Hyperglycemia: GDNF-EGR1 Pathway Target Renal Epithelial Cell Migration and Apoptosis in Diabetic Renal Embryopathy

Maternal hyperglycemia can inhibit morphogenesis of ureteric bud branching, Glial cell line-derived neurotrophilic factor (GDNF) is a key regulator of the initiation of ureteric branching. Early growth response gene-1 (EGR-1) is an immediate early gene. Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment. To evaluate the potential relationship of hyperglycemia-GDNF-EGR-1 pathway, in vitro human renal proximal tubular epithelial (HRPTE) cells as target and in vivo streptozotocin-induced mice model were used. Our in vivo microarray, real time-PCR and confocal morphological observation confirmed apoptosis in hyperglycemia-induced fetal nephropathy via activation of the GDNF/MAPK/EGR-1 pathway at E12-E15. Detachment between ureteric branch and metanephrons, coupled with decreasing number and collapse of nephrons on Day 1 newborn mice indicate hyperglycemic environment suppress ureteric bud to invade metanephric rudiment. In vitro evidence proved that high glucose suppressed HRPTE cell migration and enhanced GDNF-EGR-1 pathway, inducing HRPTE cell apoptosis. Knockdown of EGR-1 by siRNA negated hyperglycemic suppressed GDNF-induced HRPTE cells. EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%. Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.     

绿脓菌素激活MAPKs、NF-KB信号通路诱导呼吸道上皮细胞IL-8表达

采用绿脓杆菌培养上清及绿脓菌素刺激人呼吸道上皮细胞株A549和SPC-A-1,用ELISA方法检测细胞IL-8分泌水平,并使用免疫印迹(Western blot)方法观察绿脓菌素对细胞内重要的炎症信号传导途径NF—κB及丝裂原激活蛋白激酶(MAPKs)的激活作用。实验发现,绿脓杆菌培养上清及绿脓菌素可诱导呼吸道上皮细胞株IL-8分泌增加,且具有剂量依赖效应。绿脓菌素刺激细胞可使细胞内IκB—α发生降解,同时使MAPK家族蛋白分子(ERK1／2、p38、JNK)发生磷酸化。MEK1／2(ERK1／2激酶)抑制剂U0126(10μmol／L)和p38MAPK抑制剂SB203580(10μmol／L)可降低绿脓菌素诱导A549细胞IL-8的合成。以上结果显示绿脓菌素通过MAPK信号传导通路增强呼吸道上皮细胞IL-8的表达;NF-κB通路也参与了绿脓菌素调控细胞IL-8表达的过程。     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

LIF upregulates expression of NK-1R in NHBE cells

Leukemia inhibitory factor (LIF), a cytokine at the interface between neurobiology and immunology, is mainly mediated through JAK/STAT pathway and MAPK/ERK pathway. Evidence suggested LIF is related to the higher expression of neurokinin-1 receptor (NK-1R) in asthma. In this study, the immunohistochemistry stain showed the expressions of NK-1R, LIF, p-STAT3, and p-ERK1/2 in the lung tissues of allergic rats were increased compared with the controls, and the main positive cell type was airway epithelial cell. Normal human bronchial epithelial cells were treated with LIF in the presence or absence of AG490 (JAK2 inhibitor), PD98059 (MEK inhibitor), and the siRNA against STAT3. Western blot and RT-PCR indicated that LIF induced the expression of NK-1R, which was inhibited by the inhibitors mentioned above. No significant interaction was found between JAK/STAT pathway and MAPK/ERK pathway. In summary, bronchial epithelial cell changes in asthma are induced by LIF which promotes the expression of NK-1R, and JAK/STAT pathway and MAPK/ERK pathway may participate in this process.     

Role of SIRT1 in Streptococcus pneumoniae-induced human β-defensin-2 and interleukin-8 expression in A549 cell

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human β-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.     

Gfi1-mediated stabilization of GATA3 protein is required for Th2 cell differentiation

The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.     

Mitogen-activated protein kinase pathway was significantly activated in human bronchial epithelial cells by nicotine

Nicotine is potentially associated with the onset of chronic obstructive pulmonary disease (COPD) and lung cancer. To gain insights into the molecular mechanism underlying such nicotine-induced conditions, microarray- bioinformatics analysis was carried out in the present study to explore the gene expression profiles in human bronchial epithelial cells (HBECs) treated with 5 microM nicotine for 4, 8, and 10 h. Of 1,800 assessed genes overall, 260 (14.4%) were upregulated and 17 (0.9%) down regulated significantly. Gene ontology analysis demonstrated that most of the differentially expressed genes belonged to the category of molecular function, especially to the subcategories of enzyme activity. The integration of obtained information with bioinformatics tools in DAVID and KEGG databases indicated that the greatest number of overexpressed genes was involved in mitogen-activated protein kinase (MAPK) pathway. Membrane array analysis subsequently suggested that both extracellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH(2)-terminal kinase (JNK) signalings but not p38 MAPK signaling were activated in response to nicotine. Pretreatment of HBECs with specific inhibitors against ERK 1/2 and JNK but not p38 could significantly inhibit nicotine-induced interleukin- 8 production. These results suggest that MAPK pathway may mediate the effect of nicotine through ERK 1/2 and JNK but not p38 in HBECs treated with nicotine.     

pORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells

Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy.Pathogenesis of infection has been mostly attributed to excessive cytokine production.However,precise mechanisms on how C.trachomatis triggers this production,and which protein(s) stimulate inflammatory cytokines remains unknown.In the present study,the C.trachomatis pORF5 protein induced tumor necrosis factor alpha(TNF-α),interleukin-1 beta(IL-1β) and interleukin-8(IL-8) in dose-and time-dependent manners in the THP-1 human monocyte cell line.We found that intracellular p38/mitogen-activated protein kinase(MAPK) and extracellular signal-regulated kinase(ERK)/MAPK signaling pathways were required for the induction of TNF-α,IL-1β and IL-8.Blockade of toll-like receptor 2(TLR2) signaling reduced induction levels of TNF-α,IL-8 and IL-1β.We concluded that the C.trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.     

Catalase potentiates interleukin-1beta-induced expression of nitric oxide synthase in rat vascular smooth muscle cells

The role of reactive oxygen species (ROS) in regulating the expression of the inducible nitric oxide synthase (iNOS) was studied in rat aortic vascular smooth muscle cells (VSMC). We hypothesized that ROS regulate iNOS expression through the mitogen-activated protein kinases ERK and p38(MAPK). We found that interleukin-1beta (IL-1beta) stimulated the production of hydrogen peroxide (H2O2) which could be inhibited by loading the cells with the H2O2-scavenging enzyme catalase. Inhibition of the upstream ERK1,2 activator MEK1,2 with U0126 prevented IL-1beta-stimulated iNOS expression, while the p38MAPK inhibitor SB03580 potentiated iNOS expression. Loading the cells with catalase enhanced ERK activation and iNOS expression but had no effect on p38MAPK activation or PDGF-induced ERK activation. These data indicated that H2O2 negatively regulates iNOS expression through ERK inhibition independently of p38MAPK. The present results outline a novel role for H2O2 in suppressing signaling pathways leading to gene expression such as iNOS in VSMC in response to cytokines.     

A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.     

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.     

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.     

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells

A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.     

Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos, and RasGRP1

Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.