Induction Of C-fos Protein Research Articles

The association of thirst, sodium appetite and vasopressin release with c-fos expression in the forebrain of the rat after intracerebroventricular injection of angiotensin II, angiotensin-(1-7) or carbachol.

The effect intracerebroventricular injections of angiotensin II (0.1 nm), angiotensin-(1-7) (1 or 100 nm) and carbachol (500 ng) on c-fos expression was examined in the forebrain of Lister hooded rats. Intense staining of the c-Fos protein was found in the median preoptic nucleus, organum vasculosum of the lamina terminalis, subfornical organ, paraventricular nucleus and supraoptic nucleus after angiotensin II and carbachol Angiotensin II caused significantly more c-fos expression in the ventral median preoptic nucleus and organum vasculosum of the lamina terminalis than carbachol, whereas in the paraventricular and supraoptic nuclei this was reversed, with carbachol having a greater effect on c-fos expression in these areas. Angiotensin-(1-7), however, only induced c-Fos protein in the organum vasculosum of the lamina terminalis and median preoptic nucleus with the number and the intensity of staining of the nuclei significantly less in both areas than after angiotensin II or carbachol. Separate groups of Lister rats were given i.c.v. injections of the same substances at the same doses, but excluding the lower dose of angiotensin-(1-7), and the intakes of water and 1.8% NaCl over 60 min were measured. Angiotensin II stimulated intakes of both water and NaCl. The effect on water intake was almost immediate (<1 min), whereas NaCl intake did not usually start until at least 5 min after injection. Over 60 min, water (12.4 +/- 1.0 ml) and NaCl (4.2 +/- 0.9 ml) intakes were significantly greater than water (1.1 +/- 0.2 ml) and NaCl (0.6 +/- 0.5 ml) intakes of the controls. Carbachol caused less drinking than angiotensin II, the water intake over 60 min being significantly less (4.8 +/- 0.7 ml) and the latency of response greater (>5 min). Carbachol, unlike angiotensin II, had little effect on NaCl intake (0.7 +/- 0.4 ml). Angiotensin-(1-7) had no effect on water (1.1 +/- 0.3 ml) or NaCl (0.3 +/- 0.3 ml) intakes. The plasma levels of vasopressin were measured after i.c.v. injection of the same three substances in the same doses, again excluding the lower dose of angiotensin-(1-7), in further groups of rats. Angiotensin II and carbachol caused an approximate five-fold increase in plasma vasopressin levels compared to cerebrospinal fluid-injected rats, but angiotensin-(1-7) had no effect on vasopressin release. Therefore, three compounds with widely differing effects on thirst, sodium appetite and vasopressin release induce distinctive patterns of c-fos protein expression in the forebrain. By combining experimental approaches in this way it is possible to determine areas of the brain which are involved in certain behavioural and endocrine responses.

Amphetamine induction of c- fos in the nucleus accumbens is not inhibited by glutamate antagonists

Systemic administration of relatively high doses of amphetamine or cocaine induces expression of c- fos in the rat striatum and nucleus accumbens. The doses of drugs used in such experiments are substantially higher than those needed to produce reward or sensitization. Therefore, it was determined if low doses of amphetamine capable of eliciting reward and sensitization increase levels of c-Fos protein in the nucleus accumbens. Amphetamine, 1 mg/kg, stimulated locomotor activity and increased the number of nucleus accumbens cells immunohistochemically positive for c-Fos protein to approximately 800 cells per section from a control of approximately 100 cells per section. Since glutamate antagonists modify various responses to amphetamine, it was then determined whether activation of glutamate receptors is involved in the induction of c-Fos protein by low doses of amphetamine. The NMDA receptor antagonist MK-801 by itself stimulated locomotr activity but did not alter baseline levels of c-Fos protein. When given before amphetamine, both locomotor activity and extent of c- fos induction were greater than from amphetamine alone. The AMPA receptor antagonist NBQX by itself had no effect on locomotor activity but increased slightly the number of cells positive for c-Fos protein in the nucleus accumbens. When given before amphetamine, locomotor activity was completely attenuated, and the extent of c- fos induction was greater than from amphetamine alone. We conclude that low doses of amphetamine do increase abundance of c-Fos protein in the nucleus accumbens. This action does not correlate with locomotor activity and is independent of activation of glutamate receptors.

Nicotine elicits changes in expression of adrenal catecholamine biosynthetic enzymes, neuropeptide Y and immediate early genes by injection but not continuous administration

Nicotine, a major pharmacologically active component of tobacco smoke, is generally believed to be one of the factors responsible for the deleterious consequences of cigarette smoking. Nicotine activates the sympathoadrenal system and increases the synthesis and release of catecholamines into circulation. In this study we show that single and repeated injections of nicotine increase the expression of tyrosine hydroxylase (TH), a rate limiting enzyme in the catecholamine biosynthetic pathway. These treatments also regulated the expression of dopamine beta-hydroxylase (DBH) and neuropeptide Y (NPY) in rat adrenals. The effect of nicotine on several transcription factors in the adrenal medulla was examined. Nicotine administration by injection increased the phosphorylation of CREB and induced c-Fos protein without affecting members of the jun family. In contrast to the results with injections, continuous infusion via osmotic pumps did not affect any of these parameters. These data indicate that activation of several transcription factors and increased expression of TH, DBH, and NPY is dependent on the mode of nicotine administration.

Resistance of growth hormone secretion to hypoglycemia in the mouse.

Although previous studies have demonstrated that acute hypoglycemia inhibits growth hormone (GH) secretion due to stimulation of hypothalamic somatostatin (SS) neurones in the rat, the effect of hypoglycemia on GH secretion has not yet been elucidated in the mouse. In this study, the effects of insulin-induced hypoglycemia on mouse GH secretion, hypothalamic c-fos expression, GH-releasing hormone (GRH) and SS mRNA levels were investigated in conscious male mice. Seven days after implantation of chronic atrial catheters, blood samples were taken every 20 min from 1200-1600 h under unrestrained conditions. Insulin was administered iv every 20 min from 1200-1240 h to induce moderate hypoglycemia (MH) and severe hypoglycemia (SH), respectively. Expression of hypothalamic c-fos protein was examined 30 min and 60 min after induction of hypoglycemia by immunohistochemistry. Hypothalamic GRH and SS mRNA levels were examined 1 h and 3 h after induction of hypoglycemia by Northern blot analysis. The lowest mean plasma glucose levels after insulin injections were 49.1 +/- 4.1 mg/dl and 34.2 +/- 5.6 mg/dl in conscious mice, respectively. However, pulsatile GH secretion was not significantly altered in either group. Although both MH and SH markedly stimulated c-fos expression in specific hypothalamic nuclei including the paraventricular nucleus, they did not induce c-fos protein in the periventricular nucleus. Neither MH nor SH altered hypothalamic GRH or SS mRNA levels. These results suggest that hypoglycemia does not activate SS neurons which inhibit GH secretion in the mouse.

Induction of immediate early gene mRNAs and proteins by hCG in interstitial cells of rat testis

In the present study we have investigated the induction of immediate early gene (IEG) c-fos, c-jun, junB and junD mRNAs and proteins in interstitial cells of rat testis after hCG treatment in vivo using in situ hybridization and immunocytochemistry. The basal levels of all IEGs were barely detectable with our methods. A prominent induction of c-fos, c-jun and junB mRNAs and proteins in Leydig cells was seen after hCG treatment. The induction of these IEG mRNAs and proteins demonstrates that their expression is an early transcriptional response to the extracellular stimulus of hCG and that they are involved in transcriptional processes which may mediate the effects of LH on cellular adaptive responses or steroid biosynthesis.

Expression of epidermal ornithine decarboxylase and nuclear proto‐oncogenes in phorbol ester tumor promotion‐sensitive and ‐resistant mice

This study was undertaken to assess the effects of a single or two sequential topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of c-fos, c-jun, junB, c-myc, and ornithine decarboxylase (ODC) in promotion-sensitive SSIN mice and the relatively promotion-resistant C57BL/6 strain. Northern blot analysis demonstrated that a single promoting dose of TPA induced ODC mRNA expression 10- to 15-fold in both strains. Treatment of each strain with a second dose of TPA, 48 h (in C57BL/6 mice) or 72 h (in SSIN mice) after the first, led to hyperinduction of ODC activity. Although this involved transcription of new ODC mRNA, the hyperinduction of ODC enzyme activity was primarily posttranscriptional. Induction of c-fos mRNA or protein was maximal about 3 h after a single treatment in either strain but was sustained for at least 6 h in C57BL/6 mice. In contrast, two treatments of SSIN mice with TPA caused a rapid, strong c-fos induction 1-2 h after treatment, whereas C57BL/6 mice responded no more strongly than after a single treatment. c-jun mRNA and protein were induced only slightly in either strain, but junB was induced about fivefold in SSIN mice and tenfold in C57BL/6 mice. Although c-myc was induced to comparable levels in both strains, the response was more prolonged in C57BL/6 mice. Compared with SSIN mice, C57BL/6 mice responded to TPA treatment, in general, with changes in proto-oncogene mRNA to a higher level or for longer or both. Thus, although small differences in the expression of these genes were observed, they were not positively correlated with the differential sensitivity of SSIN and C57BL/6 mice toward tumor promotion by phorbol esters, with the possible exception of c-fos.

Lignocaine-induced convulsion does not induce c-fos protein (c-Fos) in rat hippocampus.

Recent studies have shown that proto-oncogene c-fos mRNA is induced in the central nervous system by a variety of stimuli including generalised convulsions. In this study, the expression of c-fos protein (c-Fos) following lignocaine-induced convulsions was examined and compared with that following convulsions induced by non-anesthetic convulsants, such as pentylenetetrazol, kainic acid and electroconvulsive shocks, in rat brain. Administration of 120 mg.kg-1 lignocaine by the intraperitoneal route induced generalised convulsions in all rats examined within 10 min. C-Fos was markedly induced in the piriform cortex and amygdala, and slightly induced in the neocortex and thalamus, while no c-Fos expression was observed in the hippocampus. In contrast, c-Fos expression following generalised convulsions induced by non-anaesthetic convulsants was very marked in the hippocampal region, piriform cortex and amygdala, and extended to the thalamus and neocortex. These results contradict those of previously reported local cerebral metabolic studies using 2-deoxyglucose as a metabolic marker, and suggest that lignocaine-induced convulsions, unlike those induced by non-anaesthetic convulsants, may not cause severe sequelae (plastic changes) in the hippocampus.

The effects of reduced perfusion and reperfusion on C-fos and HSP-72 protein immunohistochemistry in gestational day 21 rat brains

Metabolic stressors such as hyperthermia, seizures and ischemic hypoxia result in the induction of C-fos and heat-shock proteins (HSP) in affected brain cells of the adult rodent, especially within the hippocampal region, which normally has high metabolic demands. Here we ligated the uterine vessels of gestational day (GD) 21 rat pups to produce ischemic hypoxia. We confirmed that HSP-72 protein, as previously reported, was activated in the perinatal rat pup, especially in the hippocampal CA3 region. However, the capability of hippocampal cells to produce C-fos protein following drug-induced seizures has been reported to develop only after postnatal day 13. Here, ischemic hypoxia caused CA1 hippocampal cells to produce immunohistochemically detectable C-fos protein in GD-21 rats. These results seem to contradict the previous reports of no C-fos induction in rats this young by demonstrating a functional C-fos translational mechanism by GD-21. However, seizure vs ischemic hypoxia-induced C-fos expression may involve several different pre-translational pathways. A delayed development of a receptor, second messenger, or genomic element for regulating C-fos transcription remain as possible explanations for the late maturity of responsivity to seizures.

Immediate-early gene protein expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ischaemic injury to the young rat brain

A unilateral hypoxia-ischaemia (HI) 21-day-old rat preparation was used to assess the effects of HI on the expression of the immediate-early gene proteins (IEGPs) c-Fos/FRAs, Fos B, c-Jun, Jun B, Jun D, Krox 20, Krox 24, and on the mRNA for the neurotrophic factor, brain-derived neurotrophic factor (BDNF). Moderate HI (15 min hypoxia) produced delayed, selective neuronal death and was associated with a rapid induction of c-Fos, Fos B, Jun B, Jun D, and c-Jun proteins, but not Krox 20 protein or BDNF mRNA, in neurons on the side of HI and also a delayed expression of c-Jun (and to a lesser extent c-Fos/FRA's and Fos B) 24–48 h after HI in neurons that underwent delayed neuronal death. Krox 24 showed an initial induction followed by a long-lasting suppression of its expression in regions undergoing cell loss. Severe HI (60 min hypoxia) resulted in seizures and rapid neuronal loss and infarction (necrotic cell death) on the side of HI, and was associated with early induction of c-Fos, Fos B, c-Jun, Jun B, Jun D, Krox 20 and Krox 24 protein and BDNF mRNA in neurons on the non-ligated side of the brain. Fos, c-Jun, Jun B, Jun D and Krox 24, but not Krox 20, Fos B, or BDNF mRNA, were also induced in non-nerve cells on the damaged side of the brain after both moderate and severe HI, and many of these cells appeared to be dividing. Thus, moderate HI induces IEGP's in neurons and non-nerve cells in damaged regions, whereas severe HI induces IEGP's and BDNF in non-damaged regions. c-Jun (and to a lesser extent c-Fos/FRA's) showed a prolonged expression in neurons undergoing delayed, but not necrotic, cell death suggesting that they may be involved in the biochemical cascade that causes selective delayed neuronal death. BDNF was not induced by HI, and therefore, does not appear to play an endogenous neuroprotective role in the CNS.

Ciliary neurotrophic factor enhances the survival of Purkinje cells in vitro.

We have examined the effects of ciliary neurotrophic factor (CNTF) on the development of rat Purkinje cells in vitro. Cerebellar cells, derived from embryonic day 16 rat fetuses, were found to respond rapidly to CNTF treatment by induction of c-Fos protein, such that 40% of the cells were immunopositive after 60 min. Treatment with low doses of CNTF (10-100 pg/ml) for 8 days resulted in an approximately 1.6-fold increase in the number of Purkinje cells, identified by immunohistochemical staining for calbindin. Immunohistochemical staining for other Purkinje cell markers--cyclic-GMP-dependent protein kinase and the low-affinity nerve growth factor receptor--verified increased Purkinje cell survival following CNTF treatment. In addition, CNTF increased specific high-affinity GABA uptake by 45%, and the number of GABAergic neurons by 70%. A maximal increase in the number of Purkinje cells and GABA-uptake was only achieved if CNTF was added within 48 h of plating the cells, further suggesting that CNTF enhances Purkinje cell survival in vitro. These results taken together strongly support a direct effect of CNTF in promoting the survival of Purkinje cells and possibly other GABAergic cerebellar neurons.

C-Fos induction in response to a conditioned stimulus after single trial taste aversion learning

Taste aversion conditioning is characterized by its prompt acquisition despite the introduction of long delays between CS (taste) and UCS (toxic drug). Although the dramatic changes in behavioral response to a taste after this conditioning are well documented, relatively little is known about the changes in neural activity that accompany this learning. c-Fos immunohistochemical staining was employed to define brain regions activated during the expression of a conditioned taste aversion (CTA). The present studies examined c-Fos immunoreactivity in the brains of rats after i.p. injection of LiCl or NaCl or after intraoral infusion of a saccharin CS. LiCl administration, a common unconditioned stimulus (UCS) in CTA experiments, was found to induce c-Fos protein in a number of brainstem regions, including the nucleus of the solitary tract (NTS), medial and lateral pontine parabrachial nucleus (PBN), and hypoglossal nucleus. Conditioned animals received a single pairing of the CS saccharin with the UCS LiCl, while controls were exposed to the CS saccharin but received non-contingent LiCl 24 h after saccharin exposure. Following saccharin re-exposure, conditioned animals showed patterns of neuronal activation to a taste which were similar to those activated by the UCS drug. Specifically, the pattern of c-Fos expression in conditioned animals was confined to the same region of the NTS which showed the most activation following the UCS LiCl. This pattern of activation was not evident in controls re-exposed to saccharin. These results indicate that one-trial taste aversion conditioning alters the pattern of neuronal activity triggered by the CS taste such that it comes to activate some of the same pathways originally activated by the UCS.

Effects of electro-acupuncture on C-FOS expression in gerbil hippocampus during transient global ischemia.

We studied the effects of electro-acupuncture (EA) on C-FOS expression as well as on the histological changes in various regions of hippocampus in the gerbil acute global ischemia model. EA was administered at points of 'Feng-fu' and 'Jin-suo' with a frequency of 7 Hz and an intensity of 6 mA for 30 minutes. EA can substantially potentiate the induction of C-FOS protein like immunoreactivity (CFPLI) in neurons of various regions in hippocampus following transient global ischemia, especially in the CA1 subfield. At the same time EA can prevent most of the CA1 cells from delayed degeneration after ischemia. These results indicate that EA has the protective effect on neurons of hippocampus after cerebral ischemia and C-FOS may be involved in this process.

Domoic acid-treated cynomolgus monkeys ( M. fascicularis): effects of dose on hippocampal neuronal and terminal degeneration

Domoic acid is a tricarboxylic amino acid (structurally related to kainic acid and glutamic acid) that is found in the environment as a contaminant of some seafood. To determine the nature of any neurological damage caused by domoate, as well as the minimum neurotoxic dose, juvenile and adult monkeys were dosed intravenously with domoate at one of a range of doses from 0.25 to 4 mg/kg. When animals were perfused one week later, histochemical staining using a silver method to reveal degenerating axons and cell bodies showed two distinct types of hippocampal lesions. One lesion, termed ‘Type A’, was a small focal area of silver grains restricted to CA2 stratum lucidum, the site of greatest kainic acid receptor concentration in the brain. Type A lesions occurred over a dose range of 0.5 to 2.0 mg/kg in juvenile animals and 0.5 to 1.0 mg/kg in adult animals. No mortality occurred in any of the juvenile monkeys, but one juvenile animal that received 4.0 mg/kg sustained a second type of lesion, termed ‘Type B’, characterized by widespread damage to pyramidal neurons and axon terminals of CA4, CA3, CA2, CA1, and subiculum subfield of the hippocampus. Doses of more than 1.0 mg/kg in the adult monkeys either proved lethal or resulted in Type B lesions. Induction of c-fos protein had occurred in the hippocampal dentate gyrus and CA1 regions of moribund animals perfused within hours of their initial dose. Our results show that relatively low doses of domoic acid selectively effect mossy fiber terminals, that larger (near lethal) doses damage hippocampal pyramidal neurons and their axons, that some moribund monkeys had experienced limbic seizures prior to death, and that adult animals are more sensitive to domoic acid than juveniles.

Cultured hippocampal neurons show responses to BDNF, NT-3, and NT-4, but not NGF

To investigate the possibility of neurotrophins acting directly on hippocampal neurons, we first examined expression of the trk receptors in sections of adult rat brain and in cultures of embryonic rat hippocampus, and then investigated general and specific responses of cultured hippocampal neurons to each of the neurotrophins. In situ hybridization studies indicated high levels of expression of trkB and trkC but not trkA in pyramidal cells, dentate granule neurons, and scattered interneurons. Cultures of embryonic day 18 (E18) hippocampal neurons were also found by Northern analysis to express trkB and trkC but not trkA, indicating potential responsiveness to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4, but not NGF. Phosphorylation experiments indeed showed that BDNF, NT-3, and NT-4 produced rapid tyrosine phosphorylation of Trk proteins, as detected by immunoprecipitation using a pan-Trk-specific antibody, whereas NGF produced no detectable tyrosine phosphorylation in hippocampal cultures. Similarly, all of the neurotrophins, except NGF, induced expression of c-fos mRNA and c-fos protein in these cultures. c-Fos protein induction was detectable in approximately 40-50% of the cells. While we observed no major effect of any of the neurotrophins upon the survival of E18 hippocampal neurons, BDNF, NT-3, and NT-4, but not NGF, produced marked increases in the number of neurons expressing detectable levels of either calbindin or AChE. NT-3 produced the greatest increase in the number of calbindin-positive neurons, whereas BDNF and NT-4 produced the greater increase in the number of AChE-positive neurons. Our results suggest that several of the neurotrophins have important effects in the differentiation and maintenance of function of subpopulations of hippocampal neurons.

αMSH but not Org 2766 induces expression of c-fos in cultured rat spinal cord cells

Alpha-melanocyte stimulating hormone (alpha MSH) and Org 2766 (an adrenocorticotrophic hormone (ACTH) MSH4-9 analogue) stimulate sprouting after nerve damage in vivo. In cultures of rat spinal cord cells only alpha MSH enhances neurite outgrowth. In order to analyse the mode of action of alpha MSH and to get a clue why its action differs from that of Org 2766, we studied whether expression of c-fos was induced in spinal cord cells by treatment with alpha MSH or Org 2766. We found a rapid and transient induction of both c-fos mRNA and protein after treatment with 100 microM alpha MSH. Treatment with Org 2766 (100 pM-100 microM) had no effect on c-fos expression. These results suggest that c-fos is involved in the signal transduction system of alpha MSH stimulated neuronal outgrowth in vitro.

High-dose ketamine does not induce c-Fos protein expression in rat hippocampus

The effects of high-dose ketamine on the c- fos protein (c-Fos) expression were investigated in rat by an immunohistochemical technique. The administration of 100 mg/kg ketamine i.p. induced seizure-like activity (limbic seizure). No c-Fos immunoreactivity was observed in hippocampus, piriform cortex and amygdala, while it was observed in neocortex and thalamus. These findings disagree with the reports that ketamine depresses the neuronal function of the neocortex and thalamus, while it stimulates the limbic system.

Differential induction of c-Fos immunoreactivity in hypothalamus and brain stem nuclei following central and peripheral administration of endotoxin

Lipopolysaccharide (LPS), an endotoxin associated with gram-negative bacteria, is a potent activator of the immune system. We have tested the effects of ICV infusions of LPS (10 ng) or Ringer's solution on the induction of the proto-oncogene protein c-Fos in the brain as well as plasma levels of corticosterone and splenic concentrations of norepinephrine (NE) and VIP. At 3 h post-ICV infusion of LPS, numerous labeled neurons were observed in the paraventricular nucleus (PVN) of the hypothalamus and the nucleus tractus solitarius (A2) region of the brain stem. Also, corticosterone and splenic NE and VIP levels were all elevated post-ICV LPS. Analysis of the time course for the induction of c-Fos protein in the brain following IP injections of LPS indicated that, relative to control injections, increased numbers of c-Fos-positive cells were detected in the PVN 0.5 h following IP injections (100 μg), peaked at 2–3 h postinjection, and then returned to control levels at later intervals. Additional dose-response data for IP LPS indicated a small increase in the number of labeled cells at a dose of 4.0 μg, and the number and staining intensity increased up to a dose of 100 μg- Corticosterone levels followed a similar pattern and were elevated at the 4.0 μg IP dose of LPS and increased to peak levels at 40 μg and higher. In contrast to ICV injections, splenic NE levels were unaltered by IP injections of LPS. An additional difference noted between ICV and IP injections of LPS was the presence of c-Fos-labeled neurons in the supraoptic nucleus, arcuate nucleus, and the ventrolateral (A1) region of the brain stem at IP doses of 40 μg and higher. These results indicate selective and differential effects of central and peripheral LPS on the induction of c-Fos protein in hypothalamic and brain stem nuclei. The concurrent changes in corticosterone and splenic neurotransmitter levels produced by ICV LPS suggest that the endocrine and autonomie nervous systems are primary targets for these activational effects of endotoxin.

A 5′-flanking region of the bovine tyrosine hydroxylase gene is involved in cell-specific expression, activation of gene transcription by phorbol ester, and transactivation by c-Fos and c-Jun

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells and is transcriptionally activated via a protein kinase C (PKC)-dependent pathway. To identify regulatory regions of the TH gene, transfected neural crest-derived catecholaminergic cells [human neuroblastoma SH-SY5Y and bovine adrenal medullary (BAM) cells] or glia-derived SF-763 cells were used to analyze expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5′-flanking sequences of the bovine TH gene. Plasmid pTH(−245/+21)CAT was constructed by fusing the −245 to +21 region of the TH gene to CAT sequences in the promoterless pCAT basic plasmid. Cells transfected with pTH(−245/+21)CAT expressed CAT activity at levels 8- to 100-fold higher than those of cells transfected with pCAT basic. In SH-SY5Y or BAM cells, pTH(−245/+21)CAT supported the expression of CAT at levels similar to or higher than that supported by the tissue nonspecific viral SV40 promoter/enhancer. In glia-like cells, CAT expression from pTH(−245/+2 1)CAT was about 13-fold lower than that under the control of the SV40 promoter. Incubation of neuroblastoma cells with the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), increased expression of CAT from pTH(−245/+2 1)CAT over 6-fold and was accompanied by induction of c-fos and c-jun mRNAs and proteins. The regulation of TH promoter function by c-Fos/c-Jun was corroborated by transactivation of the TH promoter in SH-SY5Y cells engineered to overexpress exogenous c-Fos and c-Jun. TH gene sequences essential for c-Fos/c-Jun transactivation were located in the −245 to −52 region, upstream from a CRE-like element. Gel mobility assays demonstrated binding of c-Fos-antigen(s) to the region of the TH promoter that supports activation by PMA and c-Fos/c-Jun. Temporal patterns of nicotinic agonist-stimulated induction of c-fos and TH mRNA are also consistent with a role for c-Fos in TH gene transcription. Our results demonstrate that the 5′-flanking region of the bovine TH gene operates as an authentic promoter capable (i) of directing cell-specific expression of a bacterial CAT gene and (ii) of conferring responsivity to PKC-stimulation. The results also suggest that the nuclear proteins c-Fos and c-Jun contribute to PKC pathway-mediated activation of the TH gene via direct interaction with the TH gene promoter. The activation of TH gene expression by PKC/c-Fos/c-Jun may serve as an additional or alternative mechanism to activation by the cAMP-CRE pathway.

Chapter 16: C-fos induction occurs in cultured cortical neurons and astrocytes via multiple signaling pathways

Publisher Summary The best characterized immediate early gene, c - fos, is rapidly and transiently induced by a variety of environmental signals. In the central nervous system, many types of stimuli, some linked to neuronal excitation, and some associated with the process of glial mitogenesis and differentiation, elicit a transient induction of c-fos mRNA and protein. Multiple signaling pathways appear to mediate the gene expression. Cultured cortical cells are used to investigate what kind of stimulants induces c-fos in neurons and astrocytes. This chapter shows that N-methyl- D -aspartate (NMDA) receptor activation might be essential for translation of c-fos mRNA to Fos protein in cortical neurons. Dibutyryl-cyclic AMP (db-CAMP, 1 mM) and 12-0-tetra-decanoyl-phorbol- 13-acetate (TPA, 50 nM) induced morphological changes in astrocytes by converting them from flat, non-process-bearing cells to star-shaped, process-bearing cells, and induced c-fos mRNA and FLP expression. Growth factors for astrocytes, such as epidermal growth factor (EGF, Sigma, 20ng/ml), basic fibroblast growth factor, and platelet-derived growth factor also induced c-fos mRNA and FLP expression.

Neurotrophic activity of platelet-derived growth factor (PDGF): Rat neuronal cells possess functional PDGF beta-type receptors and respond to PDGF.

The different isoforms of PDGF bind with different affinities to two different receptor types. Previously, these receptors have been demonstrated mainly on mesenchymal and glial cells. We show here that PDGF beta-type receptors are present also on rat brain neurons in vivo as well as in vitro. Immunohistochemical analysis of a sequentially sectioned brain of a newborn rat revealed the presence of PDGF beta receptors in neurons throughout the central nervous system. These beta-receptor-bearing cells were shown to contain neurofilaments by double immunofluorescence staining. Furthermore, the PDGF-BB isoform bound to cells in a neuron-enriched culture from newborn rat brains and induced c-Fos protein in these cells. Continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival. These results indicate a neurotrophic effect of PDGF-BB.