Tumor Necrosis Factor-alpha Treatment Research Articles

Repression of hepatitis B viral gene expression by transcription factor nuclear factor-kappaB

Infection of human hepatitis B virus (HBV) causes acute hepatitis. Its persistent infection leads to a high risk of developing chronic hepatitis, cirrhosis and hepatocellular carcinoma. The levels of HBV 3.5 kb and 2.4/2.1 kb RNAs transcribed from a replicating HBV expression plasmid in human hepatoma HuH-7 cells are repressed by tumour necrosis factor alpha treatment or overexpressed p65 in a dose-dependent manner. The diminished expression of endogenous p65 by a p65-specific siRNA or IkappaB-alpha overexpression enhances the HBV gene expression. The protein bound to the Specificity protein 1 (Sp1) binding sites (nt 1733-1753) of HBV core promoter is reduced by either tumour necrosis factor alpha treatment or overexpressed p65. The N-terminal 43-amino-acid region of p65, which is required to interact with Sp1, is essential to repress the Sp1-mediated transactivation. The binding of Sp1 to Sp1 site and the Sp1-dependent reporter expression are inhibited by p65 in a dose-dependent manner. Furthermore, nuclear factor-kappa B-mediated repression of HBV gene expression is abolished by deletion of Sp1 sites of HBV gene promoter. Together, these results demonstrate that nuclear factor-kappa B represses the HBV gene expression through its interaction with Sp1 and repression of Sp1-mediated transcriptional activation.

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF‐α

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.

Decreased Expression of Cytochromes P450 1A2, 2E1, and 3A4 and Drug Transporters Na+-Taurocholate-Cotransporting Polypeptide, Organic Cation Transporter 1, and Organic Anion-Transporting Peptide-C Correlates with the Progression of Liver Fibrosis in Chronic Hepatitis C Patients

Patients with chronic hepatitis C viral infection underwent liver biopsies and laboratory studies for evaluation and to determine subsequent treatment. Changes in status of drug metabolism and disposition may vary with chronic hepatitis C stage and should be assessed. Total RNA was extracted from liver biopsy specimens (n = 63) and reverse transcribed to yield cDNA. Relative mRNA levels of drug-metabolizing enzymes, transporters, nuclear receptors, and proinflammatory cytokines were analyzed with normalization to glyceraldehyde 3-phosphate dehydrogenase expression. mRNAs encoding cytochromes P450 1A2, 2E1, and 3A4, and drug transporters, Na(+)-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1 showed remarkable decreases, and tumor necrosis factor-alpha showed an increase according to fibrosis stage progression. HepG2 cells and primary hepatocytes of two human individuals were treated with interleukin 1beta, interleukin 6, or tumor necrosis factor-alpha. CYP1A2 and Na(+)-taurocholate-cotransporting polypeptide mRNA levels significantly decreased in HepG2 cells with interleukin 1beta and interleukin 6 treatments. CYP2E1 and organic cation transporter 1 mRNA levels significantly decreased with tumor necrosis factor-alpha treatment only in HepG2. These results suggested that down-regulation of CYP1A2, 2E1, and 3A4, and drug transporters, Na(+)-taurocholate-cotransporting polypeptide, organic anion-transporting peptide-C, and organic cation transporter 1, manifested in livers of patients with chronic hepatitis C viral infection, was associated, at least in part, with the elevated production of proinflammatory cytokines, including tumor necrosis factor-alpha.

An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor‐ᆇ

Tumor necrosis factor-alpha (TNF) is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival following partial hepatectomy, but also acts in concert with other inflammatory cytokines of the innate immune response during viral infection to induce apoptosis in hepatocytes. In other epithelial cell types, TNF has recently been shown to stimulate autocrine release of transforming growth factor-alpha (TGF-alpha) and interleukin-1 (IL-1) family ligands. Here, we examine the role of these autocrine ligands in modulating TNF-induced proliferation and apoptosis in primary hepatocytes. We show that TNF-induced hepatocyte proliferation is regulated by an inducible, coupled, and self-antagonizing autocrine cascade involving the pro-proliferative TGF-alpha and IL-1 receptor antagonist (IL-1ra) ligands and antiproliferative IL-1alpha/beta ligands. Moreover, cooperative stimulation of hepatocyte proliferation by combined TNF and TGF-alpha treatment is self-limited through antiproliferative autocrine IL-1alpha/beta feedback. We show that TNF potently induces apoptosis of adenovirus-infected hepatocytes in a manner similarly determined through the integrated activity of a coupled TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade. Exogenous TGF-alpha can either enhance or diminish apoptosis in adenoviral vector-treated and TNF-treated hepatocytes, in a biphasic relationship also mediated by autocrine IL-1alpha/beta feedback. We demonstrate that TNF-induced hepatocyte proliferation and apoptosis are both governed by a self-antagonizing TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade in vitro, and thus identify multiple molecular targets for control of TNF-regulated hepatocyte phenotypic responses related to liver regeneration and adenoviral gene therapy.

Correlation of the Tight Junction-like Distribution of Claudin-1 to the Cellular Tropism of Hepatitis C Virus

Claudin-1 (CLDN1), a tight junction (TJ) protein, has recently been identified as an entry co-receptor for hepatitis C virus (HCV). Ectopic expression of CLDN1 rendered several non-hepatic cell lines permissive to HCV infection. However, little is known about the mechanism by which CLDN1 mediates HCV entry. It is believed that an additional entry receptor(s) is required because ectopic expression of CLDN1 in both HeLa and NIH3T3 cells failed to confer susceptibility to viral infection. Here we found that CLDN1 was co-immunoprecipitated with both HCV envelope proteins when expressed in 293T cells. Results from biomolecular fluorescence complementation assay showed that overexpressed CLDN1 also formed complexes with CD81 and low density lipoprotein receptor. Subsequent imaging analysis revealed that CLDN1 was highly enriched at sites of cell-cell contact in permissive cell lines, co-localizing with the TJ marker, ZO-1. However, in both HeLa and NIH3T3 cells the ectopically expressed CLDN1 appeared to reside predominantly in intracellular vesicles. The CLDN1-CD81 complex formed in HeLa cells was also exclusively distributed intracellularly, co-localizing with EEA1, an early endosomal marker. Correspondingly, transepithelial electric resistance, obtained from the naturally susceptible human liver cell line, Huh7, was much higher than that of the HeLa-CLDN1 cell line, suggesting that Huh7 is likely to form functional tight junctions. Finally, the disruption of TJ-enriched CLDN1 by tumor necrosis factor-alpha treatment markedly reduced the susceptibility of Huh7.5.1 cells to HCV infection. Our results suggest that the specific localization pattern of CLDN1 may be crucial in the regulation of HCV cellular tropism.

Evaluation and Management of Severe Asthma

Severe and difficult-to-treat asthma patients have impaired health status and account for over half of the cost of the disease and probably all of its mortality. Guideline-based treatment for these refractory asthma patients includes high doses of inhaled steroids and long acting beta2-agonists (LABA) as well as additional medication such as theophylline, oral steroids and leukotriene-antagonists. However, management regimens are rarely successful in these patients, the treating physicians are often at a loss and there are still many areas of uncertainty regarding steroid-responsiveness, safety issues and maximal dosing of inhaled corticosteroids (ICS) and bronchodilators and possible differences between ICS preparations. Furthermore, there are many other classes of medication that have been or are currently tested in severe asthma: Anti - Immunoglobulin E (IgE) seems to have a good clinical effect while results on anti - Interleukin 5 (IL-5) are less promising and anti - tumor necrosis factor alpha (TNFalpha) treatment is currently being tested in controlled studies. Other steroid sparing drugs (cyclosporine, tacrolimus, methotrexate, gold) have been used but results are unsatisfactory and side-effects are notable so steroids remain the cornerstone of severe asthma treatment. The addition of macrolides is beneficial in many cases and this is in step with the evidence of chronic chlamydia infection in severe asthma. Although there are case reports supporting the use of Immunoglobulin G (IgG), there are no controlled studies supporting this type of treatment. In this report, the authors review the various issues of guideline based therapy but also new approaches that include anti-IgE antibodies, anti-cytokines (anti-IL-5, anti-IL9, anti-TNF).

E74-like factor 2 regulates valosin-containing protein expression

Enhanced expression of valosin-containing protein (VCP) correlates with invasion and metastasis of cancers. To clarify the transcription mechanism of VCP, human and mouse genomic sequence was compared, revealing a 260 bp DNA sequence in the 5′-flanking region of VCP gene to be highly conserved between the two, in which binding motif of E74-like factor 2/new Ets-related factor (ELF2/NERF) was identified. Chromatin immunoprecipitation assay showed binding of ELF2/NERF to the 5′-flanking region of VCP gene. Knock-down of ELF2/ NERF by siRNA decreased expression level of VCP. Viability of cells under tumor necrosis factor-alpha treatment significantly reduced in ELF2/ NERF-knock-down breast cancer cell line. Immunohistochemical analysis on clinical breast cancer specimens showed a correlation of nuclear ELF2/NERF expression with VCP expression and proliferative activity of cells shown by Ki-67 immunohistochemistry. These findings indicate that ELF2/NERF promotes VCP transcription and that ELF2/NERF–VCP pathway might be important for cell survival and proliferation under cytokine stress.

Opioid receptor expression on neutrophils: effect of tumour necrosis factor alpha treatment

Opioids and endogenous opioid peptides possess immunomodulating properties and are involved in the regulation of immunocyte activity. These effects are mediated by opioid receptors (OR) on peripheral white blood cells that can be detected and quantified by flow cytometry. We investigated OR expression on neutrophils using polyclonal antibodies against δ-opioid, κ-opioid and μ-opioid receptors (DOR, KOR, MOR) to assess spontaneous and TNFα-induced OR expression on neutrophils.

Unloading kinesin transported cargoes from the tubulin track via the inflammatory c‐Jun N‐terminal kinase pathway

Axonal transport of mitochondria and synaptic vesicle precursors via kinesin motor proteins is essential to keep integrity of axons and synapses. Disturbance of axonal transport is an early sign of neuroinflammatory and neurodegenerative diseases. Treatment of cultured neurons by the inflammatory cytokine tumor necrosis factor-alpha (TNF) stimulated phosphorylation of c-Jun N-terminal kinase (JNK) in neurites. TNF treatment induced dissociation of the heavy chain kinesin family-5B (KIF5B) protein from tubulin in axons but not cell bodies as determined by lifetime-based Förster resonance energy transfer (FRET) analysis. Dissociation of KIF5B from tubulin after TNF treatment was dependent on JNK activity. Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK. Thus, TNF produced by activated glial cells in inflammatory or degenerative neurological diseases acts on neurites by acting on the kinesin-tubulin complex and inhibits axonal mitochondria and synaptophysin transport via JNK.

Critical Role for Mitochondrial Oxidative Phosphorylation in the Activation of Tumor Suppressors Bax and Bak

Activation of Bax and Bak, which act to permeabilize the mitochondrial membrane, is an essential step in the cell death response and therefore in the suppression of tumorigenesis. However, the mechanisms that regulate activation are poorly understood. Bax and Bak activation (conformational change and dimerization) was monitored in Rat-1 fibroblasts and human cancer cells subjected to endoplasmic reticulum (ER) stress, DNA damage, or tumor necrosis factor-alpha (TNF-alpha) treatment. Pharmacologic inhibitors of reactive oxygen species production, electron transport in the respiratory chain, oxidative phosphorylation, and appropriate controls were used to identify potential modes by which Bax and Bak activation and the cell death response are controlled. The oligomerization state of Bax and Bak was determined by cross-linking and subsequent immunoblot analysis; Bax conformational change was analyzed by immunoprecipitation and immunoblotting with an antibody specific for the active conformation. Cell death was evaluated by dye exclusion. In both fibroblasts and human cancer cells subjected to cell death stimuli, inhibition of oxidative phosphorylation by use of antimycin A or oligomycin prevented ER stress-, DNA damage-, and TNF-alpha-induced Bax and Bak activation and cell death (UV-induced Rat-1 cell death at 15 hours: control, mean = 33.6%, 95% confidence interval [CI] = 18.8% to 48.4%; antimycin A, mean = 10.0%, 95% CI = 0% to 21.7%; oligomycin, mean = 13.1%, 95% CI = 5.7% to 20.5%; tunicamycin-induced MCF-7 cell death at 9 hours: control, mean = 29.2%, 95% CI = 21.6% to 36.8%; antimycin A, mean = 15.3%, 95% CI = 0.8% to 29.8%; oligomycin, mean = 11.5%, 95% CI = 3.9% to 19.1%; TNF-alpha-induced MCF-7 cell death at 6 hours: control, mean = 24.0%, 95% CI = 12.6% to 35.4%; antimycin A, mean = 8.9%, 95% CI = 3.9% to 13.9%; oligomycin, mean = 13.3%, 95% CI = 10.4% to 16.2%). Increasing and decreasing glycolytic adenosine triphosphate production, by adding glucose and 2-deoxy-D-glucose to the cell growth medium, respectively, neither reversed nor recapitulated, respectively, the effect of compromised oxidative phosphorylation on Bax and Bak activation. Oxidative phosphorylation is required for the activation of Bax and Bak and cell death triggered by disparate death stimuli. The reliance of tumor cells on glycolysis in preference to oxidative phosphorylation even under normoxic conditions (Warburg effect) may therefore be a potential means by which these cells evade programmed cell death.

EMAP-II facilitates TNF-R1 apoptotic signalling in endothelial cells and induces TRADD mobilization

Endothelial monocyte-activating polypeptide-II (EMAP-II), a proinflammatory cytokine with antiangiogenic properties, renders tumours sensitive to tumour necrosis factor-alpha (TNF) treatment. The exact mechanisms for this effect remain unclear. Here we show that human endothelial cells (EC) are insensitive to TNF-induced apoptosis but after a short pre-treatment with EMAP-II, EC quickly undergo TNF-induced apoptosis. We further analysed this EMAP-II pre-treatment effect and found no increase of TNF-R1 protein expression but rather an induction of TNF-R1 redistribution from Golgi storage pools to cell membranes. In addition, we observed EMAP-II induced mobilization and membrane expression of the TNF-R1-Associated Death Domain (TRADD) protein. Immunofluorescence co-staining experiments revealed that these two effects occurred at the same time in the same cell but TNF-R1 and TRADD were localized in different vesicles. These findings suggest that EMAP-II sensitises EC to apoptosis by facilitating TNF-R1 apoptotic signalling via TRADD mobilization and introduce a molecular and antiangiogenic explanation for the TNF sensitising properties of EMAP-II in tumours.

Ginsenoside Rg1 inhibits proliferation of vascular smooth muscle cells stimulated by tumor necrosis factor-α1

To investigate the proliferation of vascular smooth muscle cells (VSMC) affected by ginsenoside Rg1 and further explore the molecular mechanism of ginsenoside Rg1 using proteomics. The proliferation of VSMC was measured by MTS assay kit and flow cytometry. Proteomic alterations were analyzed using two-dimensional electrophoresis and peptide mass fingerprinting. Differential proteins found in proteomics were confirmed by RT-PCR. The proliferation of VSMC was enhanced significantly after tumor necrosis factor-alpha (TNF-alpha) treatment, and ginsenoside Rg1 treatment inhibited proliferation in a dose-dependent manner. Proteomic analysis showed 24 protein spots were changed, including 17 spots that were increased and 7 spots that were decreased. Ginsenoside Rg1 could restore the expression levels of these proteins, at least partly, to basic levels of untreated cells. The expression of G-protein coupled receptor kinase, protein kinase C (PKC)-zeta, N-ras protein were decreased, while cycle related protein p21 was increased by ginsenoside Rg1 in TNF-alpha treated VSMC. PKC-zeta and p21 pathway might be the mechanism for inhibitory effects of ginsenoside Rg1 on proliferation of VSMC.

Effects of indomethacin and dexamethasone on mechanical scratching‐induced cutaneous barrier disruption in mice

Effects of indomethacin and dexamethasone on recovery of cutaneous barrier disruption induced by mechanical scratching were examined. Cutaneous barrier was disrupted by scratching using a stainless-steel wire brush (mechanical scratching) and compared to cutaneous application of acetone/ether (1:1) mixture (AE) and tape-stripping. Increase of transepidermal water loss (TEWL), as an indicator of a broken skin barrier, and recovery period for mechanical scratching were higher and longer than those for AE treatment and tape-stripping and we also confirmed the severity of skin damage in a histological study. Topical application of moisturizers showed a temporal effect, rapidly decreased TEWL on mechanical scratching- or AE treatment-induced cutaneous barrier disruption, and gradually increased base levels from 4 to 12 h after treatment. Topical application of indomethacin or dexamethasone prolonged the recovery period for the cutaneous barrier, and concomitant use further worsened the status of the barrier. Additionally, we examined the effects of prostaglandins (PGs) and inflammatory cytokine on mechanical scratching-induced cutaneous barrier disruption pretreated with indomethacin and dexamethasone. As a results, PGD2 and interleukin (IL)-1beta significantly accelerated the recovery of cutaneous barrier disruption by mechanical scratching but such was not the case with PGE2, IL-1alpha, and tumor necrosis factor-alpha treatment. These results suggest that indomethacin and dexamethasone prolonged the recovery period caused by inhibition of PGD2 and IL-1beta. Mechanical scratching-induced cutaneous barrier disruption may be a useful method for evaluating means of recovery from skin damage.

Glycogen Synthase Kinase 3β Functions To Specify Gene-Specific, NF-κB-Dependent Transcription

Loss of glycogen synthase kinase 3beta (GSK-3beta) in mice results in embryonic lethality via hepatocyte apoptosis. Consistent with this result, cells from these mice have diminished nuclear factor kappaB (NF-kappaB) activity, implying a functional role for GSK-3beta in regulating NF-kappaB. Here, we have explored mechanisms by which GSK-3beta may control NF-kappaB function. We show that cytokine-induced IkappaB kinase activity and subsequent phosphorylation of IkappaBalpha, p105, and p65 are not affected by the absence of GSK-3beta activity. Furthermore, nuclear accumulation of p65 following tumor necrosis factor treatment is unaffected by the loss of GSK-3beta. However, NF-kappaB DNA binding activity is reduced in GSK-3beta null cells and in cells treated with a pharmacological inhibitor of GSK-3. Expression of certain NF-kappaB-regulated genes, such as IkappaBalpha and macrophage inflammatory protein 2, is minimally affected by the absence of GSK-3beta. Conversely, we have identified a subset of NF-kappaB-regulated genes, including those for interleukin-6 and monocyte chemoattractant protein 1, that require GSK-3beta for efficient expression. We show that efficient localization of p65 to the promoter regions of the interleukin-6 and monocyte chemoattractant protein 1 genes following tumor necrosis factor alpha treatment requires GSK-3beta. Therefore, GSK-3beta has profound effects on transcription in a gene-specific manner through a mechanism involving control of promoter-specific recruitment of NF-kappaB.

Identification and expression profiling of a human C‐type lectin, structurally homologous to mouse dectin‐2

A number of C-type lectins on antigen-presenting cells play an important role in regulating innate immunity. Previously, we identified the mouse C-type lectins (dectin-1, and dectin-2) and human DECTIN-1. To identify human DECTIN-2, we employed degenerative polymerase chain reaction-based cDNA cloning using RNA from human Langerhans cell (LC)-like dendritic cells (DCs). This process yielded a cDNA encoding a C-type lectin with 66.5% amino acid sequence homology to mouse dectin-2, the same gene reported by Kanazawa et al. (J Invest Dermatol 2004: 122: 1522-1524) using the disparate approach of analyzing coding sequences in chromosome 12. Similar to their findings, we found gene expression in lung, spleen, and lymph node. Among resting leukocytes, it was expressed at highest levels by CD14+ monocytes, at lower levels by CD19+ B cells, and not at all by CD4+ T cells. Activation of CD19+ B cells with pokeweed mitogen down-regulated gene expression, whereas expression in CD4+ T cells was induced by Con A. Among our novel findings are an alternatively spliced transcript lacking exon 2, expression in bone marrow and tonsil, expression in CD8+ T cells that is abrogated following activation with phytohemagglutinin, restricted expression to CD1a+ LC within epidermis, and preferential expression by plasmacytoid (rather than myeloid) DC. Finally, we found that treatment with interleukin-4 (IL-4), IL-10, or UVB down regulated gene expression in CD14+ monocytes, whereas granulocyte-macrophage colony-stimulating factor, transforming growth factor-beta1, or tumor necrosis factor-alpha treatment up-regulated it. Our findings may form the basis for understanding the function of human DECTIN-2 in innate immunity.

Neuregulins Rescue PC12-ErbB-4 Cells From Cell Death Induced by β-Amyloid Peptide: Involvement of PI3K and PKC

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. We previously showed that NRG mediates survival of PC12-ErbB-4 cells from apoptosis induced by serum deprivation, tumor necrosis factor-alpha treatment, or H2O2. These effects of NRGs are mediated by the phosphoinositide 3-kinase (PI3K) signaling pathway. In the present study, we show that NRG induces a significant protective effect from beta-amyloid 25-35 (Abeta[25-35]) peptide-induced cell death. The PI3K signaling pathway might be involved in this effect of NRG as the downstream effector of PI3K, protein kinase B (PKB/AkT), is activated by NRG in the presence of Abeta, and PKB/AkT activation is inhibited by the PI3K inhibitor, LY294002. In addition, our results demonstrate that Abeta-induced cell death is reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways might regulate the toxic effect of Abeta. In addition, Abeta induced alteration in the levels of the proapoptotic protein Bax. Neuregulin (NRG) treatment however, induced elevation in the levels of the antiapoptotic protein BclxL. The NRG-mediated BclxL elevation is regulated by protein kinase C (PKC), as NRG failed to elevate BclxL in the presence of the PKC inhibitor, GF109203X. Moreover, activation of PKC by phorbol 12-myristate 13-acetate markedly attenuated cell death induced by Abeta and induced elevation in BclxL levels. The results suggest that NRG might affect cell viability using two signaling pathways: activation of PI3K/PKB/AkT pathway and activation of PKC, which results in increasing levels of the antiapoptotic protein BclxL.

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Protective effects of a free radical scavenger, MCI‐186, on high‐glucose–induced dysfunction of human dermal microvascular endothelial cells

Functional damage to microvascular endothelial cells by hyperglycemia is thought to be one of the critical risk factors in the impaired wound healing seen with diabetes mellitus. It is also thought that free radical stress plays a significant role in this endothelial cell dysfunction. In the present study, the effect of a free radical scavenger, MCI 186, on the endothelial cell dysfunction of cultured cells induced by high-glucose conditions was studied. Human dermal microvascular endothelial cells were cultured with high-glucose medium (50 mM) with or without MCI-186 (10 microM) for 7 days. Fifty mM mannitol was used as an osmotic control in this study. After this treatment, cell proliferation, activation of mitogen-activated protein kinase (MAPK), the level of apoptosis, and caspase-3 activation induced by removal of growth factors or tumor necrosis factor-alpha treatment were studied. High-glucose conditions significantly decreased cell proliferation and increased apoptosis levels with the activation of caspase-3 induced by growth factor removal. The high-glucose condition significantly activated MAPK. MCI-186 treatment improved cellular proliferation and reduced apoptosis and caspase-3 activation induced by high-glucose conditions. MCI-186 also inhibited the activation of MAPK. On the other hand, MCI-186 did not alter the level of apoptosis and caspase-3 activation induced by TNF-alpha treatment. In conclusion, we suggest that MCI-186 may be beneficial for improving the endothelial cell dysfunction induced by hyperglycemia.

MUC1 expression in human prostate cancer cell lines and primary tumors.

MUC1 expression was evaluated in normal prostate epithelial cells (PrEC), and prostate cancer cell lines in response to dihydrotestosterone (DHT), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment. Expression of MUC1 core protein was stimulated in PrEC and PC-3 cells after cytokine treatment, but was highly and constitutively expressed by DU-145 cells. MUC1 was not expressed by LNCaP, C4-2 or C4-2B cells under any condition. DHT alone or in combination with cytokines had no effect on MUC1 expression in any cell line tested. Using antibodies capable of detecting all isoforms of MUC1 core protein independent of their glycosylation state, immunohistochemical staining of tissue microarrays containing both nontumor and tumor tissue revealed that only 17% of tumor tissues and 41% of nontumor tissues stained positively for MUC1. Staining patterns in tumor tissue varied from focal apical staining to diffuse cytoplasmic staining. Neither the presence of MUC1 core protein nor its subcellular distribution correlated with Gleason grade. These data indicate that MUC1 is a poor marker of prostate cancer progression. Furthermore, IFN-gamma and TNF-alpha strongly induce MUC1 expression in both normal prostate epithelia and certain prostate tumor cell lines and may exacerbate pathologies associated with MUC1-positive prostate cancers.

A Novel Human Phosphatidylethanolamine-binding Protein Resists Tumor Necrosis Factor α-induced Apoptosis by Inhibiting Mitogen-activated Protein Kinase Pathway Activation and Phosphatidylethanolamine Externalization

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.