Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA

The hormone bombesin(BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) actthrough specific GRP receptors (GRP-R) to affect multiple cellularfunctions in the gastrointestinal tract; the intracellular signalingpathways leading to these effects are not clearly defined. Previously,we demonstrated that the human gastric cancer SIIA possesses GRP-R andthat BBS stimulates activator protein-1 (AP-1) gene expression. Thepurpose of our present study was to determine the signaling pathwaysleading to AP-1 induction in SIIA cells. A rapid induction ofc-jun and jun-B gene expression was noted afterBBS treatment; this effect was blocked by specific GRP-R antagonists,indicating that BBS is acting through the GRP-R. The signaling pathwaysleading to increased AP-1 gene expression were delineated using phorbol12-myristate 13-acetate (PMA), which stimulates protein kinase C(PKC)-dependent pathways, by forskolin (FSK), which stimulates proteinkinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSKstimulated jun-B expression but produced only minimalincreases of c-jun mRNA and AP-1 binding activity.Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKCinhibitors) inhibited the induction of c-jun andjun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor)exhibited only minimal effects. Pretreatment with tyrphostin-25, aprotein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediatedinduction of c-jun and jun-B, but the effect wasnot as pronounced as with H-7. Collectively, our results demonstratethat BBS acts through its receptor to produce a rapid induction of bothc-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction ofAP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimalinvolvement of PKA.

     

Expression of c-fos and jun protooncogenes in ovine trophoblasts in relation to interferon-tau expression and early implantation process

Expression of c-fos and jun protooncogenes was analyzed in the ovine extraembryonic trophoblast from days 14–18 of gestation, using Northern and Western blotting and immunohistochemistry. This study was carried out in relation to the early implantation process and the expression of interferon-tau, which is secreted in large amounts for a few days before implantation. Our results demonstrated that c-fos, c-jun, and junB were differently expressed in the ovine trophoblast around the time of implantation. The c-fos mRNA and protein were detected at high levels prior to attachment and decreased thereafter, following the pattern of expression of interferon-tau, whereas c-jun expression was maintained at relatively high levels during the implantation process. By contrast, the levels of junB mRNA and protein decreased prior to attachment. Immunohistochemical studies indicated that JunB, like C-Fos and interferon tau, was no longer expressed in the trophoblastic cells which had established cellular contacts with the uterine epithelium. A striking finding in this study is the temporal correlation between the accumulation of c-Fos and c-Jun proteins and the expression of the interferon-tau (days 14 and 15 of gestation). We also showed by gel-retardation assays that an AP-1-like site present in the promoter of one interferon-tau gene was functional in vitro, as judged by its ability to bind day-15 trophoblast nuclear protein extracts. Nuclear proteins binding to this site had the characteristics of AP-1, as judged by the ability to be competed efficiently by a consensus TRE (12.0-tetradecanoyl phorbol 13-acetate-responsive element)-site oligonucleotide and by antibodies to c-Fos and Jun proteins. These results suggest that Fos and Jun could form regulatory complexes of interferon-tau expression and/or are regulated by common mechanisms which are still unknown. Mol Reprod Dev 46:127–137, 1997. © 1997 Wiley-Liss, Inc.     

Epilepsy, Neurodegeneration, and Extracellular Glutamate in the Hippocampus of Awake and Anesthetized Rats Treated with Okadaic Acid

We have previously shown that the intrahippocampal microinjection of okadaic acid (OKA), a potent inhibitor of serine/threonine protein phosphatases, induces epileptic seizures, neuronal death, and the hyperphosphorylation of the NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor. We administered OKA by reverse microdialysis in the hippocampus of awake and halothane-anesthetized rats, with simultaneous collection of microdialysis fractions and recording of the EEG activity, and subsequent histological analysis. OKA produced intense behavioral and persistent EEG seizure activity in the awake rats but not in the anesthetized animals, and did not significantly alter the extracellular concentration of glutamate and aspartate detected in the microdialysis fractions. One day after the experiment a remarkable neurodegeneration of CA1 hippocampal region was observed in both the awake and the anesthetized rats. We conclude that the OKA-induced epilepsy cannot be ascribed to increased extracellular glutamate, but to an increased sensitivity of NMDA receptor. We propose that halothane protected against the epilepsy because it blocks NMDA receptor overactivation, and that the neurodegeneration of CA1 region is independent of this overactivation and due probably to alterations of cytoskeletal proteins consequent to the OKA-induced hyperphosphorylation.     

Regulation of spermidine/spermine N1-acetyltransferase expression by cytokines and polyamines in human hepatocarcinoma cells (HepG2)

Spermidine/spermine N¹-acetyltransferase (cSAT), a key enzyme in polyamine degradation, is induced by various hepatotoxins and liver tumor promoters. In this paper we demonstrate that physiological factors, such as cytokines, control cSAT expression in HepG2 human hepatocarcinoma cells. Hepatocyte growth factor (HGF) induced the cSAT mRNA precursor (3.5 kb) at 4 h. The mature form of mRNA (1.3 kb) increased 6–8-fold between 8 and 10 h, and remained elevated until 18 h. An increase in cSAT activity (2-fold) and high levels of N¹-acetylspermidine were observed concomitantly. Interleukin-1β (IL-1β) enhanced cSAT expression (both mRNA and enzyme activity) similar to HGF, while tumor necrosis factor-α (TNFα) was less effective. This system also provides a useful means for examining the involvement of negative and positive changes of polyamines in the induction of cSAT and c-jun, a gene that participates in the control of cSAT expression. α-Difluoromethylornithine (DFMO) pretreatment, by lowering putrescine and spermidine in HGF- or IL-1β-treated cells, prevented the induction of cSAT. This effect was reversed by exogenous putrescine or spermidine. IL-1β induced c-jun mRNA more than HGF. DFMO prevented almost completely the enhancement of c-jun mRNA expression by IL-1β, and this effect was reversed by exogenous putrescine or spermidine. Therefore, we suggest that cSAT and c-jun expression is specifically regulated by polyamine-mediated mechanisms in IL-1β treated HepG2 cells. Since cSAT is inducibile by cytokines that control tumor metabolism and growth as well as tumor-host interaction, we hypothesize an involvement of cSAT in hepatoma growth. J. Cell. Physiol. 174:125–134, 1998. © 1998 Wiley-Liss, Inc.     

Hepatocyte Growth Factor-Induced Expression of Ornithine Decarboxylase, c-met,and c-mycIs Differently Affected by Protein Kinase Inhibitors in Human Hepatoma Cells HepG2

Binding of hepatocyte growth factor (HGF) to its receptor Met induces autophosphorylation and activation of the tyrosine kinase activity. In HGF-treated HepG2 cells, we studied: (i) the expression patterns of early(c-myc,c-jun,and c-fos) and delayed-early (ornithinedecarboxylase and c-met) response genes and (ii) thepossible involvement of protein kinase transducersin the control of the expression of c-metand of other genes eventually induced downstream. c-metand c-mycmRNAs peaked 1–2 h after HGF, while c-junandc-fosmRNAs slightly increased at 1 h. Ornithinedecarboxylase activity was induced earlier (4 h) thanthe mRNA (8–10 h). The transducers involved in HGF-triggered gene inductions were investigated using different protein kinase inhibitors: genistein for the receptor tyrosine kinase, herbimycin A for the nonreceptor tyrosine kinase (pp60^c-src), wortmannin for phosphatidylinositol 3-kinase (PI3K) and H7 for protein kinase C (PKC). The similarity of responses to PKC inhibition led to suppose that c-mycand ornithinedecarboxylase mRNAs were induced sequentially along the same transduction pathway triggered by HGF. Ornithine decarboxylase activity seemed to be largely regulated by phosphorylation(s). The mRNA expression of c-junwas likely to undergo a negative regulation through a mechanism involving PI3K, while that ofc-metseemed to be almost independent from various protein kinases (PI3K, pp60^c-src, and PKC).     

Cyclooxygenase-2 Induction in Cerebral Cortex: An Intracellular Response to Synaptic Excitation

Abstract: We have characterised the induction of the mitogen-inducible form of cyclooxygenase, COX-2, in the rat cerebral cortex in response to excitotoxin injection into the nucleus basalis. This model is associated with intense stimulation of the ascending pathway to the cerebral cortex, seizure activity, and subsequent ipsilateral cortical induction of various immediate early genes (IEGs), including c-fos, c-jun, and zif268, and ornithine decarboxylase enzyme activity and mRNA, all of which processes are sensitive to treatment with the N-methyl-d -aspartate (NMDA) receptor antagonist MK-801. In this study we show that excitotoxin injection also causes a marked induction of COX-2 mRNA in ipsilateral cortex detectable at 1 h and peaking at 4 h, where COX-2 mRNA levels were 19 times those in unoperated animals. Levels of COX-2 mRNA remained significantly elevated at 24 h. The early induction of COX-2 at 1 h was also seen in sham-operated animals, but at 4 h the COX-2 mRNA level was significantly increased (4.4-fold) in animals injected with excitotoxin compared with sham-operated animals. The induction at this time point (4 h) was explored pharmacologically and found to be significantly attenuated by treatment with MK-801 (1.5 mg/kg), lamotrigine (10 mg/kg), which prevents presynaptic glutamate release by blocking voltage-sensitive Na⁺ channels, and the glucocorticoid dexamethasone (3 mg/kg), which has an indirect inhibitory effect on phospholipase A₂ and COX activity. These results demonstrate that the induction of COX-2 mRNA occurs by two distinct mechanisms: the rapid and transient response to tissue damage and a second delayed and more substantial response, which is initiated by excitotoxin stimulation and is mediated by presynaptic glutamate release, NMDA receptor activation, and subsequent phospholipase A₂ activity. We propose a model to demonstrate the similarities between COX-2 and IEG mRNA induction and highlight possible mechanistic differences in the nature of the induction by the phospholipase A₂ pathway.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.