Impaired cAMP and Rac 1 Signaling Contribute to TNF‐α‐induced Endothelial Barrier Breakdown in Microvascular Endothelium

Objective: In sepsis, tumor necrosis factor‐alpha (TNF‐α) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling. Materials and Methods: For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate–dextran flux, Western blotting, immunostaining, and enzyme‐linked immunosorbent assay–based measurements of cAMP levels and Rho‐GTPase activity in human microvascular endothelial cells. Results: TNF‐α disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF‐α‐induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF‐α‐induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R‐mediated increase of cAMP, whereas Rho A activation was only partially inhibited. Conclusion: Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF‐α‐induced endothelial barrier breakdown in vivo and in vitro.     

Interleukin-1β induces cyclo-oxygenase-2 expression in gastric cancer cells by the p38 and p44/42 mitogen-activated protein kinase signaling pathways

BACKGROUND AND AIMS: Cyclo-oxygenase-2 (COX-2) is the inducible enzyme in the gastric mucosa responsible for prostaglandin production during inflammation and ulcer healing. The regulation of COX-2 gene expression in gastric epithelial cells is not well understood. In this study, we investigated the effect of interleukin (IL)-1beta on COX-2 expression in the human gastric cancer cell, and explored the signaling pathways involved. METHODS: Gastric cancer cell line AGS was treated with IL-1beta or the inhibitors of mitogen-activated protein-Erk kinase (MEK) and p38 mitogen-activated protein (MAP) kinase prior to the addition of IL-1beta. The COX-2 mRNA or protein levels were measured by using RT-PCR or western blot analysis, respectively. Prostaglandin E2 (PGE2) production/secretion was determined by using the prostaglandin E2 EIA assay. The phosphorylation/activation of p44/42 and p38 MAP kinases were determined by using western blot analysis and using phospho-specific antibodies. RESULTS: Interleukin-1beta treatment dose- and time-dependently increased COX-2 mRNA and protein expression levels, and enhanced PGE2 production/secretion in AGS cells. In contrast, IL-1beta had no effect on the level of the constitutively expressed COX-1. In parallel to the increase of COX-2, we showed that p44/42 and p38 MAP kinase activities were also upregulated by IL-1beta treatment. To demonstrate the cause-effect relationship, we showed that inhibition of MEK and p38 MAP kinase with specific inhibitors suppressed IL-1beta-mediated increases in COX-2 mRNA and protein levels, and the PGE2 production. CONCLUSIONS: Our results demonstrated that in human gastric cancer cells, IL-1beta upregulates the COX-2 gene expression through the activation of MEK/p44/42 and p38 MAP kinases pathway.     

Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor,induces pleiotropic anti‐myeloma activity in vitro and in vivo

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine‐induced osteoclast differentiation more potently (9‐ to 10‐fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G₀‐G₁ cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti‐MM therapies. Significant tumour growth reduction in AS703026‐ vs. vehicle‐treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC‐induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti‐MM agents, to improve patient outcome.     

Coxsackievirus A6 Recombinant Subclades D3/A and D3/H Were Predominant in Hand-Foot-And-Mouth Disease Outbreaks in the Paediatric Population,France, 2010–2018

Coxsackievirus A6 (CVA6) emerged as the most common enterovirus of seasonal outbreaks of hand-foot-and-mouth disease (HFMD). We investigated CVA6 genetic diversity among the clinical phenotypes reported in the paediatric population during sentinel surveillance in France between 2010 and 2018. CVA6 infection was confirmed in 981 children (mean age 1.52 years [IQR 1.17–2.72]) of whom 564 (58%) were males. Atypical HFMD was reported in 705 (72%) children, followed by typical HFMD in 214 (22%) and herpangina in 57 (6%) children. Throat specimens of 245 children were processed with a target-enrichment new-generation sequencing approach, which generated 213 complete CVA6 genomes. The genomes grouped within the D1 and D3 clades (phylogeny inferred with the P1 genomic region). In total, 201 genomes were classified among the recombinant forms (RFs) A, B, F, G, H, and N, and 12 genomes were assigned to 5 previously unreported RFs (R–V). The most frequent RFs were A (58%), H (19%), G (6.1%), and F (5.2%). The yearly number of RFs ranged between 1 (in 2012 and 2013) and 6 (2018). The worldwide CVA6 epidemic transmission began between 2005 and 2007, which coincided with the global spread of the recombinant subclade D3/RF-A.