Increase In C-fos mRNA Levels Research Articles

Effects of the putative lithium mimetic ebselen on pilocarpine-induced neural activity

Lithium, commonly used to treat bipolar disorder, potentiates the ability of the muscarinic agonist pilocarpine to induce seizures in rodents. As this potentiation by lithium is reversed by the administration of myo-inositol, the potentiation may be mediated by inhibition of inositol monophosphatase (IMPase), a known target of lithium. Recently, we demonstrated that ebselen is a ‘lithium mimetic’ in regard to behaviours in both mice and man. Ebselen inhibits IMPase in vitro and lowers myo-inositol in vivo in the brains of mice and men, making ebselen the only known inhibitor of IMPase, other than lithium, that penetrates the blood-brain barrier. Our objective was to determine the effects of ebselen on sensitization to pilocarpine-induced seizures and neural activity. We administered ebselen at different doses and time intervals to mice, followed by injection of a sub-seizure dose of pilocarpine. We assessed seizure and neural activity by a subjective seizure rating scale, by monitoring tremors, and by induction of the immediate early gene c-fos. In contrast to lithium, ebselen did not potentiate the ability of pilocarpine to induce seizures. Unexpectedly, ebselen inhibited pilocarpine-induced tremor as well as pilocarpine-induced increases in c-fos mRNA levels. Both lithium and ebselen inhibit a common target, IMPase, but only lithium potentiates pilocarpine-induced seizures, consistent with their polypharmacology at diverse molecular targets. We conclude that ebselen does not potentiate pilocarpine-induced seizures and instead, reduces pilocarpine-mediated neural activation. This lack of potentiation of muscarinic sensitization may be one reason for the lack of side-effects observed with ebselen treatment clinically.

Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats

The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3, 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats.

Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in dorsal and ventral hippocampus (VH). Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day) during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH). By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR) subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR) subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.

P.2.011 GluA1 and its Postsynaptic density protein 95 (PSD-95)/Discs large/Zonula occludens-1 (PDZ)-interaction: a role in experience-dependent behavioural plasticity in the forced swim test

treatment with neither L-DOPA nor L-DOPA/sarizotan induced any significant behavioural sensitisation. Chronic treatment with L-DOPA and L-DOPA/sarizotan induced AIMs in both lines of rats. However, FRL rats treated with L-DOPA spent more time in AIMs compared to FSL rats. Moreover, co-administration with sarizotan reduced significantly L-DOPA-induced AIMs in FRL, but not in FSL rats. In situ hybridisation analysis showed that treatment with L-DOPA and L-DOPA/sarizotan significantly increased c-fos mRNA levels in the lesioned side as compared to the nonlesioned side of saline treated FRL and FSL rats. Interestingly, c-fos mRNA level in FSL group after chronic L-DOPA treatment was significantly lower than in FRL group receiving the same treatment. Moreover, co-administration with sarizotan reduced significantly L-DOPA-induced c-fos in FRL, but not in FSL, rats. The results suggest that parkinsonian animals with distinct depressive-like phenotypes react differently to PD treatments in terms of dyskinetic behaviour and striatal neuronal-like activity.

Urocortin 1 reduces food intake and ghrelin secretion via CRF2 receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.

Molecular cloning of pit-1 cDNA from porcine anterior pituitary and its involvement in pituitary stimulation by growth hormone-releasing factor.

Pit-1/GHF-1 (hereafter Pit-1) is a pituitary-specific transcription factor that participates in growth hormone (GH), prolactin and thyroid-stimulating hormone gene expression and in proliferation of the cells that produce these hormones. In this study, we determined the nucleotide sequence of porcine Pit-1 cDNA, which shows high homology (95%) with the known mammalian Pit-1s. Some differences in amino acid sequence were found in the amino terminal region, but the POU-specific and POU-homeo domains were well conserved. Porcine anterior pituitary cells in primary culture were treated for 24 h with GH-releasing factor (GRF), forskolin and phorbol ester (TPA). These agents stimulated progressive secretion of GH into the culture media. The levels of Pit-1, GH, cJun and cFos mRNAs were examined using the reverse transcription-polymerase chain reaction. Stimulation with GRF for 2 h increased the Pit-1 mRNA level 3-fold. This response was transient, as the level returned to the control level after 6 h. Forskolin and TPA evoked similar increases, but their effects appeared after 30 min and reached their maxima after 2 h. In contrast, GRF and forskolin, but not TPA, increased the GH mRNA level 2-fold after 24 h. The cJun mRNA level showed no significant change in response to these agents over 24 h and GRF and TPA increased the cFos mRNA level 1.4 and 2.3-fold, respectively, after 30 min. These results suggest that increasing Pit-1 mRNA level in response to GRF plays a role in the early event of the GH gene expression or with the assistance of other transcription factors that modulate GH gene. Furthermore, the GRF-induced increase in cFos mRNA level by GRF and TPA did not appear to be participated straightforward in the GH gene expression, suggesting that cFos alone is insufficient to the activation.

Centrifugal forces within usually-used magnitude elicited a transitory and reversible change in proliferation and gene expression of osteoblastic cells UMR-106

Centrifugation is an important step in biochemical and molecular biological researches. But the effects of centrifugal stress on cells are still unclear. In this study, osteoblastic cells UMR-106 were subjected to a moderate centrifugal stress at 209 x g for 10 min. Then the cell proliferation and gene transcription after centrifugation were analyzed with flow cytometry and Real-time RT-PCR techniques, respectively. The result showed that the cell proliferation and mRNA expression of Runx2/Cbfa1, Collagen I and osteocalcin changed shortly after centrifugal loading, but recovered to pre-load levels within 24 h. A dose-response study of exposure cells to centrifugal force at 209, 253 and 301 x g showed that the centrifugal forces within usually-used range can rapidly influenced the mRNA expression of the osteoblast-specific genes, but no statistical differences were found among the three centrifugal magnitudes. And the fast regulation in the investigated genes was proved to be related to increased c-fos mRNA levels and subsequent activation of RTK and integrity of cytoskeleton construction. The result showed that the osteoblastic cells displayed a fast auto-regulation to usually-used centrifugal stress through multiple signal pathways.

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Nitric Oxide Inhibits Endothelin-1-Induced Cardiomyocyte Hypertrophy through cGMP-mediated Suppression of Extracellular-Signal Regulated Kinase Phosphorylation

Cardiac hypertrophy is a compensatory mechanism in response to a variety of cardiovascular diseases. Recently, reactive oxygen species and nitric oxide (NO) have been demonstrated to be involved in the pathogenesis of atherosclerosis; however, the role of these free radicals in the development of cardiac hypertrophy remains unclear. In this study, we investigate NO modulation of cellular signaling in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy in culture. ET-1 treatment of cardiomyocytes increased constitutive NO synthase activity and induced NO production via the stimulation of ET-receptor subtype ET(B). Using Northern blot analysis and chloramphenicol acetyltransferase assay, we found that NO suppressed the ET-1-induced increase in c-fos mRNA level and promoter activity. In contrast, ET-1 stimulation of c-fos expression was augmented by depletion of endogenous NO generation with the addition of NO scavenger PTIO into cardiomyocytes. Cells cotransfected with the dominant negative and positive mutants of signaling molecules revealed that the Ras/Raf/extracellular-signal regulated kinase (ERK) signaling pathway is involved in ET-induced c-fos gene expression. Furthermore, NO directly inhibited ET-1-induced ERK phosphorylation and activation in a cGMP-dependent manner, indicating that NO modulates ET-1-induced c-fos expression via its inhibitory effect on ERK signaling pathway. The ET-1-stimulated activator protein-1 (AP-1) DNA binding activity and AP-1-mediated reporter activity were attenuated by NO. In addition, NO also significantly inhibited ET-1-stimulated promoter activity of hypertrophic marker gene beta-myosin heavy chain and the enhanced protein synthesis. Taken together, our findings provide the molecular basis of NO as a negative regulator in ET-1-induced cardiac hypertrophy.

Force‐independent expression of c‐fos mRNA by endothelin‐1 in rat intact small mesenteric arteries

Wall stress-independent signalling pathways were studied for endothelin-1 (ET-1)-induced c-fos expression in rat intact mesenteric small arteries. Arteries were kept unmounted in Krebs buffer, equilibrated for 1 h and stimulated with vasoactive substances for 15-60 min. The c-fos mRNA expression was determined by real-time polymerase chain reaction. Stimulation with fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and ET-1 caused about a doubling of c-fos mRNA. The ET-1-induced c-fos expression was steady (15-60 min) and was inhibited by the inhibitor of the ET(A) receptor, BQ-123. Platelet-derived growth factor-B, angiotensin II and U46619 did not cause increased c-fos mRNA levels. The broad specificity inhibitor staurosporine inhibited the response to ET-1, but inhibitors of Rho-A kinase and phosphatidylinositol 3-kinase had no effect. However, inhibitors to tyrosine kinases, the MAP kinases [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase, p38], and to conventional protein kinase C showed no inhibition. Consistent with these findings, ET-1 did not cause activation of ERK1/2, a finding also seen in vessels held under pressure. In contrast, ET-1-induced c-fos expression was inhibited by the calcium chelator BAPTA, suggesting a role for intracellular calcium. This possibility was supported by the finding that raising the extracellular K(+) concentration caused increased expression of c-fos in a concentration-dependent manner. The results suggest that in the absence of wall stress, ET-1 is able to induce increased expression of c-fos independent of traditional growth pathways, such as MAP kinase. The mechanism appears to be calcium-dependent.

Phosphorylation of cAMP response element-binding protein in cultured striatal neurons by metabotropic glutamate receptor subtype 5.

The metabotropic glutamate receptor subtype 5 (mGluR5) is densely expressed in striatal projection neurons. As a G protein-coupled receptor, mGluR5 may initiate an intracellular cascade that conveys extracellular signals to gene expression. This study investigated the possible role of mGluR5 in the inducible phosphorylation of a nuclear transcription factor, cAMP response element-binding protein (CREB), in primary cultures of striatal neurons from rat E19 embryos or neonatal day-1 pups. We found that selective activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) induced a rapid and transient increase in phosphorylated CREB immunoreactivity in striatal neurons as analyzed by both immunocytochemistry and western blot. The increase in CREB phosphorylation was concentration-dependent, and seen in neurochemically identified GABAergic neurons. Pre-treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) blocked the CHPG phosphorylation of CREB. In contrast, the mGluR1 antagonist, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOet) did not alter CHPG-stimulated CREB phosphorylation. The mGluR5 antisense oligonucleotides, but not their controls, selectively reduced basal mGluR5 levels as well as CREB phosphorylation in response to CHPG addition. Lastly, using an immediate early gene c-fos as a reporter of inducible gene expression downstream to phosphorylated CREB, we found that CHPG induced a rapid and transient increase in c-fos mRNA levels in cultured neurons as revealed by quantitative in situ hybridization. The increase in c-fos was kinetically correlated well with the CREB phosphorylation and blocked by MPEP and the CREB antisense oligonucleotides. These results demonstrate a positive linkage from surface mGluR5 to CREB phosphorylation, which is able to facilitate immediate gene expression in striatal neurons.

Effects of the adenosine A 1 receptor agonist N 6-cyclopentyladenosine on phencyclidine-induced behavior and expression of the immediate-early genes in the discrete brain regions of rats

Because of the possible interaction between adenosine receptors and dopaminergic functions, the compound acting on the specific adenosine receptor subtype may be a candidate for novel antipsychotic drugs. To elucidate the antipsychotic potential of the selective adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA), we examined herein the effects of CPA on phencyclidine (PCP)-induced behavior and expression of the immediate-early genes (IEGs), arc, c-fos and jun B, in the discrete brain regions of rats. PCP (7.5 mg/kg, s.c.) increased locomotor activity and head weaving in rats and this effect was significantly attenuated by pretreatment with CPA (0.5 mg/kg, s.c.). PCP increased the mRNA levels of c-fos and jun B in the medial prefrontal cortex, nucleus accumbens and posterior cingulate cortex, while leaving the striatum and hippocampus unaffected. CPA pretreatment significantly attenuated the PCP-induced increase in c-fos mRNA levels in the medial prefrontal cortex and nucleus accumbens. CPA also significantly attenuated the PCP-induced arc expression in the medial prefrontal cortex and posterior cingulate cortex. When administered alone, CPA decreased the mRNA levels of all IEGs examined in the nucleus accumbens, but not in other brain regions. Based on the ability of CPA to inhibit PCP-induced hyperlocomotion and its interaction with neural systems in the medial prefrontal cortex, posterior cingulate cortex and nucleus accumbens, the present results provide further evidence for a significant antipsychotic effect of the adenosine A 1 receptor agonist.

Neuronal activation and corticotropin-releasing hormone expression in the brain of obese (fa/fa) and lean (fa/?) Zucker rats in response to refeeding.

The present study was conducted to investigate the pattern of neuronal activation and corticotropin-releasing hormone (CRH) expression in fed, food deprived and refed lean (Fa/?) and obese (fa/fa) Zucker rats. The pattern of neuronal activation was studied by measuring the expression of the immediate-early gene c-fos. Expression of c-fos and CRH mRNA was determined by in situ hybridization histochemistry. In both lean and obese rats, one hour of refeeding led to a transient increase in c-fos mRNA levels which was detected in the paraventricular hypothalamic nucleus (PVH), the dorsomedial hypothalamic nucleus, the supraoptic nucleus, the paraventricular thalamic nucleus, the central nucleus of amygdala (CeA), the lateral and medial parabrachial nuclei, the nucleus of the solitary tract, and the area postrema. In addition, refeeding led to strong activation of the arginine-vasopressin neurons located in the magnocellular part of the PVH. Following 24 h of food deprivation, CRH expression in the parvocellular division of the PVH was significantly higher in obese rats compared to lean animals. During refeeding, PVH CRH mRNA levels in obese rats decreased to reach control values. The decrease in CRH expression in obese rats was accompanied by the alleviation of the hypercorticosteronemia that characterized obese Zucker rats. CRH mRNA levels in the central nucleus of the amygdala were significantly higher in lean rats than in obese animals, when the rats were fed ad libitum During food deprivation, CeA CRH mRNA levels decreased in lean rats and gradually returned to predeprivation values during refeeding. In refed obese rats, CeA levels of CRH mRNA were higher than those of ad libitum fed or food-deprived obese mutants. In the perifornical region of the lateral hypothalamic area (LHA), the expression of CRH mRNA rose significantly in response to refeeding in lean rats, but not in obese animals. Following the first hour of refeeding, the number of neurons expressing CRH mRNA in the LHA in lean rats almost doubled. The present results demonstrate that refeeding has a stimulating effect in obese Zucker rats in a pattern of activation similar to that seen in lean Fa/? rats. They also demonstrate differences in CRH expression between Fa/? and fa/fa rats after refeeding. The most apparent of these differences was seen in the lateral hypothalamus in which refeeding failed to up-regulate CRH expression in obese rats.

Naloxone-Precipitated Morphine Withdrawal Elicits Increases in c-fos mRNA Expression in Restricted Regions of the Infant Rat Brain

This paper is the first report of a genetic index for morphine withdrawal in infant rats. We examined the effects of naloxone (2 mg/kg) on c-fos mRNA levels in brains of infant and adult rats following repeated treatment with morphine (20 mg/kg, once daily for 5 days). One hour after a single administration of naloxone (naloxone challenge), an increase in c-fos mRNA was observed in the olfactory bulb, hypothalamus and medulla oblongata of infant rats, and in the olfactory bulb and hypothalamus, but not in the medulla oblongata of adult rats. The c-fos mRNA levels returned to control levels 6 h after the naloxone challenge. The increase in c-fos mRNA levels was followed by body weight loss in both infant and adult rats. When MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, was co-administered along with morphine, it inhibited the naloxone-induced increases in c-fos mRNA levels in infant rats following repeated morphine administration. These results suggest that physical dependence develops in infant rats following repeated morphine administration and that the increment of c-fos mRNA levels is a useful indicator for naloxone-precipitated morphine withdrawal in infant as well as in adult rats.

Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.

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.

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes.

Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

The Neurotrophic Activity of PACAP on Rat Cerebellar Granule Cells Is Associated with Activation of the Protein Kinase A Pathway and c‐fos Gene Expressiona,

In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth in immature cerebellar granule cells. In the present study, we have examined the transduction pathways involved in the neurotrophic activity of PACAP. Incubation of cultured granule cells with graded concentrations of PACAP produced a dose-dependent increase in c-fos mRNA level. The effects of PACAP on c-fos gene expression and granule cell survival were both mimicked by dbcAMP but not by PMA. The maximum effect of PACAP on c-fos gene expression was observed after 1 h of treatment. Similar effects of the peptide on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The PKA inhibitor H89 significantly reduced the effect of PACAP on c-fos mRNA level, whereas the specific PKC inhibitor chelerytrine had no effect. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/PKA pathway.

Regulation of c-fos expression by EGF and GnRH in specific anterior pituitary cells from proestrous female rats.

C-fos is an early expression oncogene that can be stimulated by a variety of regulators. It is expressed by subsets of all pituitary cells, with increased expression seen in proestrous rats. However, in freshly dispersed pituitary cells studied during different stages of the cycle, there is limited expression of fos by luteinizing hormone (LH) cells and little basal expression by cells with follicle-stimulating hormone (FSH) antigens. Proestrus is a time during which pituitary gonadotropes express peak levels of receptors for gonadotropin-releasing hormone (GnRH) and epidermal growth factor (EGF). We hypothesized that if GnRH or EGF stimulated fos activity in gonadotropes they would be most effective during the peak expression of their receptors. Anterior pituitaries were removed, cut into small pieces, and stimulated for 30 min. Total RNA was then collected and analyzed by Northern analysis. Both EGF and GnRH caused an increase in c-fos mRNA levels in the anterior pituitary gland compared with unstimulated pituitary glands assayed immediately after removal from the pituitary. However, the stimulatory effects were no greater than those seen with medium alone. This suggested that fos expression could be stimulated by local factors either in the pituitary or the medium itself. The second phase of the study focused on pituitary cells plated for 1 hr and then stimulated with EGF and GnRH for 15 min. Dual immunocytochemistry was done to learn which cell types expressed the fos proteins. After 15 min, EGF and GnRH both increased the percentages of fos-bearing cells above levels seen in medium alone. EGF stimulated fos proteins in subsets of FSH, adrenocorticotropin (ACTH), and growth hormone (GH) cells. GnRH increased fos proteins in subsets of ACTH and GH cells. These results suggest that EGF and GnRH may regulate fos expression, but not necessarily in gonadotropes. They also highlight the need for carefully timed experiments because endogenous factors in the pituitary itself may stimulate immediate early gene expression. (J Histochem Cytochem 46:935-943, 1998)

Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.