Regulation Of C-fos Expression Research Articles

Endogenous SOLOIST/MRTFB i4, a Neuronal Isoform of MKL2/MRTFB, Positively and Negatively Regulates SRF Target Immediate Early Genes in Neuro-2a Cells.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

Cyclolinopeptide F, a cyclic peptide from flaxseed inhibited RANKL-induced osteoclastogenesis via downergulation of RANK expression.

Previously, we reported that cyclolinopeptides (CLs) extracted from flaxseed inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis from mouse bone marrow cells in vitro. However, mode of action involved in CLs-inhibited osteoclastogenesis has been yet unknown. Therefore, in this study, we investigated the details of inhibitory activity of cyclolinopeptide-F (CL-F) in osteoclastogenesis, as a representative of CLs. CL-F dose-dependently inhibited RANKL-induced osteoclastogenesis (IC50 0.58µM) without cytotoxic effects. The inhibition by CL-F was mainly observed in macrophage colony-stimulating factor (M-CSF)-induced proliferation/differentiation phase from M-CSF responsive immature myeloid cells to monocyte/macrophage (M/Mϕ) lineage. Additionally, CL-F also slightly inhibited RANKL-induced differentiation phase from M/Mϕ to mature osteoclasts. Expression of RANKL receptor, RANK, in M-CSF-induced M/Mϕ, i.e. osteoclast progenitor cells, was decreased by CL-F treatment. Furthermore, RT-PCR analysis revealed that CL-F inhibited c-fos gene expression, which is reported to be crucial for RANK expression in osteoclast progenitor cells induced with M-CSF from myeloid lineage cells. These results suggested that CL-F inhibits osteoclastogenesis via down regulation of c-fos expression, which leads to the down-regulation of RANK expression in M-CSF-induced osteoclast progenitors.

Effect of P. ginseng on the expression of c-Fos in the brain of Wistar rats with testosterone induced benign prostatic hyperplasia

The lower urinary tract symptoms (LUTS) show in benign prostatic hyperplasia (BPH). The three major micturition centers in brain are pontine micturition center (PMC), ventrolateral periaqueductal gray (vlPAG), and medial preopticnucleus (MPA) regions. Previous study showed that c-Fos expression change was associated with LUTS. In present study, the effect of P. ginseng on c-Fos expression in PMC, vlPAG, and MPA regions in rat brain was tested. P. ginseng is the four year-old Korean ginseng. It was collected at the department of medicinal crop research (Eumsunggun, Chungbuk, Korea) in September 2010. The four groups (n = 6) are control group, BPH-induced group, BPHinduced and P. ginseng-treated group, and BPH-induced and finasteride-treated group. BPH in rats was induced by testosterone. After 4 weeks, all animals were sacrificed to evaluate c-Fos expression in PMC, vlPAG, and MPA regions in rat brain. The c-Fos expression was evaluated in the regions of rat brain by immunohistochemistry (IHC). Present results showed that c-Fos expressions in PMC, vl-PAG, and MPA regions in brain of rats in the BPH-induced group were higher compared to c-fos expression of the control group. The increased c-Fos expression in three regions (PMC, vlPAG, and MPA) were decreased by treatment with P. ginseng (200 mg/kg). These results suggest that P. ginseng has an inhibitory effect on the symptoms of BPH and is associated with regulation of c-Fos expression in the brain in a testosterone induced BPH rat model.

Cyclin-dependent kinase 2 (CDK2) is a key mediator for EGF-induced cell transformation mediated through the ELK4/c-Fos signaling pathway.

Cyclin dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression dramatically decreases EGF-induced cell transformation mediated through the down-regulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is over-expressed in melanoma and knocking down ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for chemoprevention and therapy against skin cancer.

Regulation of c-fos expression by the dopamine D1-D2 receptor heteromer

The dopamine D1 and D2 receptors form the D1-D2 receptor heteromer in a subset of neurons and couple to the Gq protein to regulate intracellular calcium signaling. In the present study the effect of D1-D2 heteromer activation and disruption on neuronal activation in the rat brain was mapped. This was accomplished using the dopamine agonist SKF 83959 to activate the D1-D2 heteromer in combination with a TAT-D1 disrupting peptide we developed, and which has been shown to disrupt the D1/D2 receptor interaction and antagonize D1-D2 heteromer-induced cell signaling and behavior. Acute SKF 83959 administration to rats induced significant c-fos expression in the nucleus accumbens that was significantly inhibited by TAT-D1 pretreatment. No effects of SKF 83959 were seen in caudate putamen. D1-D2 heteromer disruption by TAT-D1 did not have any effects in any striatal subregions, but induced significant c-fos immunoreactivity in a number of cortical regions including the orbitofrontal cortex, prelimbic and infralimbic cortices and piriform cortex. The induction of c-fos by TAT-D1 was also evident in the anterior olfactory nucleus, as well as the lateral habenula and thalamic nuclei. These findings show for the first time that the D1-D2 heteromer can differentially regulate c-fos expression in a region-dependent manner either through its activation or through tonic inhibition of neuronal activity.

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF-α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)-induced cardiomyocyte TNF-α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS-induced TNF-α production in a dose-dependent manner. α1-adrenoceptor (AR) antagonist (prazosin), but neither β1-nor β2-AR antagonist, abrogated the inhibitory effect of NE on LPS-stimulated TNF-α production. Furthermore, phenylephrine (PE), an α1-AR agonist, also suppressed LPS-induced TNF-α production. NE inhibited p38 phosphorylation and NF-κB activation, but enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and c-Fos expression in LPS-treated cardiomyocytes, all of which were reversed by prazosin pre-treatment. To determine whether ERK1/2 regulates c-Fos expression, p38 phosphorylation, NF-κB activation and TNF-α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c-Fos expression, p38 mitogen-activated protein kinase (MAPK) phosphorylation and TNF-α production, but not NF-κB activation in LPS-challenged cardiomyocytes. In addition, pre-treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS-induced TNF-α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c-Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF-α production and prevented LPS-provoked cardiac dysfunction. Altogether, these findings indicate that activation of α1-AR by NE suppresses LPS-induced cardiomyocyte TNF-α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF-κB activation.

Regulation of Primary Response Genes in B Cells

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

Kinetics of c-Fos induction depends upon strength of TCR stimulation. (63.13)

Abstract Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation with different MHC-peptide (MHC-p) dose and potency is not well understood. Our aim is to study the kinetics of c-Fos induction in T cells in response to antigen dose and potency. Our model system is AND TCR transgenic mice, whose T cells are specific for moth Cytochrome-C (MCC) peptide. We study the impact of MHC-p potency using altered peptide ligands (APLs). To study c-Fos induction we crossed AND mice to c-Fos-EGFP transgenic mice that express a fusion protein of c-Fos and EGFP driven by the Fos promoter. We observed that upon stimulation of TCR, expression of c-Fos starts after ~30 min, peaks at 3hrs and plateaus for another 2hrs. Strong agonists induce c-Fos even without co-stimulatory molecules where as weak agonists fail to do so. Co-stimulation enhances the percentage of cells responding as well as the level of c-Fos expression. Highering or lowering the peptide dose reduces the expression level of c-Fos. Inhibiton of Calcineurin has no effect on c-Fos expression. Experiment with MAPK inhibitors indicates that all the three MAP Kinases- ERK, p38 and JNK regulate c-Fos induction. ERK primarily regulate c-fos expression in the case of strong agonists (MCC and L98A) where as JNK primarily regulates c-fos expression in case of weak agonists (K99A). We are looking at other regulators of c-Fos induction in response to APLs.

Role of central nervous system glucagon-like Peptide-1 receptors in enteric glucose sensing.

OBJECTIVE—Ingested glucose is detected by specialized sensors in the enteric/hepatoportal vein, which send neural signals to the brain, which in turn regulates key peripheral tissues. Hence, impairment in the control of enteric-neural glucose sensing could contribute to disordered glucose homeostasis. The aim of this study was to determine the cells in the brain targeted by the activation of the enteric glucose-sensing system.RESEARCH DESIGN AND METHODS—We selectively activated the axis in mice using a low-rate intragastric glucose infusion in wild-type and glucagon-like peptide-1 (GLP-1) receptor knockout mice, neuropeptide Y–and proopiomelanocortin–green fluorescent protein–expressing mice, and high-fat diet diabetic mice. We quantified the whole-body glucose utilization rate and the pattern of c-Fos positive in the brain.RESULTS—Enteric glucose increased muscle glycogen synthesis by 30% and regulates c-Fos expression in the brainstem and the hypothalamus. Moreover, the synthesis of muscle glycogen was diminished after central infusion of the GLP-1 receptor (GLP-1Rc) antagonist Exendin 9-39 and abolished in GLP-1Rc knockout mice. Gut-glucose–sensitive c-Fos–positive cells of the arcuate nucleus colocalized with neuropeptide Y–positive neurons but not with proopiomelanocortin-positive neurons. Furthermore, high-fat feeding prevented the enteric activation of c-Fos expression.CONCLUSIONS—We conclude that the gut-glucose sensor modulates peripheral glucose metabolism through a nutrient-sensitive mechanism, which requires brain GLP-1Rc signaling and is impaired during diabetes.

HnRNP-R regulates the PMA-induced c-fos expression in retinal cells

This study focused on the function of hnRNP-R in the regulation of c-fos expression. We demonstrated that hnRNP-R accelerated the rise and decline phases of c-fos mRNAs and Fos proteins, allowing PMA to induce an augmented pulse response of c-fos expression. Then, we examined the role of the c-fos-derived AU-rich element (ARE) in hnRNP-R-regulated mRNA degradation. Studies with the ARE-GFP reporter gene showed that hnRNP-R significantly reduced the expression of GFP with an inserted ARE. Moreover, immunoprecipitation-RT-PCR analysis demonstrated that in R28 cells and rat retinal tissues, the c-fos mRNA was co-immunoprecipitated with hnRNP-R. These findings indicate that hnRNP-R regulates the c-fos expression in retinal cells, and that the ARE of c-fos mRNAs contributes to this regulation.

The activation of the ERK pathway contributes to the spinal c-fos expression observed after noxious bladder stimulation

C-fos is an immediate-early gene whose expression in the spinal cord has been extensively used as a marker of peripheral noxious stimulation. The Fos protein accumulates in the nuclei of spinal neurons, reaching detectable levels 2 h after stimulation. The ERK pathway is an important signalling pathway in spinal cord neurons. ERK is activated upon phosphorylation on specific amino acid residues. Its activation in the spinal cord, following noxious stimulation, has been shown to contribute to the establishment and maintenance of long-term neuronal alterations associated with chronic pain. Phosphorylated ERK can target several cellular elements, including transcription factors, which indicates that ERK participates in the regulation of gene expression. The relation between ERK and c-fos is at present still unclear. Some in vitro studies have reached the conclusion that ERK contributes to c-fos regulation whereas others have provided evidence of ERK-independent c-fos expression. In fact, in the spinal cord the occurrence of c-fos expression in the absence of ERK phosphorylation has been reported. In this study we investigated in vivo the contribution of ERK to c-fos expression in the spinal cord. By inhibiting spinal ERK activation with intrathecal administration of PD98059, we verified that ERK phosphorylation does contribute to regulate c-fos expression upon noxious bladder stimulation.

Nitric oxide regulates c-fos expression in osteoblastic cells induced by wall-shear

To observe regulation of nitric oxide on c-fos expression in osteoblastic cells in response to changes in wall-shear stress in vitro. Isolated and purified osteoblastic cells from the calvaria of newborn SD rats were cultured and passaged. The third generation cells, pre-treated with 10% FBS DMEM, 0.3 mmol/L L-NMMA DMEM and 0.1 mmol/L SNP DMEM separately, were subjected to wall-shear stress of 1.2 Pa. Gene expression of the c-fos and NOS activity were studied before (0 min) and 10 min, 15 min, 30 min, 60 min after treated with wall-shear stress. The expression of c-fos mRNA was increased transiently after application of 1.2 Pa wall-shear stress in osteoblastic cells and peaked at 15 min. The expression of c-fos mRNA was decreased after pre-application with L-NMMA and increased after use of SNP. Changes in the osteoblastic cells mechanical environment may cause a dramatic induction of NO and c-fos expression.

Photic regulation of c-Fos expression in the protocerebrum of crayfish

In various species of crayfish it has been proposed that different extra-retinal photoreceptors and pacemakers could be involved in the photo entrainment mechanisms. The identity of the neural structures responsible for this phenomenon is not known. The objective of this work was to contribute to the knowledge of the neurophysiological mechanisms and structures underlying the biological clock of crayfish by studying c-fos induction in one of the neural structures proposed as a pacemaker, the brain. Using qualitative – quantitative immunohistochemical and image methods we identified light-induced c-Fos immunoreactivity in cells located in the median protocerebrum of the supraesophagic ganglion. The immunoreaction varied with circadian time. The protocerebrum showed a statistically significant c-Fos induction, especially in neurons of the dorsal anterior region and the protocerebral bridge. A statistically significant difference between the control and the experimental conditions indicated a differential sensitivity in the early subjective night. This is the first report on c-Fos expression in crayfish and indicates that this marker may be a useful tool for studying the circadian clock in this organism.

Chinese medicine Banxia-houpu decoction regulates c-fos expression in the brain regions in chronic mild stress model in rats.

Banxia-houpu decoction is a safe and effective traditional Chinese medicinal formula used in the treatment of mild and manic-depressive disorders for centuries. There has been increasing interest in its therapeutic application in depression. However, the mechanisms behind behavioural changes are still poorly understood. Chronic mild stress (CMS)-induced preference behaviour change has been used as a model to predict the clinical efficacy of many types of antidepressant treatment. Both EtOH and water extracts (AE and WE) of Banxia-houpu decoction exhibited a significantly increased sucrose intake in the CMS model in rats, but there was no effect in unstressed animals. In the present study, it was found that the c-fos expression in cerebral cortex, hippocampus and striatum corpora were very high in the CMS model in rats. WE and AE at a dose of 130 mg/kg exhibited a significantly decreased c-fos expression in the cerebral regions in CMS model in rats, respectively. The former was more potent than the latter. However, no significant changes in the c-fos expression were observed in unstressed rats treated with the decoction. Fluoxetine not only significantly reduced c-fos expression in all regions in the CMS model in rats, but only showed a marked decrease in c-fos expression in the hippocampus in unstressed animals. A different molecular mechanism of Banxia-houpu decoction and fluoxetine may be implied. The cerebral cortex, hippocampus and striatum conpora might be important structural substrates in the central nervous system mediating the section of the Banxia-houpu decoction on preference behaviour in CMS-induced rats, and fos protein might be the common substrate of the signal transduction process of the decoction.

Regulation of c-fos expression by static stretch in rat myometrial smooth muscle cells

Objective: We investigated whether mechanical stretch stimulates expression of the immediate early gene c-fos messenger RNA in rat myometrial smooth muscle cells. Study Design: Freshly isolated myometrial smooth muscle cells (from virgin rats) were plated on flexible culture plates that were coated with either collagen I, laminin, elastin, or pronectin and were subjected to a static stretch with a computerized strain unit. Immunocytochemistry was used to define smooth muscle cell phenotype, and c-fos expression levels were assessed by Northern blotting. Results: Myometrial smooth muscle cells maintained their phenotype for at least 7 days in culture. Stretch induced a dramatic increase in levels of c-fos messenger RNA, which peaked after 30 minutes. Stimulation of c-fos gene expression was dependent on the degree of stretch (maximum induction at 25% elongation). Interestingly, as little as 1 minute of stretch was sufficient to induce maximal c-fos expression at 30 minutes. The most dramatic induction of c-fos was observed in smooth muscle cells that were stretched on collagen I-coated plates. Conclusion: Myometrial smooth muscle cells can directly respond to mechanical stretch with a strength- and matrix component-dependent increase in c-fos messenger RNA. The time delay between the initial stimulus and the cumulative elevation of c-fos messenger RNA suggests a complex regulation from mechanoreception to c-fos gene expression. (Am J Obstet Gynecol 2002;186:1358-65.)

Bifurcation of osteoclasts and dendritic cells from common progenitors

AbstractOsteoclasts and dendritic cells are derived from monocyte/macrophage precursor cells; however, how their lineage commitment is regulated is unknown. This study investigated the differentiation pathways of osteoclasts and dendritic cells from common precursor cells at the single-cell level. Osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor–κB ligand (RANKL) or tumor necrosis factor-α (TNF-α) is completely inhibited by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 at early stages of differentiation. GM-CSF–treated cells express both c-Fms and RANK and also low levels of CD11c and DEC205, which are detected on dendritic cells. Addition of GM-CSF also reduces expression of both c-Fos and Fra-1, which is an important event for inhibition of osteoclastogenesis. Overexpression of c-Fos by retroviral infection or induction in transgenic mice can rescue a failure in osteoclast differentiation even in the presence of GM-CSF. By contrast, differentiation into dendritic cells is inhibited by M-CSF, indicating that M-CSF and GM-CSF reciprocally regulate the differentiation of both lineages. Dendritic cell maturation is also inhibited when c-Fos is expressed at an early stage of differentiation. Taken together, these findings suggest that c-Fos is a key mediator of the lineage commitment between osteoclasts and dendritic cells. The lineage determination of osteoclast progenitors seen following GM-CSF treatment functions through the regulation of c-Fos expression.

Pharmacological antagonism of tyrosine kinases and MAP kinase in brainstem blocks taste aversion learning in mice

Although c-Fos induction in the brainstem is a reliable correlate of taste aversion learning and appears necessary for the encoding of the unconditioned stimulus, little is known about the intracellular signaling pathways in the brainstem that regulate c-Fos expression during taste aversion learning. Infusion of the tyrosine kinase inhibitor genistein and the MAP kinase kinase (MEK) inhibitor PD98059 into the fourth ventricle of mice potently blocks acquisition of a learned taste aversion. The unconditioned stimulus LiCl produces a rapid and robust phosphorylation of MAP kinase, as revealed by immunohistochemistry with an antibody specific to the dually phosphorylated active form of MAP kinase. This immunoreactivity is localized to the same region of the intermediate nucleus tractus solitarius in which we have shown large increases in c-Fos immunoreactive cells, suggesting that in at least a subset of these cells, MAP kinase activation may lead to c-fos induction.

Cleavage of the Serum Response Factor during Death Receptor-induced Apoptosis Results in an Inhibition of the c-FOS Promoter Transcriptional Activity

The c-FOS protooncogene is rapidly induced by a wide variety of extracellular stimuli including mitogenic signals. Regulation of c-FOS expression is tightly dependent on the serum response element localized within its promoter. Two transcription factors, the serum response factor (SRF) and the ternary complex factor, bind to the serum response element and play a key role in the regulation of the c-FOS promoter activity. In the present study, we show that two death effectors (CH11 and TRAIL) severely impaired the transcriptional activity of the c-FOS promoter in Jurkat T cells. This inhibition can be accounted for by the specific cleavage by caspase 3 of the SRF both in vitro and in vivo, since acetyl-DEVD-aldehyde prevented SRF cleavage and abolished the inhibitory effect of CH11 and TRAIL on the c-FOS promoter activity. Moreover, phorbol myristate acetate, a potent anti-apoptotic effector, was found to protect SRF completely from cleavage by caspase 3 and also to prevent the inhibition of the c-FOS promoter activity by death effectors. Survival factors play an essential function in the regulation of cell growth mainly by regulating the expression of immediate early gene such as c-FOS. In this line, cleavage of SRF at the onset of apoptosis could abrogate the ability of the cell to induce inappropriate survival pathways. All together, our results are consistent with a role of SRF at the interface between cell survival and death pathways.

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 expression by RNA polymerase elongation competence

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.