Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis

PROBLEM: Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. METHOD OF STUDY: Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. RESULTS: IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. CONCLUSIONS: Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.     

Differential activation of mitogen-activated protein kinases and glial cells in the trigeminal sensory nuclear complex following lingual nerve injury

Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signaling molecules. The current study demonstrates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in each subdivision of the trigeminal sensory nuclear complex (TSNC) following lingual nerve injury. Immunohistochemical labeling for phosphorylated ERK (p-ERK) or phosphorylated p38 (p-p38) MAPK was performed in histological sections of the brainstem. A transient increase in the immunoreactivity for p-ERK was found in each subdivision of the TSNC followed by a prolonged increase in the immunoreactivity for p-p38 MAPK after nerve injury. Double immunofluorescence labeling with cell-specific markers revealed that ERK and p38 MAPK were phosphorylated predominantly by OX-42-positive microglia or GFAP-positive astrocytes. Increased immunofluorescence labeling for OX-42 and GFAP indicated that microglia and astrocytes were activated by nerve injury in the TSNC. Activation of MAPKs and glial cells in the rostral subdivisions of the TSNC was comparable with that in the subnucleus caudalis of the trigeminal spinal tract nucleus (Vc). We conclude that differential activation of MAPKs and glial cells in the rostral subdivisions of the TSNC as well as the Vc may have a substantial role in the pathogenesis of neuropathic pain following trigeminal nerve injury.     

Role of mitogen-activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells

BACKGROUND: Influenza virus (IV) infection causes airway inflammation; however, it has not been determined whether IV infection could catabolize arachidonic acid cascade in airway epithelial cells. In addition, the responsible intracellular signalling molecules that catabolize arachidonic acid cascade have not been determined. OBJECTIVE: In the present study, to clarify these issues, we examined the cyclooxygenase (COX) expression, cytosolic phospholipase A2 (cPLA2) phosphorylation and prostaglandin E2 (PGE2) release in human bronchial epithelial cells (BEC) upon IV infection, and the role of mitogen-activated protein kinase (MAPK) including extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-NH2-terminal kinase (JNK) in catabolizing arachidonic acid cascade in BEC. METHODS: COX-2 expression, phosphorylation of cPLA2 and phosphorylation of ERK, JNK and p38 MAPK were determined by Western blot. The concentrations of PGE2 were determined by ELISA. PD 98059 as a specific inhibitor of MAPK kinase-1 (MEK-1), an up-stream kinase of ERK, SB 203580 as a specific inhibitor of p38 MAPK and CEP-11004 as a specific inhibitor of JNK cascade were used to investigate the role of ERK, p38 MAPK and JNK in catabolizing arachidonic acid cascade in BEC. RESULTS: The results showed that (1) IV infection increases COX-2 expression, cPLA2 phosphorylation and PGE2 release, (2) ERK, p38 MAPK and JNK were phosphorylated, (3) CEP-11004 and PD 98059 predominantly attenuated COX-2 expression and cPLA2 phosphorylation, respectively, (4) SB 203580 did not remarkably affect COX-2 expression and cPLA2 phosphorylation, and (5) each inhibitor dose-dependently attenuated PGE2 release by various extents. CONCLUSION: These results indicate that IV infection activates three distinct MAPKs, ERK, p38 MAPK and JNK, to participate to various extents in the induction of PGE2 synthesis from arachidonic acid in BEC.     

NGF protects Dorsal Root Ganglion neurons from oxaliplatin by modulating JNK/Sapk and ERK1/2

The involvement of the Mitogen-Activated Protein Kinases (MAPKs) family in platinum derivative-induced peripheral neuropathy has already been demonstrated. In particular, it has been evidenced that in Dorsal Root Ganglion (DRG) neurons prolonged exposure to oxaliplatin (OHP) induces early activation of p38 and ERK1/2, which mediate neuronal apoptosis, while the neuroprotective action of JNK/Sapk is downregulated by the drug treatment. In this study, the exposure of OHP-treated neurons to a neuroprotective stimulus, represented by a high dose of NGF, counteracts OHP-induced neuronal mortality. This effect was achieved by restoring the MAPK activation existing in untreated control cells. Increased viability occurred also after the administration of retinoic acid (RA), a pro-differentiative agent able to activate both JNK/Sapk and ERK1/2. The use of specific chemical inhibitors of MAPKs confirms the importance of this class of proteins for the neuroprotective pathway, since they reverse the protective effect. In summary, our findings assess the validity of MAPKs as the target of neuroprotective therapies during chemotherapeutic treatment. Moreover they also describe a double role for ERK1/2, depending on cellular stimulation, since it mediates neuronal apoptosis after OHP exposure. However, it is also important, as is JNK/Sapk, in preserving the correct cellular differentiation that is pivotal for neuronal survival.     

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.     

MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis

Three major MAP kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase (p38 MAPK), are involved in the regulation of lung inflammation and injury. This study investigated whether MAPKs are activated and associated with lung injury in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The expression of the active ERK, JNK, and p38 MAPK was examined using western blot analysis and immunohistochemistry and apoptosis was also examined by the TUNEL method, in lung tissues from ten patients with IPF obtained by thoracoscopic biopsy and in eight normal lung parenchyma specimens obtained by lobectomy for lung cancer. Activated MAPKs are significantly increased in lung homogenates from patients with IPF compared with controls. Activated ERK in epithelial and endothelial cells, but not in fibroblasts or smooth muscle cells, was decreased, accompanied by the progression of fibrosis. Activated JNK in epithelial and endothelial cells, but not in fibroblasts, was increased, accompanied by the progression of fibrosis. Activated p38 MAPK in epithelial, endothelial, smooth muscle cells, and fibroblasts was increased at the intermediate stage of fibrosis, in which the TUNEL-positive cells were predominantly detected. This is the first study to suggest that MAPKs may be associated with the regulation of inflammation and lung injury in IPF.     

MKP-1: A critical phosphatase in the biology of macrophages controlling the switch between proliferation and activation

Macrophages play a central role in the immune response. These cells either proliferate in response to, for example, growth factors or become activated in response to, for example, LPS and develop functional activities. Experiments carried out in mice showed that macrophage proliferation requires a short period of ERK phosphorylation, while an extended period is required for macrophage activation. The length of phosphorylation is controlled by the MAPK phosphatase-1 (MKP-1), a nuclear-localized dual-specificity phosphatase that dephosphorylates the MAPKs ERK, p38, and c-Jun NH(2) -terminal kinase (JNK). MKP-1 is induced in macrophages by growth factors, as well as by activators such as LPS, but with different kinetics; to achieve the different functional outcomes (proliferation versus activation), the inhibition of MKP-1 by cytokines such as IFN-γ blocks macrophage proliferation and induces activation. The data presented in this review show that this phosphatase is the switch between macrophage proliferation and activation.     

Regulation of proliferation/apoptosis equilibrium by mitogen-activated protein kinases in normal, hyperplastic, and carcinomatous human prostate

This study investigate the expression of the mitogen-activated protein kinases (MAPKs) in normal prostate, benign prostatic hyperplasia (BPH), and prostatic cancer (PC), and also the possible relationship between the activity of these MAPKs and the apoptosis/proliferation index. Immunochemical techniques were carried out using 2 mouse monoclonal antibodies against human extracellular signal-regulated protein kinase (ERK) and Jun N-terminal kinase (JNK), and 1 goat polyclonal antibody against mouse p38. To compare the results obtained in the 3 specimens, the average percentages of both epithelial and stromal immunostained cells were calculated on immunostained sections. For each of the 3 kinases studied, the percentage of immunostained stromal cells did not change with prostatic alterations. For both ERK and p38, the percentage of immunostained epithelial cells increased significantly in BPH and even more so in PC. For JNK, the percentage of immunostained epithelial cells increased significantly only in PC. These results suggest that ERK could be involved in the elevated proliferation indexes reported in BPH and PC, whereas p38 might contribute to the increased apoptotic index reported in PC. The most probable action of JNK in PC would be cell proliferation stimulation. Overexpression of MAPKs, involved in the development of prostatic hyperplasia and neoplasia, might be secondary to the overexpression of several growth factors.     

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.     

Curcumin attenuates ethanol-induced toxicity in HT22 hippocampal cells by activating mitogen-activated protein kinase phosphatase-1

Ethanol causes neurotoxicity through formation of reactive oxygen species and activation of mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase-1 (MKP-1) is one of the phosphatases responsible for dephosphorylation/deactivation of MAPKs. In this report, we examined the potential involvement of MKP-1 in cytoprotective effects of the well-known antioxidant curcumin. In HT22 hippocampal cells, ethanol caused cell death and activation of p38 MAPK and other two kinases. Blockage of p38 MAPK by its inhibitor protected HT22 cells against ethanol-induced toxicity. Curcumin attenuated ethanol-induced cell death, inhibited activation of p38 MAPK, and activated MKP-1. In HT22 cells transiently transfected with small interfering RNA against MKP-1, curcumin failed to inhibit ethanol-induced activation of p38 MAPK and to protect HT22 cells from ethanol-induced toxicity. Our results suggest that curcumin can attenuate ethanol-induced neurotoxicity by activating MKP-1 which acts as the negative regulator of p38 MAPK. This novel pathway may contribute to and explain at least one of the cytoprotective actions of curcumin.     

三叉神经根慢性压迫损伤对三叉神经节细胞髓鞘碱性蛋白及P75表达的影响

目的 探讨三叉神经节中卫星胶质细胞与神经元相互作用对三叉神经痛（TN）的影响。 方法 在SD大鼠假手术组（n=24）和三叉神经根慢性压迫诱导的TN大鼠模型组（n=24）中,运用免疫荧光和Western blotting方法,研究髓鞘碱性蛋白（MBP）、胶质纤维酸性蛋白（GFAP）及P75神经营养因子受体（P75NTR）在三叉神经节中的表达情况。 结果 TN模型组P75NTR 表达较假手术组增高,术后第7天和第14 天的表达增高有统计学意义（P<0.01）;MBP在TN模型组的表达较假手术组均降低,特别在第7和14天的表达降低有统计学意义（P<0.05）;卫星胶质细胞标志物GFAP在TN模型组术后第7、14和21天表达增高（P<0.01）。 结论 大鼠三叉神经根区慢性压迫损伤引起三叉神经节神经元MBP和P75NTR 表达改变和卫星胶质细胞的活化,可能影响神经元-卫星胶质细胞的相互作用,参与TN大鼠口面部周围伤害性信息向中枢的传递。     

Involvement of ILK/ERK1/2 and ILK/p38 pathways in mediating the enhanced osteoblast differentiation by micro/nanotopography

The hierarchical micro/nanotextured topography (MNT) on titanium (Ti) implant surface significantly enhances osteoblast differentiation. We have demonstrated that integrin-linked kinase (ILK) is a key underlying signal molecule and β-catenin is one of its downstream mediators in MNT-regulated osteoblast behavior. Here we propose that mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun NH₂-terminal kinase (JNK), are other mediators downstream of ILK, and this study aims to confirm this. Firstly, the levels of ILK and MAPK activity in MG63 cells on MNT are examined by Western blot analysis. The ILK, ERK1/2 and p38 signals are significantly up-regulated by MNT, whereas the JNK activity is undetectable by Western blot. The MG63 cell morphology, proliferation and differentiation are studied in the absence and presence of the MAPK subgroup inhibitors to confirm their roles in cell functions on the Ti surface. The MAPK subgroup inhibitors obviously change the cell shape and depress cell proliferation. Blocking the ERK1/2 or p38 signaling, but not the JNK signaling, significantly down-regulates the cell osteogenesis-related gene expression, ALP production, collagen secretion and matrix mineralization. Afterwards, the ILK expression is down-regulated using ILK-specific siRNA (ILKsi) and then the MAPK activity is determined. ILKsi significantly attenuates the phosphorylated ERK1/2 and p38 levels on MNT, explicitly demonstrating that the ERK1/2 and p38 signalings are downstream effectors of ILK. In conclusion, these data demonstrate that both ILK/ERK1/2 and ILK/p38 pathways are involved in the mechanisms mediating the enhanced osteoblast differentiation by biomaterial surface topography, hopefully directing the biomaterial modification and biofunctionalization.     

Noxious cold stimulation induces mitogen-activated protein kinase activation in transient receptor potential (TRP) channels TRPA1- and TRPM8-containing small sensory neurons

Two cold-sensitive transient receptor potential (TRP) channels, TRPA1 and TRPM8, have been identified and considered interesting because of their possible roles in thermosensation, nociception and other functions. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase occurred in primary afferent neurons in response to noxious heat stimulation of the peripheral tissue, i.e. activity-dependent activation of extracellular signal-regulated protein kinase and p38 in dorsal root ganglion neurons. In the present study, we investigated the phosphorylation of extracellular signal-regulated protein kinase, p38, and c-Jun N-terminal kinase in the rat dorsal root ganglion by cold stimulation using immunohistochemistry. Cold stimuli (28-4 °C) were applied by immersion of the hind paw into a water bath (six times of 10 s stimulation and 10 s interval, total 2 min). Noxious cold stimulation induced phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38, but not phosphorylated-c-Jun N-terminal kinase, in small to medium diameter sensory neurons with a peak at 2 min after stimulation. We found that a cold stimulation at 4 °C showed a marked increase in the number of activated neurons. Furthermore, double staining for phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38 showed no colocalization in the dorsal root ganglion neurons. We then performed double-labeling experiments for TRPA1 and TRPM8 mRNA and phosphorylation of mitogen-activated protein kinase. The majority of phosphorylated-extracellular signal-regulated protein kinase-positive neurons also expressed TRPM8 mRNA, whereas phosphorylated-p38 heavily colocalized with TRPA1 mRNA after noxious cold stimulation. Our data suggest that the noxious, but not innocuous, cold stimulation in vivo induced differential activation of extracellular signal-regulated protein kinase and p38 pathways in each subpopulation containing TRPA1 or TRPM8 in dorsal root ganglion.     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

Regulation of cytokine and chemokine expression by the ribotoxic stress response elicited by Shiga toxin type 1 in human macrophage-like THP-1 cells

Shiga toxins (Stxs) are cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC). Stxs bind to a membrane glycolipid receptor, enter cells, and undergo retrograde transport to ultimately reach the cytosol, where the toxins exert their protein synthesis-inhibitory activity by depurination of a single adenine residue from the 28S rRNA component of eukaryotic ribosomes. The depurination reaction activates the ribotoxic stress response, leading to signaling via the mitogen-activated protein kinase (MAPK) pathways (Jun N-terminal protein kinase [JNK], p38, and extracellular signal-regulated kinase [ERK]) in human epithelial, endothelial, and myeloid cells. We previously showed that treatment of human macrophage-like THP-1 cells with Stxs resulted in increased cytokine and chemokine expression. In the present study, we show that individual inactivation of ERK, JNK, and p38 MAPKs using pharmacological inhibitors in the presence of Stx1 resulted in differential regulation of the cytokines tumor necrosis factor alpha and interleukin-1β (IL-1β) and chemokines IL-8, growth-regulated protein-β, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. THP-1 cells exposed to Stx1 upregulate the expression of select dual-specificity phosphatases (DUSPs), enzymes that dephosphorylate and inactivate MAPKs in mammalian cells. In this study, we confirmed DUSP1 protein production by THP-1 cells treated with Stx1. DUSP1 inhibition by triptolide showed that ERK and p38 phosphorylation is regulated by DUSP1, while JNK phosphorylation is not. Inhibition of p38 MAPK signaling blocked the ability of Stx1 to induce DUSP1 mRNA expression, suggesting that an autoregulatory signaling loop may be activated by Stxs. Thus, Stxs appear to be capable of eliciting signals which both activate and deactivate signaling for increased cytokine/chemokine production in human macrophage-like cells.     

酪氨酸激酶在TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用

目的 研究在类风湿关节炎成纤维样滑膜细胞（RA FLS）信号转导中,酪氨酸激酶在TNF－α刺激下丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPKs)活化中的作用。方法 原代培养类风湿关节炎成纤维样滑膜细胞。应用Western blot检测TNF－α短时间内引起RA FLS蛋白质酪氨酸磷酸化状态改变,及其对MAPKs家族成员活化的浓度效应和时相特点;并应用genistein,酪氨酸激酶（PTK）抑制剂观察对MAPKs活化的抑制情况。结果 TNF－α可以瞬时引起RA FLS蛋白质酪氨酸磷酸化程度增加;并在短时间内激活MAPKs通路（ERK2、JNK2、P38）。不同浓度梯度TNF－α作用显示：10IU／ml时对ERK2、JNK2即可达到峰值活化,100IU／ml时P38达到最大活化。时间上,ERK2、JNK2、P38的活化分别在TNF－α作用后5min、15min、15min最明显;genistein对TNF－α诱导的ERK2活化抑制作用显著,而对于JNK2、P38的抑制则较弱。结论 TNF－α在RA FLS信号转导中,可以瞬时导致蛋白质酪氨酸磷酸化程度增加,并同时激活MAPKs 3条通路,但是对MAPKs 3个亚家族成员的活化具有异质性;PTK在TNF－α导致ERK活化中发挥作用,对JNK、P38活化无明显影响。     

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.     

LPS影响p38蛋白在单核细胞内定位

目的： 探讨 LPS作用下p38蛋白激酶激活的动力学特点及其在细胞内超微结构中的定位。方法： 应用激酶活性测定、胶体金标记的免疫电镜技术观察LPS刺激前后p38蛋白激酶的动力学特点及在单核细胞株Raw264.7中的分布特征。结果： 动力学检测结果显示，LPS作用后15 min，p38磷酸化活性明显升高，30 min达到高峰，2 h达基线水平；p38在LPS浓度为100 μg/L时达最大激活效应。超微定位结果显示，未受刺激的及EGF刺激的细胞，p38在胞浆和胞核中金颗粒呈弥散性分布，金颗粒弥散在细胞的各个部分，如细胞质中线粒体、内质网、溶酶体；单核细胞株受到LPS刺激后，细胞核区的金颗粒明显增多，而胞浆区域的金颗粒显著减少。结论： 单核细胞株Raw264.7受LPS刺激后，其p38蛋白激酶由胞浆移位到胞核。     

Differential modulation of MAP kinases by zinc deficiency in IMR-32 cells: role of H(2)O(2)

The influence of zinc deficiency on the modulation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) was studied. Using human IMR-32 cells as a model of neuronal cells, the role of oxidants on MAPKs and activator protein-1 (AP-1) activation in zinc deficiency was investigated, characterizing the participation of these events in the triggering of apoptosis. Relative to controls, cells incubated in media with low zinc concentrations showed increased cell oxidants and hydrogen peroxide (H(2)O(2)) release, increased JNK and p38 activation, high nuclear AP-1-DNA binding activity, and AP-1-dependent gene expression. Catalase addition to the media prevented the increase of cellular oxidants and inhibited JNK, p38, and AP-1 activation. Low levels of ERK1/2 phosphorylation were observed in the zinc-deficient cells in association with a reduction in cell proliferation. Catalase treatment did not prevent the above events nor the increased rate of apoptosis in the zinc-deficient cells. It is first demonstrated that a decrease in cellular zinc triggers H(2)O(2)-independent, as well as H(2)O(2)-dependent effects on MAPKs. Zinc deficiency-induced increases in cellular H(2)O(2) can trigger the activation of JNK and p38, leading to AP-1 activation, events that are not involved in zinc deficiency-induced apoptosis.