Piperlonguminine Downregulates Endothelial Protein C Receptor Shedding In Vitro and In Vivo

Endothelial cell protein C receptor (EPCR) plays an important role in coagulation and inflammation. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α-converting enzyme (TACE). Piperlonguminine (PL), an important component of Piper longum fruits, is known to exhibit antihyperlipidemic, antiplatelet, and antimelanogenesis activities. However, little is known about the effects of PL on EPCR shedding. Here, we investigated this issue by monitoring the effects of PL on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. PL induced potent inhibition of PMA, and CLP induced EPCR shedding through suppression of TACE expression. And treatment with PL resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, PL might have potential as an anti-sEPCR shedding reagent against PMA- and CLP-mediated EPCR shedding.     

Inhibitory effects of epi-sesamin on endothelial protein C receptor shedding in vitro and in vivo

Objective and design

Endothelial protein C receptor (EPCR) plays a pivotal role in augmenting Protein C activation by the thrombin–thrombomodulin complex. The activity of EPCR is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). The EPCR shedding is mediated by the tumor necrosis factor-α converting enzyme (TACE). Epi-sesamin (ESM), from the roots of Asarum siebodlii, is known to exhibit anti-allergic and anti-fungal activities. However, little is known about the effects of ESM on EPCR shedding.

Methods

We investigated this issue by monitoring the effects of ESM on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-mediated EPCR shedding.

Results

Data showed that ESM induced potent inhibition of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding, likely through suppression of TACE expression. In addition, treatment with ESM resulted in a reduction of PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK).

Conclusions

Given these results, ESM should be viewed as a candidate therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of EPCR shedding.     

Gö6976 inhibits LPS-induced microglial TNFalpha release by suppressing p38 MAP kinase activation

Microglial responses to endotoxin, including the synthesis of inflammatory factors, contribute to gliosis and neuron degeneration in cultured brain tissue. We have previously shown that G?6976, a protein kinase C (PKC) inhibitor, suppressed the lipopolysaccharide (LPS)-induced production of inflammatory factors in microglia and afforded marked protection of neurons from glia-mediated cytotoxicity. The purpose of this study was to identify the signal transduction pathway underlying the neuroprotective effect of G?6976. G?6976 suppressed the LPS-induced release of tumor necrosis factor alpha (TNFalpha) in the microglial cell line, BV2. We show in this study the inhibitory effect of G?6976 on TNFalpha release occurring through suppression of p38 mitogen-activated protein kinase (MAPK) phosphorylation and not through a PKC mechanism. While G?6976 did not inhibit the activity of p38 MAPK directly, it did suppress its activation by phosphorylation, indicating the target of action of G?6976 is a signaling event upstream of p38 MAPK. Although G?6976 is considered a selective inhibitor of certain PKC isozymes, suppression of TNFalpha production was not mediated through inhibition of PKC activity. G?6976 appears to play a novel role in neuroprotection by suppressing the release of pro-inflammatory factors by inhibiting the activation of p38 MAPK in microglia, rather than a PKC isoform.     

Inhibitory Effects of Rutin on the Endothelial Protein C Receptor Shedding In Vitro and In Vivo

Endothelial cell protein C receptor (EPCR) has important functions in regulation of coagulation and inflammation. EPCR shedding from the cell surface is mediated by tumor necrosis factor-α converting enzyme (TACE). Rutin is one of the major flavonoids from the buckwheat plant Fagopyrum tataricum. In this study, we investigated the effects of rutin on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding. We used a CLP model because this model more closely resembles human sepsis. Data showed rutin was a potent inhibitor of PMA, TNF-α, IL-1β, and CLP-induced EPCR shedding by suppression of TACE expression. Treatment with rutin resulted in a decrease of PMA-stimulated phosphorylation of p38, extracellular regulated kinases 1/2, and c-Jun N-terminal kinase. These results suggest the potential application of rutin for treatment of PMA and CLP-mediated EPCR shedding.     

Mitogen-Activated Protein Kinase Is Activated During IgG-Mediated Phagocytosis, But Is Not Required for Target Ingestion

Arachidonic acid (AA) release is required for IgG-mediated phagocytosis in human monocytes. AA release is mediated by a calcium-independent phospholipase A₂ (PPL) that is in turn regulated by protein kinase C (PKC). As mitogen-activated protein kinase (MAPK) activates cytosolic phospholipase A₂, we examined the activation and involvement of MAPK in IgG-mediated phagocytosis. MAPK activity was assessed in immunoprecipitates; tyrosine phosphorylation was detected by immunoblotting. Ingestion of IgG-opsonized glass beads, or treatment with phorbol myristate acetate, increased enzymatic activity and tyrosine phosphorylation of p42 MAPK. This MAPK activation was attenuated by PKC inhibitors staurosporine or calphostin C. Treatment with PD98059, a p42/p44 MAPK kinase (MEK) inhibitor, decreased BIgG-stimulated p42 MAPK activity by >90% with no significant effect on phagocytosis or pPL activity. These results suggest that p42 MAPK is activated in a PKC-dependent manner during IgG-dependent phagocytosis but is not required for target ingestion.     

蛋白激酶C在脂多糖诱导内皮细胞β-1,4-半乳糖基转移酶-Ⅰ表达中的作用

目的 研究蛋白激酶c(PKC)对脂多糖(LPS)诱导内皮细胞β-1,4-半乳糖基转移酶-Ⅰ(β-1,4-galactosyltransferase-Ⅰ,β-1,4-GalT-Ⅰ)表达的调节作用,以及对内皮细胞骨架结构改变及其黏附能力的影响.方法 分别用PKC激动剂或几种不同类型的PKC抑制剂预处理人脐静脉内皮细胞(HUVECs)30 min,LPS刺激HUVECs 4 h后,RT-PCR、Western blot方法 检测β-1,4一GalT-Ⅰ表达变化,细胞荧光染色观察β-1,4-GalT-Ⅰ催化的糖链表达变化及细胞骨架结构的改变,通过内皮-单核细胞黏附试验观察HUVECs黏附能力的改变.结果 几种不同类型的PKC抑制剂均能不同程度地抑制LPS刺激HUVECs引起的β-1,4-GalT-Ⅰ表达的上调,PKC激动剂能够使上调的β-1,4-GalT-Ⅰ的表达进一步增加;在HUVECs中β-1,4-GalT-Ⅰ与细胞骨架有共同定位,PKC抑制剂显著抑制LPS诱导的内皮细胞骨架蛋白的重构和β-1,4-GalT-Ⅰ细胞内的再分布;PKC抑制剂显著抑制LPS诱导的内皮-单核细胞黏附能力的上调.结论 PKC可能参与调节LPS诱导的HUVECs β-1,4-GalT-Ⅰ的表达,可能多种类型的PKC参与了这一调节过程;PKC可能通过对β-1,4-GalT-Ⅰ的调节进而影响炎症过程中内皮细胞骨架蛋白的重构及内皮细胞与单核细胞的黏附能力.     

Antigen receptor-mediated activation of extracellular related kinase (ERK) in B lymphocytes of teleost fishes.

In mammalian B lymphocytes, engagement of the B cell antigen receptor (BCR) activates several parallel intracellular signaling pathways which ultimately lead to expression of differentiated functions such as cell proliferation and antibody production or to cellular apoptosis. BCR engagement stimulates the classical mitogen activated protein kinase (MAPK) pathway, also called the extracellular-related kinase (ERK) pathway, resulting in activation of the signature terminal enzyme in the pathway, MAPK (or ERK). BCR signaling also activates the phosphatidyl inositol pathway and its key enzyme protein kinase C (PKC). To investigate the ERK pathway in cells of the teleost immune system, peripheral blood leukocytes from red drum or channel catfish were treated with PKC activators or antibodies which crosslink the BCR. Proteins were identified in both red drum and catfish B cells that resembled mammalian ERKs in molecular weight and in their possessing a distinctive pTEpY dual phosphorylation site. BCR-mediated activation of these presumptive teleost ERKs depended in part (red drum) or in total (catfish) on PKC. To our knowledge this represents the first report of a functional MAPK kinase pathway in teleost fish.     

A novel role of andrographolide,an NF-kappa B inhibitor,on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP

Andrographolide is a novel NF-κB inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25–75 μΜ) exhibited a more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca²⁺ mobilization; thromboxane A₂ formation; and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless, a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH^⋅) formation. In vivo studies revealed that andrographolide (22 and 55 μg/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism and significantly prolonged platelet plug formation in mice. This study demonstrates for the first time that andrographolide possesses a novel role of antiplatelet activity, which may involve the activation of the eNOS-NO/cyclic GMP pathway, resulting in the inhibition of the PI3 kinase/Akt-p38 MAPK and PLCγ2–PKC cascades, thereby leading to inhibition of platelet aggregation.     

Regulation of adhesion of AML14.3D10 cells by surface clustering of beta2-integrin caused by ERK-independent activation of cPLA2

We examined the role of cell surface clustering of beta2-integrin caused by protein kinase C (PKC)-activated-cPLA2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against beta2-integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A2 (cPLA2) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA2 activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA2 inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA2- or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through beta2-integrin by PKC activation of cPLA2. This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering - rather than conformational change or up-regulation of CD11b/CD18 - causes PMA stimulated adhesion of AML cells.     

Role of PKC-delta during hypoxia in heart-derived H9c2 cells

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.     

miR-92a-3p promotes ox-LDL induced-apoptosis in HUVECs via targeting SIRT6 and activating MAPK signaling pathway

Atherosclerosis could be induced by multiple factors, including hypertension, hyperlipidemia, and smoking, and its pathogenesis has not been fully elucidated. MicroRNAs have been shown to possess great anti-atherosclerotic potential, but the precise function of miR-92a-3p in atherosclerosis and its potential molecular mechanism have not been well clarified. Flow cytometry assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay were performed to evaluate effects of oxidized low-density lipoprotein (ox-LDL) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs), respectively. Malondialdehyde and superoxide dismutase levels in cell lysate were assessed with biochemical kits. The expression levels of miR-92a-3p and Sirtuin6 (SIRT6) in HUVECs exposed to ox-LDL were estimated by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the protein levels of SIRT6, c-Jun N-terminal kinase (JNK), phosphorylation JNK (p-JNK), p38 mitogen activated protein kinase (p38 MAPK), and phosphorylation p38 MAPK (p-p38 MAPK) were measured by western blot assays. The relationship between miR-92a-3p and SIRT6 was confirmed by dual-luciferase reporter assay. Ox-LDL induced apoptosis and oxidative stress in HUVECs in concentration- and time-dependent manners. Conversely, miR-92a-3p silencing inhibited apoptosis and SIRT6 expression in HUVECs. The overexpression of miR-92a-3p enhanced apoptosis and phosphorylation levels of JNK and p38 MAPK as well as inhibited proliferation in ox-LDL-induced HUVECs. In addition, SIRT6 was a target of miR-92a-3p. miR-92a-3p negatively regulated SIRT6 expression in ox-LDL-induced HUVECs to activate MAPK signaling pathway in vitro. In summary, miR-92a-3p promoted HUVECs apoptosis and suppressed proliferation in ox-LDL-induced HUVECs by targeting SIRT6 expression and activating MAPK signaling pathway.     

葡萄糖对人脐静脉内皮细胞蛋白C受体表达的影响及吡格列酮的干预作用

目的:研究葡萄糖对人脐静脉内皮细胞蛋白C受体(EPCR)mRNA 表达的影响,以及吡格列酮的干预作用。方法:体外培养人脐静脉内皮细胞(HUVECs),分别以流式细胞术和RT-PCR技术确认HUVECs膜上EPCR的表达水平和 mRNA 水平的表达。再分别以含不同浓度D-葡萄糖(5、10、30、50 mmol/L)的培养基以及含吡格列酮(5、10、20 μmol/L)或不含吡格列酮的高糖(50 mmol/L)培养基孵育HUVECs 24 h,行剂量和时间依赖性实验,并采用实时定量PCR技术测定HUVECs细胞 EPCR mRNA 的表达。结果:随着培养基D-葡萄糖浓度的增加,HUVECs培养24 h后其EPCR mRNA 的表达逐渐下调。在采用吡格列酮干预后, 50 mmol/L高糖处理的HUVECs EPCR mRNA 表达的下调得到明显改善。结论:(1)EPCR 在HUVECs上高表达,高糖可通过下调EPCR mRNA 的表达而损伤内皮细胞功能。(2)吡格列酮可阻止高糖诱导的HUVECs EPCR mRNA表达的下调,从而保护内皮细胞功能。     

重组梅毒螺旋体蛋白Tp47通过诱导uPA增加血管内皮细胞通透性

目的探讨重组梅毒螺旋体蛋白Tp47(rTpp47)对单核-巨噬细胞系THP-1合成尿激酶型纤溶酶原激活物(uPA)的调控及其对人脐静脉/血管内皮细胞(HUVECs)通透性的影响。方法用rTpp47刺激THP-1细胞24 h后,分别收集细胞培养上清和THP-1细胞,用ELISA和Western blot检测THP-1细胞表达的uPA含量;用THP-1细胞培养上清刺激人单层血管内皮细胞后,使用FITC-葡聚糖评价单层内皮细胞通透性的变化,用Western blot检测uPA对HUVECs细胞紧密连接蛋白claudin-5表达的影响以及PKC信号通路是否参与rTpp47诱导的THP-1细胞表达uPA。结果重组蛋白rTpp47刺激THP-1细胞合成和分泌的uPA显著高于对照组(P<0.05,P<0.001);用rTpp47与THP-1细胞共培养24 h后收集的细胞培养上清刺激单层血管内皮细胞12和24 h,实验组血管内皮细胞相对通透性显著高于对照组(P<0.05,P<0.000 1);uPA活性抑制剂阿米洛利(amiloride)抑制了rTpp47刺激THP-1细胞分泌...     

Sevoflurane stimulates MAP kinase signal transduction through the activation of PKC alpha and betaII in fetal rat cerebral cortex cultured neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral -cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (alpha, betaI, betaII, gamma, delta, varepsilon and zeta) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC alpha and PKC betaII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC alpha and betaII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5-10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10-15 min after sevoflurane treatments. That of ERK proteins was induced at 15-60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the -localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC alpha and PKC betaII species.     

Titanium implants alter endothelial function and vasoconstriction via a protein kinase C-regulated pathway

The application of titanium (Ti) alloy in joint prostheses is a good choice in orthopedic reconstruction. An elevated serum concentration of Ti has been shown in the patients with loosened knee prostheses. The precise actions of elevated Ti on the circulation remain unclear. In this study the maximal contractile responses elicited by phenylephrine in the aortas of rats 4 weeks after Ti alloy implantation and in cultured rat aortas treated with a soluble form of Ti for a period of 18 h were significantly decreased as compared with controls. Aortas isolated from rats with Ti alloy implants or aortas treated with a soluble form of Ti had enhanced protein expression of endothelial nitric oxide synthase (eNOS) and protein kinase C (PKC)-α and enhanced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Treatment of human umbilical vein endothelial cells (HUVECs) with a soluble form of Ti for 24 h dose-dependently increased eNOS protein expression. Short-term treatment of HUVECs with Ti for 1 h effectively enhanced the phosphorylation of eNOS, PKC (pan) and ERK1/2. PKC inhibitors RO320432 and chelerythrine effectively inhibited Ti-enhanced phosphorylation of eNOS and PKC (pan). These results indicate that Ti in the circulation may alter endothelial function and reduce vasoconstriction.     

Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells

BACKGROUND: Flavonoids have a variety of activities including anti-allergic activities, and are known to inhibit histamine release from human basophils and murine mast cells. OBJECTIVE: The effects of luteolin, a flavone, on the immunoglobulin (Ig) E-mediated allergic mediator release from human cultured mast cells (HCMCs) were investigated and compared with those of baicalein and quercetin. METHODS: HCMCs were sensitized with IgE, and then treated with flavonoids before challenge with antihuman IgE. The amount of released mediators was determined as was mobilization of intracellular Ca2+ concentration, protein kinase C (PKC) translocation and phosphorylation of intracellular proteins were detected after anti-IgE stimulation. RESULTS: Luteolin, baicalein and quercetin inhibited the release of histamine, leukotrienes (LTs), prostaglandin D2 (PGD2), and granulocyte macrophage-colony stimulating factor (GM-CSF) from HCMC in a concentration-dependent manner. Additionally, the three flavonoids inhibited A23187-induced histamine release. As concerns Ca2+ signalling, luteolin and quercetin inhibited Ca2+ influx strongly, although baicalein did slightly. With regard to PKC signalling, luteolin and quercetin inhibited PKC translocation and PKC activity strongly, although baicalein did slightly. The suppression of Ca2+ and PKC signallings might contribute to the inhibition of mediator release. The activation of extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinase (JNK), that were activated just before the release of LTs and PGD2 and GM-CSF mRNA expression in IgE-mediated signal transduction events, were clearly suppressed by luteolin and quercetin. In contrast, the flavonoids did not affect the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway. CONCLUSION: These results indicate that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation.     

Histamine H1 receptor-stimulated interleukin 8 and granulocyte macrophage colony-stimulating factor production by bronchial epithelial cells requires extracellular signal-regulated kinase signaling via protein kinase C

BACKGROUND: Histamine stimulates the release of several cytokines, such as interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor, from bronchial epithelial cells. However, the functional individual histamine receptor subtype and intracellular signaling in bronchial epithelial cells are poorly defined. METHODS: Using human primary epithelial cells and the NCI-H292 cell line, we examined the expression of histamine receptor subtypes and histamine-induced second messenger. We also evaluated the involvements of mitogen-activated protein kinase, protein kinase C (PKC) and epidermal growth factor receptor in cytokine expression caused by histamine. RESULTS: Histamine H1 receptor (H1R) was the only subtype expressed in both types of cells. Histamine elevated intracellular calcium ion without affecting cAMP levels. Histamine induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Histamine also phosphorylated PKC and myristoylated alanine-rich C kinase substrate. Ro-31-8220, a PKC inhibitor, and PD98059, a mitogen-activated protein/ERK kinase inhibitor, suppressed the histamine-induced ERK activation and the production of granulocyte macrophage colony-stimulating factor and IL-8. On the contrary, histamine had no effect on the phosphorylation of epidermal growth factor receptor, and its specific inhibitor AG1478 failed to inhibit the histamine-induced ERK activation. Olopatadine, an H1 antagonist, completely blocked the histamine-related responses, whereas H2 and H3 antagonists did not. Histamine also augmented the IL-8 production caused by IL-4 or tumor necrosis factor-alpha. CONCLUSIONS: The H1R-PKC-ERK pathway may play crucial roles in eliciting cytokine production from bronchial epithelial cells stimulated by histamine, leading to airway inflammation.     

Protein kinase C mediates phosphorylation of the regulatory light chain of myosin-II during mitosis

Phosphorylation of the regulatory light chain (RLC) of myosin-II is cell cycle dependent. Early in mitosis the RLC is phosphorylated predominantly on Ser-1/2, while during cytokinesis the primary site of phosphorylation is Ser-19 (Yamakita et al., 1994). To identify candidate kinases likely to mediate the mitotic phosphorylation on Ser-1/2, we assayed RLC kinase activity in mitotic cell extracts and measured apparent steady-state kinetic constants using purified enzymes. The mitotic RLC kinase is distinct from cdc2 kinase, protein kinase A and protein kinase G, as activators or inhibitors specific for these kinases do not affect the mitotic kinase activity. The activity of the mitotic RLC kinase is enhanced by the addition of Ca²⁺ and DAG and/or phorbol esters, characteristics of a conventional protein kinase C (PKC). Moreover, the PKC inhibitors, Gö6983 and Gö6976, significantly attenuate the phosphorylation of the RLC in mitotic extracts. Apparent steady-state kinetic studies indicate that several PKC isoforms display high specificity for myosin-II. These results suggest that current models describing Ser-1/2 phosphorylation during mitosis need to be re-evaluated.     

Activation of extracellular signal-regulated protein kinase in the dorsal root ganglion following inflammation near the nerve cell body

Inflammation of the primary afferent proximal to the dorsal root ganglion (DRG) and the DRG itself is known to produce radicular pain. Here, we examined pain-related behaviors and the activation of extracellular signal-regulated protein kinase (ERK) in the DRG after inflammation near the DRG somata. Inflammation of the L4/5 nerve roots and DRG induced by complete Freund's adjuvant (CFA) produced mechanical allodynia on the ipsilateral hindpaw and induced an increase in the phosphorylation of ERK, mainly in tyrosine kinase (trk) A-expressing small- and medium-size neurons. This CFA-induced increase in ERK phosphorylation was mediated through trk receptors, because intrathecal treatment with the tyrosine kinase inhibitor, K252a, reduced the activation of ERK. On the other hand, an increase in brain-derived neurotrophic factor (BDNF) mRNA/protein in the DRG concomitant with the ERK activation was also observed. Furthermore, we found that nerve growth factor (NGF) injection directly into the L4/5 nerve roots and DRG produced mechanical allodynia, and an increase in the phosphorylation of ERK and BDNF expression in the DRG, but the mitogen-activated protein kinase (MAPK) kinase1/2 inhibitor, U0126, inhibited the effects induced by NGF. Therefore, we suggest that after inflammation near the cell body, NGF synthesized within the nerve root and DRG induces BDNF expression through trkA receptors and intracellular ERK-MAPK. The activation of MAPK in the primary afferents may be involved in the pathophysiological mechanisms of inflammation-induced radiculopathy and MAPK pathways in the primary afferents may be potential targets for pharmacological intervention for neuropathic pain produced by inflammation near the DRG somata.