Protease-activated receptor (PAR)-induced interleukin-8 production in airway epithelial cells requires activation of MAP kinases p44/42 and JNK

The mechanism underlying protease-activated receptor (PAR)-activation and subsequent interleukin (IL)-8 production in airway epithelial cells is not yet understood. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 airway epithelial cells. We studied the consequence of activation of PARs with simultaneous exposure to LPS. Thrombin, PAR-2-activating peptide and LPS, were tested alone and in combination. They induced significant synthesis of IL-8. However, only activation of PAR triggered phosphorylation of ERK1/2 and JNK. The application of the inhibitors of these two MAPKs resulted in reduction of IL-8 production. Thus, activation of PARs but not stimulation with LPS leads to ERK1/2 and JNK-mediated production of IL-8.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

TGFβ1 induces IL‐6 and inhibits IL‐8 release in human bronchial epithelial cells: The role of Smad2/3

Human bronchial epithelial (HBE) cells contribute to asthmatic airway inflammation by secreting cytokines, chemokines, and growth factors, including interleukin (IL)‐6, IL‐8 and transforming growth factor (TGF) β1, all of which are elevated in asthmatic airways. This study examines the signaling pathways leading to TGFβ1 induced IL‐6 and IL‐8 in primary HBE cells from asthmatic and non‐asthmatic volunteers. HBE cells were stimulated with TGFβ1 in the presence or absence of signaling inhibitors. IL‐6 and IL‐8 protein and mRNA were measured by ELISA and real‐time PCR respectively, and cell signaling kinases by Western blot. TGFβ1 increased IL‐6, but inhibited IL‐8 production in both asthmatic and non‐asthmatic cells; however, TGF induced significantly more IL‐6 in asthmatic cells. Inhibition of JNK MAP kinase partially reduced TGFβ1 induced IL‐6 in both cell groups. TGFβ1 induced Smad2 phosphorylation, and blockade of Smad2/3 prevented both the TGFβ1 modulated IL‐6 increase and the decrease in IL‐8 production in asthmatic and non‐asthmatic cells. Inhibition of Smad2/3 also increased basal IL‐8 release in asthmatic cells but not in non‐asthmatic cells. Using CHIP assays we demonstrated that activated Smad2 bound to the IL‐6, but not the IL‐8 promoter region. We conclude that the Smad2/3 pathway is the predominant TGFβ1 signaling pathway in HBE cells, and this is altered in asthmatic bronchial epithelial cells. Understanding the mechanism of aberrant pro‐inflammatory cytokine production in asthmatic airways will allow the development of alternative ways to control airway inflammation. J. Cell. Physiol. 225: 846–854, 2010. © 2010 Wiley‐Liss, Inc.     

Upregulation of Fas and FasL in Taiwan cobra phospholipase A2‐treated human neuroblastoma SK‐N‐SH cells through ROS‐ and Ca2+‐mediated p38 MAPK activation

The aim of the present study is to elucidate the signaling pathway involved in death of human neuroblastoma SK‐N‐SH cells induced by Naja naja atra phospholipase A₂ (PLA₂). Upon exposure to PLA₂, p38 MAPK activation, ERK inactivation, ROS generation, increase in intracellular Ca²⁺ concentration, and upregulation of Fas and FasL were found in SK‐N‐SH cells. SB202190 (p38MAPK inhibitor) suppressed upregulation of Fas and FasL. N‐Acetylcysteine (ROS scavenger) and BAPTA‐AM (Ca²⁺ chelator) abrogated p38 MAPK activation and upregulation of Fas and FasL expression, but restored phosphorylation of ERK. Activated ERK was found to attenuate p38 MAPK‐mediated upregulation of Fas and FasL. Deprivation of catalytic activity could not diminish PLA₂‐induced cell death and Fas/FasL upregulation. Moreover, the cytotoxicity of arachidonic acid and lysophosphatidylcholine was not related to the expression of Fas and FasL. Taken together, our results indicate that PLA₂‐induced cell death is, in part, elicited by upregulation of Fas and FasL, which is regulated by Ca²⁺‐ and ROS‐evoked p38 MAPK activation, and suggest that non‐catalytic PLA₂ plays a role for the signaling pathway. J. Cell. Biochem. 106: 93–102, 2009. © 2008 Wiley‐Liss, Inc.     

Aggravation of Alzheimer's disease due to the COX‐2‐mediated reciprocal regulation of IL‐1β and Aβ between glial and neuron cells

Alzheimer's disease (AD) is the most common form of dementia and displays the characteristics of chronic neurodegenerative disorders; amyloid plaques (AP) that contain amyloid β‐protein (Aβ) accumulate in AD, which is also characterized by tau phosphorylation. Epidemiological evidence has demonstrated that long‐term treatment with nonsteroidal anti‐inflammatory drugs (NSAIDs) markedly reduces the risk of AD by inhibiting the expression of cyclooxygenase 2 (COX‐2). Although the levels of COX‐2 and its metabolic product prostaglandin (PG)E₂ are elevated in the brain of AD patients, the mechanisms for the development of AD remain unknown. Using human‐ or mouse‐derived glioblastoma and neuroblastoma cell lines as model systems, we delineated the signaling pathways by which COX‐2 mediates the reciprocal regulation of interleukin‐1β (IL‐1β) and Aβ between glial and neuron cells. In glioblastoma cells, COX‐2 regulates the synthesis of IL‐1β in a PGE₂‐dependent manner. Moreover, COX‐2‐derived PGE₂ signals the activation of the PI3‐K/AKT and PKA/CREB pathways via cyclic AMP; these pathways transactivate the NF‐κB p65 subunit via phosphorylation at Ser 536 and Ser 276, leading to IL‐1β synthesis. The secretion of IL‐1β from glioblastoma cells in turn stimulates the expression of COX‐2 in human or mouse neuroblastoma cells. Similar regulatory mechanisms were found for the COX‐2 regulation of BACE‐1 expression in neuroblastoma cells. More importantly, Aβ deposition mediated the inflammatory response of glial cells via inducing the expression of COX‐2 in glioblastoma cells. These findings not only provide new insights into the mechanisms of COX‐2‐induced AD but also initially define the therapeutic targets of AD.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

JlpA of Campylobacter jejuni interacts with surface-exposed heat shock protein 90alpha and triggers signalling pathways leading to the activation of NF-kappaB and p38 MAP kinase in epithelial cells

Campylobacter jejuni is a leading cause of acute bacterial gastroenteritis in humans. The mechanism by which C. jejuni interacts with host cells, however, is still poorly understood. Our previous study has shown that the C. jejuni surface lipoprotein JlpA mediates adherence of the bacterium to epithelial cells. In this report, we demonstrated that JlpA interacts with HEp-2 cell surface heat shock protein (Hsp) 90alpha and initiates signalling pathways leading to activation of NF-kappaB and p38 MAP kinase. Gel overlay and GST pull down assays showed that JlpA interacts with Hsp90alpha. Geldanamycin, a specific inhibitor of Hsp90, and anti-human Hsp90alpha antibody significantly blocked the interaction between JlpA and Hsp90alpha, suggesting a direct interaction between JlpA and HEp-2 cell surface-exposed Hsp90alpha. The treatment of HEp-2 cells with GST-JlpA initiated two signalling pathways: one leading to the phosphorylation and degradation of IkappaB and nuclear translocation of NF-kappaB; and another one to the phosphorylation of p38 MAP kinase. The activation of NF-kappaB and p38 MAP kinase in HEp-2 cells suggest that JlpA triggers inflammatory/immune responses in host cells following C. jejuni infection.     

Long noncoding RNA HNF1A‐AS1 regulates proliferation and apoptosis of glioma through activation of the JNK signaling pathway via miR‐363‐3p/MAP2K4

Long noncoding RNAs (lncRNAs) have been proven to exert important functions in the various biological processes of human cancers. It has been reported that lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A‐AS1) was abnormally expressed and played a role in the initiation and development of various human cancers. In this study, we confirmed that the expression level of HNF1A‐AS1 was increased in glioma tissues and cells. Knockdown of HNF1A‐AS1 inhibited cell proliferation and promoted cell apoptosis in glioma. Then, we disclosed the downregulation of miR‐363‐3p in glioma tissues and cell lines. The interaction between HNF1A‐AS1 and miR‐363‐3p was identified in glioma cells. Furthermore, an inverse correlation between HNF1A‐AS1 and miR‐363‐3p was observed in glioma tissues. Afterwards, we recognized that MAP2K4 was a direct target of miR‐363‐3p. The expression of MAP2K4 was negatively correlated with miR‐363‐3p while positively related to HNF1A‐AS1 in glioma tissues. We also found the regulatory effect of HNF1A‐AS1 on the MAP2K4‐dependent JNK signaling pathway. All findings indicated that HNF1A‐AS1 induces the upregulation of MAP2K4 to activate the JNK signaling pathway to promote glioma cell growth by acting as a miR‐363‐3p sponge.     

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and αB-crystallin in C6 rat glioma cells, that had been heated at 43°C for 30 min with a subsequent culture for 16 h at 37°C, were markedly increased. The exposure of the cells to a low concentration (0.1–3 µg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and αB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and αB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1–3 µg/ml anisomycin. The results of a reporter assay, using an αB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of αB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 µg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO₂, or CdCl₂. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 µg/ml of anisomycin. The heat-induced accumulations of Hsp27 and αB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and αB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and αB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

Thrombin induces cyclooxygenase‐2 expression and prostaglandin E2 release via PAR1 activation and ERK1/2‐ and p38 MAPK‐dependent pathway in murine macrophages

Thrombin levels increase at sites of vascular injury and during acute coronary syndromes. It is also increased several fold by sepsis with a reciprocal decrease in the anti‐thrombin III levels. In this study we investigate the effects of thrombin on the induction of cyclooxygenase‐2 (COX‐2) and prostaglandin (PG) production in macrophages. Thrombin‐induced COX‐2 protein and mRNA expression in RAW264.7 and primary cultured peritoneal macrophages. A serine proteinase, trypsin, also exerted a similar effect. The inducing effect by thrombin in macrophages was not affected by a lipopolysaccharide (LPS)‐binding antibiotic, polymyxin B, excluding the possibility of LPS contamination. The increase of COX‐2 expression by thrombin was functionally linked to release of PGE₂ and PGI₂ but not thromboxane A₂ into macrophage culture medium. Thrombin‐induced COX‐2 expression and PGE₂ production were significantly attenuated by PD98059 and SB202190 but not by SP600125, suggesting that ERK1/2 and p38 MAPK activation were involved in this process. This was supported by the observation that thrombin could directly activate ERK1/2 and p38 MAPK in macrophages. A further analysis indicated that the proteinase‐activated receptor 1 (PAR1)‐activating agonist induced effects similar to those induced by thrombin in macrophages and the PAR1 antagonist‐SCH79797 could attenuate thrombin‐induced COX‐2 expression and PGE₂ release. Taken together, we provided evidence demonstrating that thrombin can induce COX‐2 mRNA and protein expression and PGE₂ production in macrophages through PAR1 activation and ERK1/2 and p38 MAPK‐dependent pathway. The results presented here may explain, at least in part, the possible contribution of thrombin and macrophages in these pathological conditions. J. Cell. Biochem. 108: 1143–1152, 2009. © 2009 Wiley‐Liss, Inc.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.