Clinorotation upregulates inducible nitric oxide synthase by inhibiting AP‐1 activation in human umbilical vein endothelial cells

Alterations of nitric oxide contribute to post‐flight orthostatic intolerance. The aim of this study was to investigate the changes of inducible nitric oxide synthase (iNOS) and the mechanisms underlying regulation of iNOS by simulated microgravity in human umbilical vein endothelial cells (HUVECs). Clinorotation, a simulated‐model of microgravity, increased iNOS expression and promoter activity in HUVECs. The transactivations of NF‐κB and AP‐1 were suppressed by 24 h clinorotation. A key role for AP‐1, but not NF‐κB in the regulation of iNOS was shown. (1) PDTC, a NF‐κB inhibitor, had no effect on clinorotation upregulation of iNOS. (2) SP600125, a JNK‐specific inhibitor, which resulted in inhibition of AP‐1 activity, enhanced the iNOS expression and promoter activity in clinorotation. (3) Overexpression of AP‐1 remarkably attenuated the upregulation effect of clinorotation. These findings indicate that clinorotation upregulates iNOS in HUVECs by a mechanism dependent on suppression of AP‐1, but not NF‐κB. These results support a key role for AP‐1 in the signaling of postflight orthostatic intolerance. J. Cell. Biochem. 107: 357–363, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of the hyaluronan oligosaccharides inflammatory response: reduction of adenosine 2A receptor activation by EPAC and PKA

The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4‐mer hyaluronan (HA) oligosaccharide‐induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4‐mer HA increased Toll‐like receptor‐4, TNF‐alpha and IL‐1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF‐kB) activation. Addition to NSF, previously stimulated with 4‐mer HA oligosaccharides, of ADO significantly reduced NF‐kB activation, TNF‐alpha and IL‐1beta expression. The pre‐treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC‐specific inhibitors significantly inhibited the anti‐inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO‐induced NF‐kB inhibition although EPAC seems to be more involved than PKA. Copyright © 2014 John Wiley & Sons, Ltd.     

MALT1 is a potential therapeutic target in glioblastoma and plays a crucial role in EGFR‐induced NF‐κB activation

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF‐κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF‐κB activation in GBM; however, the correlation between EGFR and the NF‐κB pathway remains unclear. In this study, we investigated the role of mucosa‐associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti‐tumour activity and effectiveness of MI‐2, a MALT1 inhibitor in a pre‐clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR‐induced NF‐kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle–associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF‐κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR‐induced NF‐kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.     

Sulforaphene enhances radiosensitivity of hepatocellular carcinoma through suppression of the NF‐κB pathway

Sulforaphene (SFE), a naturally occurring isothiocyanate found in cruciferous vegetables, has attracted increasing attention for its anti‐cancer effect in many cancers, including hepatocellular carcinoma (HCC). However, the precise role of SFE in the radiosensitivity of HCC is still unclear. Here, cell proliferation and apoptosis were detected by MTT and flow cytometry assay, respectively. The activity of NF‐κB was further evaluated by ELISA. We also observed the effect of SFE and/or radiation on tumor growth. The results showed that SFE inhibited cell proliferation and induced apoptosis in HCC cells. Radiation increased NF‐kB activity, while PDTC, a NF‐kB inhibitor, enhanced radiation‐induced cell death. SFE inhibited NF‐kB activity and the downstream gene expressions of the NF‐kB pathway in HCC cells. Moreover, SFE enhanced the inhibitory effect of radiation on tumor growth both in vitro and in vivo. This study indicated that SFE sensitized the radiosensitivity of HCC by blocking the NF‐kB pathway.     

Distortion of homeostatic signaling proteins by simulated microgravity in rat hypothalamus: A16O/18O‐labeled comparative integrated proteomic approach

Microgravity generates oxidative stress in central nervous system, causing distortion of various vital signaling cascades involved in many homeostatic functions. Here, we performed comparative ¹⁶O/¹⁸O labeled integrated proteomic strategy to observe the differential expression of signaling proteins involved in homeostasis. In this study, rat‐tail suspension model is employed to induce simulated microgravity in CNS. By wide proteomic analysis, total of 35 and 97 significantly differentially expressed proteins were found by HPLC/ESI‐TOF and HPLC‐Q‐TOF analysis, respectively. Among the total of 132 proteins quantified, 25 proteins were found related to various signaling cascades. Protein Thy‐1, 14‐3‐3 gamma, 14‐3‐3 epsilon, 14‐3‐3 theta, 14‐3‐3 eta, and 14‐3‐3 beta/alpha proteins, calmodulin and calcium/calmodulin‐dependent protein kinase type‐II subunit beta were found upregulated under the influence of simulated microgravity. These proteins are found involved in disrupting homeostatic pathways like sleep/wake cycle, drinking behavior, hypothalamic‐pituitary‐adrenocortical regulation and fight and/or flee actions under stress. Furthermore, MS results for protein Thy‐1 were verified by Western blot analysis showing the quantification accuracy of MS instruments. Results presented here will serve as means to understand the mechanism of action of microgravity and further reference for future detailed study of consequences of microgravity on astronauts and their possible countermeasures.     

Curcumin reinforces MSC‐derived exosomes in attenuating osteoarthritis via modulating the miR‐124/NF‐kB and miR‐143/ROCK1/TLR9 signalling pathways

Curcumin treatment was reported to delay the progression of OA, but its underlying mechanism remains unclear. In this study, we aimed to investigate the molecular mechanism underlying the role of curcumin in OA treatment. Accordingly, by conducting MTT and flow cytometry assays, we found that the exosomes derived from curcumin‐treated MSCs helped to maintain the viability while inhibiting the apoptosis of model OA cells. Additionally, quantitative real‐time PCR and Western blot assays showed that the exosomes derived from curcumin‐treated MSCs significantly restored the down‐regulated miR‐143 and miR‐124 expression as well as up‐regulated NF‐kB and ROCK1 expression in OA cells. Mechanistically, curcumin treatment decreased the DNA methylation of miR‐143 and miR‐124 promoters. In addition, the 3’ UTRs of NF‐kB and ROCK1 were proven to contain the binding sites for miR‐143 and miR‐124, respectively. Therefore, the up‐regulation of miR‐143 and miR‐124 in cellular and mouse OA models treated with exosomes remarkably restored the normal expression of NF‐kB and ROCK1. Consequently, the progression of OA was attenuated by the exosomes. Our results clarified the molecular mechanism underlying the therapeutic role of MSC‐derived exosomes in OA treatment.     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

Protein pattern of Xenopus laevis embryos grown in simulated microgravity

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.     

Long noncoding ribonucleic acid NKILA induces the endoplasmic reticulum stress/autophagy pathway and inhibits the nuclear factor‐k‐gene binding pathway in rats after intracerebral hemorrhage

Long noncoding RNAs (lncRNAs) have emerged as an important class of molecules that have been associated with brain function and neurological disease, but the expression profiles of lncRNAs after intracerebral hemorrhage (ICH) remain to be elucidated. In this study, we determined the expression pattern of nuclear factor‐k‐gene binding (NF‐kB) interacting lncRNA (NKILA) after ICH and examined its respective effects on the endoplasmic reticulum stress (ERS)/autophagy pathway, hippocampal neuron loss, and the NF‐kB pathway after type VII collagenase‐induced ICH in rats. The regulatory mechanisms of NKILA were investigated by an intraperitoneal injection of small interfering (siRNA) against NKILA into rats after ICH. NKILA inhibition mediated by siRNA against NKILA was shown to significantly reduce ERS and autophagy, activate the NF‐kB pathway, decrease neurological deficits, brain edema, and injury, and induce blood–brain barrier breakdown, further leading to hippocampal neuron loss and the production of inflammation cytokines. Taken together, the demonstration that NKILA induces the ERS/autophagy pathway and inhibits the NF‐kB pathway after ICH supports the concept that NKILA functions as a novel target that is required for the attenuation of brain injuries after ICH.     

A role for the non‐receptor tyrosine kinase ACK1 in TNF‐alpha‐mediated apoptosis and proliferation in human intestinal epithelial caco‐2 cells

The roles of tumor necrosis factor alpha (TNF‐alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42‐associated kinase 1 (ACK1) in TNF‐alpha‐mediated apoptosis and proliferation in Caco‐2 cells. ACK1 expression was knocked down using ACK1‐specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin‐dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1‐specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF‐alpha‐mediated anti‐apoptotic effects and proliferation of Caco‐2 cells. Interestingly, TNF‐alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco‐2 cells. ACK1‐Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down‐stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF‐alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor‐кB (NF‐кB) activity, suggesting a correlation between NF‐кB signaling and TNF‐alpha‐mediated apoptosis in Caco‐2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF‐alpha‐induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down‐stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation.