以ＰＭＡ为阳性对照,结果提示ＬＰＳ介导的抑制性巨噬细胞免疫调变机制中涉及到ＰＫＣ－α和ＰＫＣ－ε从胞质到膜组份的转位而不是ＰＫＣ－β（ＰＫＣ－βＩ或βⅡ）     

MAPK信号转导通路对炎证反应的调控

丝裂原活化蛋白激酶（ｍｉｔｏｈｅｎ－ａｃｔｅｖａｔｃｄ ｐｒｏｔｅｉｎ ｋｉｎａｓａ,ＭＡＰＫ）是生物体内重要的信号转导系统之一,参与介导生长、发育、化裂、分化、死亡以及细胞间的功能同步等多种细胞过程,在哺乳动物细胞中已发现和克隆了ＥＲＫ、ＪＮＫ／ＳＡＰＫ、ｐ３８／ＲＫ、ＥＲＫ５／ＢＭＫ１四个ＭＡＰＫ亚族。这些ＭＡＰＫ能被多种炎性刺激所激活,并对炎症的发生、发展起生重要调控作用。研究感染和炎症反应     

Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK

p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.     

Activation of the MAPK signal cascade by the neural cell adhesion molecule L1 requires L1 internalization

L1-mediated axon growth involves intracellular signaling, but the precise mechanisms involved are not yet clear. We report a role for the mitogen-activated protein kinase (MAPK) cascade in L1 signaling. L1 physically associates with the MAPK cascade components Raf-1, ERK2, and the previously identified p90(rsk) in brain. In vitro, ERK2 can phosphorylate L1 at Ser(1204) and Ser(1248) of the L1 cytoplasmic domain. These two serines are conserved in the L1 family of cell adhesion molecules, also being found in neurofascin and NrCAM. The ability of ERK2 to phosphorylate L1 suggests that L1 signaling could directly regulate L1 function by phosphorylation of the L1 cytoplasmic domain. In L1-expressing 3T3 cells, L1 cross-linking can activate ERK2. Remarkably, the activated ERK localizes with endocytosed vesicular L1 rather than cell surface L1, indicating that L1 internalization and signaling are coupled. Inhibition of L1 internalization with dominant-negative dynamin prevents activation of ERK. These results show that L1-generated signals activate the MAPK cascade in a manner most likely to be important in regulating L1 intracellular trafficking.     

Augmented lipopolysaccharide-induced TNF-alpha production by peritoneal macrophages in type 2 diabetic mice is dependent on elevated glucose and requires p38 MAPK

Dysregulated inflammation is a complication of type 2 diabetes (T2D). In this study, we show that augmented LPS-induced TNF-alpha production by resident peritoneal macrophages (PerMphi) in type 2 diabetic (db/db) mice is dependent on elevated glucose and requires p38 MAPK. Intraperitoneal LPS administered to db/db and nondiabetic (db/+) mice induced 3- and 4-fold more TNF-alpha in the peritoneum and serum, respectively, of db/db mice as compared with db/+ mice. Examination of the TLR-4/MD2 complex and CD14 expression showed no difference between db/db and db/+ PerMphi. Ex vivo stimulation of PerMphi with LPS produced a similar 3-fold increase in TNF-alpha production in db/db PerMphi when compared with db/+ PerMphi. PerMphi isolated from db/+ mice incubated in high glucose (4 g/L) medium for 12 h produced nearly 2-fold more TNF-alpha in response to LPS than PerMphi incubated in normal glucose medium (1 g/L). LPS-dependent stimulation of PI3K activity, ERK1/2 activation, and p38 kinase activity was greater in PerMphi from db/db mice as compared with db/+ mice. Only inhibition of p38 kinase blocked LPS-induced TNF-alpha production in PerMphi from db/db mice. Taken together, these data indicate that augmented TNF-alpha production induced by LPS in macrophages during diabetes is due to hyperglycemia and increased LPS-dependent activation of p38 kinase.     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.     

p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression

Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor beta-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38alpha by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b-->p38alpha blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.     

Macrophage-colony-stimulating factor-induced proliferation and lipopolysaccharide-dependent activation of macrophages requires Raf-1 phosphorylation to induce mitogen kinase phosphatase-1 expression

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of cyclin-dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions.     

Dual beta-adrenergic modulation in the immune system: stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between beta-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which beta-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that beta-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.