Amygdala Kindling Potentiates Seizure-Stimulated Immediate-Early Gene Expression in Rat Cerebral Cortex

Kindling induces long-term adaptations in neuronal function that lead to a decreased threshold for induction of seizures. In the present study, the influence of amygdala kindling on levels of mRNA for the immediate-early genes (IEGs) c-fos, c-jun, and NGF1-A were examined both before and after an acute electroconvulsive seizure (ECS). Although amygdala kindling did not significantly influence resting levels of c-fos mRNA in cerebral cortex, ECS-stimulated levels of c-fos mRNA (examined 45 min after ECS) were approximately twofold greater in the cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was dependent on the time course of kindling, as ECS-stimulated levels of c-fos mRNA were not significantly increased in stage 2 kindled animals. ECS-stimulated levels of c-jun and NGF1-A mRNA were also significantly increased in cerebral cortex of kindled rats relative to sham-treated controls. The influence of kindling on IEG expression was long-lasting because an acute ECS stimulus significantly elevated levels of c-fos and c-jun mRNA in the cerebral cortex of animals that were kindled 5 months previously. In contrast to these effects in cerebral cortex, kindling did not influence ECS-stimulated levels of c-fos mRNA in hippocampus. Finally, immunohistochemical studies revealed lamina-specific changes in the cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mouse Brain c-fos mRNA Distribution Following a Single Electroconvulsive Shock

The regional distribution of c-fos mRNA in the mouse brain has been investigated by in situ hybridization autoradiography after seizures induced by an acute electroconvulsive shock (ECS). ECS led to a widespread induction of the proto-oncogene c-fos in the brain, with highest concentrations in discrete areas within the limbic system and also in the hypothalamus and cerebellum. The mild stress of sham treatment in earclipped animals induced a weaker and qualitatively different pattern of c-fos mRNA expression involving the cortex, hippocampus, and cerebellum. These data suggest the usefulness of c-fos in situ hybridization as a marker of neuronal stimulation and in mapping a range of effects from a mild stress to the robust changes of an electroconvulsive seizure.     

Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures

G protein-coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNA's encoding regulators of G protein signaling (RGS) protein subtypes-2, -4, -7, -8 and -10 were evaluated in rat brain using northern blotting and in situ hybridization. In prefrontal cortex, RGS2 mRNA levels were increased several-fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, -7, and -10 mRNA levels were reduced by 20-30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, -8 and -10 mRNA levels were decreased in the granule cell layer, and RGS7 and -8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and -10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype-, time-, and region-specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.     

Effects of neonatal diethylstilbestrol exposure on c-fos and c-jun protooncogene expression in the mouse uterus

Quantitative and cell-type-specific expression of c-fos and c-jun genes after 17beta-estradiol (E2) stimulation, was investigated in the uteri of neonatally diethylstilbestrol (DES)-exposed and ovariectomized adult mice (neoDES-mice), employing Northern blot analysis, immunohistochemistry and in situ hybridization. The c-fos mRNA level before E2 injection (at baseline) was about 2.2-fold higher in neoDES-mice than in vehicle-treated control mice. In controls, E2 treatment transiently increased c-fos mRNA levels, showing a peak value (15.8-fold relative to the baseline) after 2 hours. In neoDES-mice, c-fos mRNA level reached a peak showing a 2.1-fold increase compared with its baseline value 1 hour after E2 injection. Immunohistochemistry and in situ hybridization revealed that c-fos protein (Fos) and mRNA are induced in the epithelium and vascular endothelium in both groups. Most uterine epithelia of neoDES-mice revealed low sensitivity to the c-fos expression after E2 administration compared with those of vehicle-treated controls, whereas few epithelia showed high c-fos mRNA expression even at baseline. The c-jun mRNA concentration in the neoDES-mice uteri at baseline was 70% of that in vehicle-treated controls. At 1 hour after E2 injection, c-jun mRNA levels increased 1.8-fold in controls and 1.3-fold in the neoDES-mice relative to each baseline value. There were no significant differences in the distribution pattern of c-jun protein (Jun) and mRNA in the uteri of either groups; E2 stimulated c-jun mRNA expression in the stromal and myometrial cells but suppressed it in the epithelial cells, whereas intensity of c-jun immunostaining increased in the three cell types. The permanent changes in the expression of estrogen-regulated protooncogenes, c-fos and c-jun genes, by neonatal DES exposure may be responsible for the wide range of abnormalities in the genital tract of mature animals.     

Induction of early response genes in trypsin inhibitor-induced pancreatic growth

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1-24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1-2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.     

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osteoblasts are target cells for transformation in c-fos transgenic mice

We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype.     

低、高频电针诱导原癌基因c－fos／c－jun和阿片肽基因表达及其关系的比较研究

本工作对低频（２Ｈｚ）和高频（１００Ｈｚ）电针引起大鼠中枢Ｆｏｓ／Ｊｕｎ表达和三类阿片肽基因表达进行了详细的比较研究,并用针对ｃ－ｆｏｓ／ｃ－ｊｕｎ的反义寡核苷酸（ＯＤＮｓ）对电针引起的Ｆｏｓ／Ｊｕｎ表达进行选择性阻断,然后观察阿片肽基因表达是否受到影响,以确定Ｆｏｓ／Ｊｕｎ复合物在电针引起阿片肽基因表达中的作用。主要结果如下：（１）２Ｈｚ和１００Ｈｚ电针引起脑内不同部位的Ｆｏｓ／Ｊｕｎ表达;（２）２Ｈｚ电针使前脑啡肽原（ＰＰＥ）ｍＲＮＡ表达大幅度增高,１００Ｈｚ电针也能增加ＰＰＥｍＲＮＡ的转录,但不如２Ｈｚ电针的作用显著;但１００Ｈｚ电针能使某些脑区的前强啡肽原（ＰＰＤ）基因转录加速,而２Ｈｚ电针没有这一作用;（３）用ｃ－ｆｏｓ／ｃ－ｊｕｎ反义ＯＤＮｓ特异地阻断Ｆｏｓ／Ｊｕｎ表达后,电针引起的ＰＰＤ转录加速被明显阻断,而ＰＰＥ表达不受影响。提示Ｆｏｓ／Ｊｕｎ是电针引起ＰＰＤ（而非ＰＰＥ）基因表达的转录因子。     

Morphine activation of c-fos expression in rat brain

The post-receptor mechanism of opiate action has been studied by examining the activation by morphine of the proto-oncogene c-fos and its encoded nucleoprotein pp55c-fos (FOS) in rat caudate-putamen, which is rich in the mu-type opiate receptor. Following an acute morphine treatment, c-fos mRNA levels in rat caudate-putamen were increased to maximum (420% of control level) at 45 minutes and returned to control levels at 90 minutes. This induction was completely abolished by naloxone, a morphine antagonist. Fos protein, detected by immunocytochemistry, was also increased 3 hours after morphine injection, in the caudate-putamen, but not in the olfactory tubercle, which does not have the mu-type opiate receptor. Upon activation of opiate receptors by morphine, the c-fos gene is activated and Fos protein may act as a signal transducer uniquely involved in the mechanism of opiate addiction at the level of gene regulation.     

Adrenalectomy Potentiates Immediate Early Gene Expression in Rat Brain

Administration of kainate or pentylenetetrazole increased c-fos, c-jun, junB, and junD mRNA levels in rat brain in a dose-dependent manner. Kainate increased these mRNA levels predominantly in the hippocampus, and pentylenetetrazole was more effective in the cortex. Adrenalectomy (3 days) was used to eliminate endogenous glucocorticoid hormones. Adrenalectomy significantly potentiated kainate-induced increases, compared with increases caused by kainate (4 mg/kg) alone, in the hippocampal mRNA levels of c-fos and junB by 6.5-fold and of junD by twofold and tended to augment c-jun mRNA. Corticosterone administration blocked the potentiated stimulation of these mRNA levels caused by adrenalectomy. Adrenalectomy also significantly increased pentylenetetrazole-induced levels of c-fos mRNA in the cortex. These results demonstrate that glucocorticoids modulate immediate early gene expression in the brain, raising the possibility that this interaction contributes to interneuronal and interindividual differences in responses to stimuli and to the effects of stress- or disease-induced changes in glucocorticoid concentrations.     

Intracellular signaling of gp34, the OX40 ligand: induction of c-jun and c-fos mRNA expression through gp34 upon binding of its receptor, OX40.

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.