敲减NADPH氧化酶4能降低STAT3活性抑制人黑色素瘤A375细胞的增殖

NADPH氧化酶参与细胞活性氧族（ROS）的生成过程,而ROS与肿瘤细胞增殖密切相关. 为了阐明NADPH氧化酶影响黑色素瘤A375细胞增殖的分子机制,本文首先应用荧光定量PCR和Western 印迹证实NOX4为人黑色素瘤A375细胞的NADPH氧化酶功能核心亚基;随后根据NOX4基因设计3条干扰序列和对照序列并连接到pSuper-retro-puro载体,经鉴定后转化E.coli DH5α感受态细胞、筛选有效干扰序列并用于逆转录病毒包装,病毒液感染A375细胞并经嘌呤霉素筛选10 d,构建了NOX4缺陷的A375稳转细胞珠（A375 NOX4Δ）,其NOX4的mRNA和蛋白表达分别下降了66.02%和77.35%,伴随NADPH氧化酶活性和ROS水平分别下降了79.17%和64.16%;MTT、EdU法检测显示,A375-NOX4Δ细胞的增殖能力比A375-WT细胞明显降低、倍增时间延长,增殖细胞数量下降了68.27%（P＜0.01）,呈现G1→S期阻滞;Western blot检测表明A375 NOX4Δ细胞的 cyclin D1、CDK4分别下降了55.7%（P＜0.01）和64.8%（P＜0.01）,而P53、P21分别增加了6.89 倍（P＜0.01）和3.27 倍（P＜0.01）,STAT3、P-STAT3分别下降了51.80%（P＜0.05）和82.58%（P＜0.01）;电泳迁移率变动分析（EMSA）表明,A375 NOX4Δ细胞的STAT3-DNA结合活性明显降低. 上述结果提示,敲减A375细胞的NOX4表达可能通过减少ROS生成使得STAT3磷酸化水平及其结合DNA的活性下降,最终导致A375-NOX4Δ细胞增殖减少、呈现G1→S期阻滞,这为黑色素瘤发病机制研究提供了新思路及可能的药物作用靶点.     

Effects of Lipids on ENaC Activity in Cultured Mouse Cortical Collecting Duct Cells

Direct effects on epithelial Na⁺ channels (ENaC) activity by lipids, e.g., arachidonic acid (AA), eicosatetraynoic acid (ETYA), linoleic acid (LA), stearic acid (SA), hydroxyeicosatetraenoic acid (HETE), 11,12–epoxyeicosatrienoic acid (EET), (PGF2), and (PGE2), in cultured mouse cortical collecting duct (M1) cells were clarified by using single-channel recordings in this study. In a cell-attached recording, a bath application of 10 μM AA significantly reduced the ENaC open probability (NPo), whereas 10 μM ETYA or 5 μM LA only induced a slight inhibition. The inside-out recording as a standard protocol was thereafter performed to examine effects of these lipids on ENaC activity. Within 10 min after the formation of the inside-out configuration, the NPo of ENaC in cultured mouse cortical collecting duct (M1) cells remained relatively constant. Application of ETYA or LA or SA exhibited a similar inhibition on the channel NPo when applied to the extracellular side, suggesting that fatty acids could exert a nonspecific inhibition on ENaC activity. 11,12-EET, a metabolite of AA via the cytochrome P450 epoxygenase pathway, significantly inhibited the ENaC NPo, whereas 20-HETE, a metabolite of AA via the hydroxylase pathway, only caused a small inhibition of the ENaC NPo, to a similar degree as that seen with ETYA and LA. However, both PGE2 and PGF2α significantly enhanced the ENaC NPo. These results suggest that fatty acids exert a nonspecific effect on ENaC activity due to the interaction between the channel proximity and the lipid. The opposite effects of 11,12-EET and prostaglandin (PG) implicate different mechanisms in regulation of ENaC activity by activation of epoxygenase and cyclooxygenase.     

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

Objective

Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.

Methods and Results

We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.

Conclusion

We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.     

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H₂O₂ production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H₂O₂ production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H₂O₂ production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.     

Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells

Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe’s equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity.     

Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22^phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.     

Resveratrol Inhibits the Epithelial Sodium Channel via Phopshoinositides and AMP-Activated Protein Kinase in Kidney Collecting Duct Cells

Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCD_c14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCD_c14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P₃ reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol''s known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4–24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP₃, and a sustained late (4–24 h) effect secondary to activation of AMPK.     

Rac1、Rac2参与调节人单核细胞趋化和NADPH氧化酶活性

为了探讨小分子GTP酶蛋白Rac1和Rac2在人单核细胞中趋化迁移以及还原型辅酶II（NADPH）氧化酶活性中的作用,采用小分子干扰siRNA对人单核细胞中RAC1、RAC2分别进行特异性抑制,采用实时定量PCR技术、免疫印迹技术在RNA和蛋白质水平上确认抑制效果,使用甲酰三肽(formyl-met-leu-phe,fMLP)、人单核细胞趋化因子（monocyte chemoattractant protein-1,MCP-1）诱导单核细胞趋化;用血清调理的酵母多糖（serum opsonized zymosan,ZOP)、佛波酯（phosphomolybdic acid, PMA）激活单核细胞NADPH氧化酶活性,诱导活性氧(Reactive oxygen species, ROS)产生,以此对Rac1和Rac2作用进行研究. 结果表明,小分子干扰siRNA能够在mRNA水平和蛋白质水平分别有效抑制目的基因表达;使用Chamber assay方法发现,仅Rac1参与了fMLP、MCP-1诱导的人单核细胞趋化. Rac激活实验确证,Rac1参与MCP-1诱导的趋化;细胞色素C还原法表明,Rac1和Rac2均参与PMA和ZOP诱导人单核细胞ROS生成. 在人单核细胞中,RAC1和RAC2基因沉默模型的成功建立以及初步研究显示,Rac1和Rac2的不同作用结果将为深入研究它们在人单核细胞中的功能奠定了良好基础.     

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.     

Aspirin-Triggered Lipoxin A4 Attenuates Lipopolysaccharide-Induced Intracellular ROS in BV2 Microglia Cells by Inhibiting the Function of NADPH Oxidase

Lipoxins have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on neuroinflammation has not been investigated. Microglia is the major cells involved in brain tissue damage during infection and neurodegenerative diseases. One of the major features shared by neuroinflammation conditions is the increased production of reactive oxygen species (ROS) generated by NADPH oxidase activation. In this study, we have examined whether aspirin-triggered lipoxin A(4) (ATL) modulates ROS generation in BV2 cells. Pre-treatment of BV2 cells with ATL blocked ROS production triggered by LPS in the time-dependent and concentration-dependent manner. ATL inhibited the translocation of the cytoplasmic NADPH oxidase subunit p47(phox) to the cell membrane as well as NADPH oxidase activity. Taken together, these results demonstrate that ATL suppresses NADPH oxidase-mediated ROS generation in BV2 microglia cells, strongly indicating that ATL may play an important role against the development and progression of neuroinflammtion.     

赖氨酰氧化酶样蛋白2在肾细胞癌中的表达及其临床意义

为探究赖氨酰氧化酶样蛋白2 (lysyl oxidase like 2, LOXL2)与肾细胞癌(renal cell carcinoma, RCC)的关系,本研究采用免疫组织化学方法检测58例RCC组织及13例癌旁组织中LOXL2的表达水平,并利用统计学方法进一步分析LOXL2与临床病理参数和预后之间的关系。研究显示,LOXL2在癌组织和癌旁组织的阳性表达率分别为68.96%和23.08%,差异具有统计学意义(p<0.05)。LOXL2阳性表达与患者的临床分期和肾包膜浸润相关(p<0.05),但是与患者的性别、年龄、吸烟、病理类型、肿瘤大小和肿瘤分级均无相关性(p>0.05)。采用Kaplan-Meier法和Log-Rank检验分析LOXL2与术后生存时间的关系,实验显示LOXL2阳性组和阴性组患者的5年生存率分别为61.58%和89.46%,差异具有统计学意义(p<0.05)。我们的研究表明LOXL2在肾癌中过表达,其可能在肾癌的侵袭和转移过程中发挥作用,可作为预后的分子标记物。     

The Effect of Copper on the mRNA Expression Profile of Xenobiotic-Metabolizing Enzymes in Cultured Rat H4-II-E Cells

Copper (Cu²⁺) is an essential element that plays important roles in physiological functions of the body. However, high Cu²⁺ levels can have toxic implications. This study aims to investigate the constitutive response to Cu²⁺ exposure of xenobiotic-metabolizing enzymes in cultured rat liver (H4-II-E) cell lines. Rat cells were exposed to copper sulfate (0–500 μM) for 24 h. The effects of Cu²⁺ on the messenger RNA (mRNA) expressions of phase I and II enzymes and regulatory elements were examined using real-time PCR. Metallothionein mRNA expression was induced in a dose-dependent manner after treatment with Cu²⁺. mRNA expressions of phase I enzymes such as cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2) were slightly induced after exposure to low concentrations of Cu²⁺; however, CYP1A1 and CYP1A2 mRNA expressions were significantly downregulated at higher Cu²⁺ concentrations. These effects corresponded with expression of aryl hydrocarbon receptor mRNA. The mRNA expressions of phase II enzymes were reduced upon exposure to Cu²⁺. In conclusion, phase I and II enzyme expressions were significantly modulated upon Cu²⁺ exposure. These results indicated that Cu²⁺ exposure had toxicological implications for cultured H4-II-E cells.     

Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells

Abstract: Metabotropic glutamate receptor (type 1; mGluR1 ) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N -methyl-D-aspartate and non- N -methyl-D-aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1 . The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.     

pEGFP-C2/LAT-ORF3真核表达载体在Vero细胞中的表达特性及其对细胞活性的影响

目的:研究单纯疱疹病毒Ⅱ型(HSV-2)潜伏相关转录体(LAT)开放读码框3(ORF3)的表达特点及其对细胞活性的影响。方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF3真核表达载体pEGFP-C2/LAT-ORF3的可用性,并将其转染入vero细胞,通过荧光和RT-PCR检验其在细胞中的表达,用MTT法进行细胞活性分析。结果:融合蛋白在细胞核中大量集中,且影响了绿色荧光蛋白在细胞中的分布;重组质粒对Vero细胞没有损伤作用。结论:HSV-2 LAT ORF3可抵消空质粒对细胞的损伤作用;其作用靶点可能主要存在于细胞核中,为阐明HSV-2 LAT ORF3在潜伏复发中的功能提供了实验基础。     

HepaCAM对肾癌786-0细胞Caspase-3活性和表达的影响

探讨肝细胞黏附分子(hepatocyte cell adhesion molecule,hepaCAM)在肾癌786-0细胞中不同表达水平对其凋亡及Caspase-3活性和表达的影响。将携带hepaCAM基因的重组质粒瞬时转染786-0细胞,RT-PCR鉴定hepaCAM基因在786-0中的表达;FCM检测细胞凋亡情况;分光光度计结合Western印迹法检测hepaCAM表达与Caspase-3活性及蛋白水平表达的关系。结果显示,上调hepaCAM基因表达能诱导肾癌786-0细胞凋亡,能上调Caspase-3活性及其蛋白水平且与hepaCAM表达水平呈正相关。上述结果表达,hepaCAM可能通过调节Caspase-3凋亡通路诱导肾癌786-0细胞凋亡。     

Uric Acid Promotes Apoptosis in Human Proximal Tubule Cells by Oxidative Stress and the Activation of NADPH Oxidase NOX 4

Mild hyperuricemia has been linked to the development and progression of tubulointerstitial renal damage. However the mechanisms by which uric acid may cause these effects are poorly explored. We investigated the effect of uric acid on apoptosis and the underlying mechanisms in a human proximal tubule cell line (HK-2). Increased uric acid concentration decreased tubule cell viability and increased apoptotic cells in a dose dependent manner (up to a 7-fold increase, p<0.0001). Uric acid up-regulated Bax (+60% with respect to Ctrl; p<0.05) and down regulated X-linked inhibitor of apoptosis protein. Apoptosis was blunted by Caspase-9 but not Caspase-8 inhibition. Uric acid induced changes in the mitochondrial membrane, elevations in reactive oxygen species and a pronounced up-regulation of NOX 4 mRNA and protein (p<0.05). In addition, both reactive oxygen species production and apoptosis was prevented by the NADPH oxidase inhibitor DPI as well as by Nox 4 knockdown. URAT 1 transport inhibition by probenecid and losartan and its knock down by specific siRNA, blunted apoptosis, suggesting a URAT 1 dependent cell death. In summary, our data show that uric acid increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signalling and URAT 1 transport. These results might explain the chronic tubulointerstitial damage observed in hyperuricaemic states and suggest that uric acid transport in tubular cells is necessary for urate-induced effects.     

质膜NADPH氧化酶的活化与H2O2的产生介导内源激发子诱导人参悬浮细胞中PAL活性的提高与人参皂甙的积累

利用纤维素酶降解人参(Panax ginseng C．A．Meyer)悬浮细胞的细胞壁制备了内源激发子(CDW)。CDW体外诱导了游离人参细胞质膜NADPH氧化酶的活性,激发了活体人参悬浮细胞产生H2O2。CDW还可以诱导提高苯丙氨酸解氨酶(PAL)活性,促进人参鲨烯环氧酶基因(sqe)的转录与人参皂甙的积累。NADPH氧化酶的抑制剂不仅可以抑制CDW体外诱导的质膜NADPH活性而且还可以抑制CDW诱导人参细胞产生H2O2。进而,这些抑制剂还可以抑制CDW诱导PAL活性的提高,以及sqe的转录与人参皂甙的合成。过氧化氢酶与H2O2的粹灭剂也可以抑制CDW激发产生的这些诱导效应。上述结果表明CDW激发质膜NADPH氧化酶的活化与H2O2的产生在介导CDW诱导人参细胞抗性反应中,包括PAL活性的提高与人参皂甙的积累,起了重要的信号转导作用。     

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).