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Currently, the human MEX3C gene is known to encode an RNA-binding protein of 659 amino acid residues. Here we show that the MEX3C gene has alternative splicing forms giving rise to multiple MEX3C variants, and some cells express MEX3C transcripts coding for short MEX3C isoforms but not transcripts for MEX3C(659AA) MEX3C(659AA) functions as an adaptor protein for Exportin 1 (XPO1)-mediated nuclear export since it increases the cytoplasmic distribution of poly(A)(+) RNA and since addition of the nuclear export signal (NES) sequence to a short MEX3C isoform MEX3C(464AA) confers similar cytoplasmic poly(A)(+) RNA accumulation activity as MEX3C(659AA) FOS mRNA is a potential MEX3C target mRNA. One mechanism by which MEX3C(659AA) could regulate FOS mRNA is by promoting its nuclear export. Overexpressing MEX3C(659AA) significantly increased FOS mRNA expression, whereas mutating the NES of MEX3C(659AA) and treating cells with leptomycin B to inhibit XPO1-mediated nuclear export attenuated FOS upregulation. FOS mRNA is unstable in somatic cells but less so in oocytes; how it is stabilized in the oocytes is unknown. Transcripts for the mouse counterpart of human MEX3C(659AA) (MEX3C(652AA)) are specifically expressed in developing oocytes in the ovary, although total Mex3c transcripts are expressed in both granulosa cells and oocytes. The specific expression of this long MEX3C isoform in oocytes and its ability to enhance FOS mRNA nuclear export and stability all suggest that MEX3C(659AA) is an RNA-binding protein that preserves maternal FOS mRNA in oocytes., (© 2016 by the Society for the Study of Reproduction, Inc.)

An inflammatory microenvironment may cause organ degenerative diseases and malignant tumors. However, the precise mechanisms of inflammation-induced diseases are not fully understood. Here, we show that the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) synergistically impair self-renewal and differentiation of mesenchymal stem cells (MSCs) via nuclear factor κB (NFκB)-mediated activation of mothers against decapentaplegic homolog 7 (SMAD7) in ovariectomized (OVX) mice. More interestingly, a long-term elevated levels of IFN-γ and TNF-α result in significantly increased susceptibility to malignant transformation in MSCs through NFκB-mediated upregulation of the oncogenes c-Fos and c-Myc. Depletion of either IFN-γ or TNF-α in OVX mice abolishes MSC impairment and the tendency toward malignant transformation with no NFκB-mediated oncogene activation. Systemic administration of aspirin, which significantly reduces the levels of IFN-γ and TNF-α, results in blockage of MSC deficiency and tumorigenesis by inhibition of NFκB/SMAD7 and NFκB/c-FOS and c-MYC pathways in OVX mice. In summary, this study reveals that inflammation factors, such as IFN-γ and TNF-α, synergistically induce MSC deficiency via NFκB/SMAD7 signaling and tumorigenesis via NFκB-mediated oncogene activation., (Copyright © 2013 AlphaMed Press.)

Objective: Obesity and type 2 diabetes are associated with an increase in the incidence and prevalence of Alzheimer's disease (AD) and an impaired cognitive function. Because peripheral blood mononuclear cells (MNC) express amyloid precursor protein (APP), the precursor of β-amyloid, which forms the pathognomonic plaques in the brain, we hypothesized that APP expression diminishes after the marked caloric restriction and weight loss associated with Roux-en-Y gastric bypass (RYGB) surgery., Research Design and Methods: Fifteen type 2 diabetic patients with morbid obesity (body mass index, 52.1 ± 13 kg/m(2)) underwent RYGB, and the expression of inflammatory and AD-related genes was examined before and after 6 months in plasma and in MNC., Results: Body mass index fell to 40.4 ± 11.1 kg/m(2) at 6 months after RYGB. There was a significant fall in plasma concentrations of glucose and insulin and in homeostasis model of assessment for insulin resistance. The expression of APP mRNA fell by 31 ± 9%, and that of protein fell by 36 ± 14%. In addition, there was a reduction in the expression of other AD-related genes including presinilin-2, ADAM-9, GSK-3β, PICALM, SORL-1, and clusterin (P < 0.05 for all). Additionally, the expression of c-Fos, a subunit of the proinflammatory transcription factor AP-1, was also suppressed after RYGB. These changes occurred in parallel with reductions in other proinflammatory mediators including C-reactive protein and monocyte chemoattractant protein-1., Conclusions: Thus, the reversal of the proinflammatory state of obesity is associated with a concomitant reduction in the expression of APP and other AD-related genes in MNC. We conclude that obesity and caloric intake modulate the expression of APP in MNC. If indeed, this effect also occurs in the brain, this may have implications for the pathogenesis and the treatment of AD. It is relevant that cognitive function has been shown to improve with weight loss following bariatric surgery.

During mouse skin wound healing, mRNAs encoding IL-1, activins, and TGF-βs significantly increased. To elucidate involvement of IL-1 in the regulation of activins and related factors in the wounded skin, human foreskin fibroblasts were stimulated with IL-1β, and levels of mRNAs encoding activins, TGF-βs, and follistatin family proteins were examined by quantitative real-time PCR. IL-1β increased activin βA (INHBA) and follistatin (FST) mRNA expression within 6 h. A p38 MAPK inhibitor, SB202190, a MAPK/ERK kinase inhibitor, U0126, and an nuclear factor κB pathway inhibitor, SC-514, significantly suppressed the IL-1β-stimulated INHBA and FST mRNA expression. A prostaglandin-endoperoxide synthase inhibitor indomethacin, a potent inhibitor of prostaglandin E(2) (PGE(2)) synthesis, also significantly suppressed the IL-1β-stimulated INHBA but not FST mRNA expression. Furthermore, stimulation of fibroblasts with PGE(2) significantly increased INHBA mRNA. The PGE(2)-induced INHBA mRNA expression was significantly suppressed by U0126 and a protein kinase C inhibitor, Gö 6983. Although IL-1β stimulated FST mRNA in an acute phase, long-term exposure of fibroblasts to IL-1β revealed time-dependent stimulatory and inhibitory effects of IL-1β on FST mRNA expression. On the other hand, coculture with keratinocytes significantly increased INHBA mRNA expression in dermal equivalents. In summary, the present study indicates that the p38 MAPK, the MAPK/ERK kinase, the nuclear factor κB pathway, and PGE(2) mediate the effects of IL-1β on INHBA mRNA expression. Furthermore, it is indicated that keratinocyte-derived factor of factors stimulate INHBA mRNA expression during wound healing.

Background: Dopamine β-hydroxylase (DBH) plays an indispensable role in catecholamine synthesis by converting dopamine into norepinephrine. Here, we characterized a DBH promoter polymorphism (C-2073T; rs1989787; minor allele frequency ~16%) that influences not only gene transcription but also enzyme secretion and blood pressure (BP) in vivo., Methods: Plasma DBH activity was measured spectrophotometrically. DBH genetic effects on BP were tested in subjects with the most extreme BP values in a large primary care population. Functional effects of promoter variants were studied by site-directed mutagenesis in DBH promoter haplotype/luciferase reporter plasmids transfected into chromaffin cells. Sequence motifs were predicted from position weight matrices, and endogenous transcription factor binding was probed by Chromatin ImmunoPrecipitation (ChIP)., Results: The T-allele of common promoter variant C-2073T was contained in a promoter haplotype that associated with plasma DBH activity, a trait also predicted by that variant itself. Promoter haplotypes including C-2073T predicted BP in the population, and the effect was also referable to C-2073T itself. Computationally, C-2073 disrupted a predicted match for transcription factor c-FOS. Site-directed mutagenesis at C-2073T altered not only basal promoter activity, but also transactivation by c-FOS, as well as the chromaffin cell secretory stimuli nicotine or pituitary adenylate cyclase-activating polypeptide (PACAP). Endogenous c-FOS bound to the motif in chromatin., Conclusions: These results suggest that DBH promoter variant C-2073T is functional in vivo: this promoter variant seems to initiate a cascade of transcriptional and biochemical changes including augmented DBH secretion, eventuating in elevation of basal BP, and hence cardiovascular risk. The observations suggest new strategies for probing the pathophysiology, risk, and treatment of hypertension.

Objectives: The aim of this study was to investigate the effect of gamma-glutamylcysteine ethylester (GCEE), a precursor of glutathione biosynthesis, on the levels of glutathione, formation of reactive oxygen species and c-fos mRNA expression in rat hippocampus and cortex in kainic acid-induced excitotoxicity., Methods: Sprague-Dawley rats were used and divided into four groups: control, kainic acid (10 mg/kg), GCEE (10 mg/kg) and kainic acid (10 mg/kg) + GCEE (10 mg/kg). Kainic acid and GCEE were administered to the rats intraperitoneally. The levels of glutathione and the expressions of c-fos mRNA in hippocampus and cortex tissues were determined using spectrophotometric and reverse transcription followed real-time PCR methods, respectively. Formation of reactive oxygen species was determined using dichlorofluorescin fluorescence in brain synaptosomes treated with kainic acid or GCEE in vitro., Key Findings: Kainic acid treatment significiantly upregulated the expression of c-fos mRNA in the hippocampus and cortex when compared to the control group. GCEE treatment significantly decreased the levels of c-fos mRNA in the cortex when compared to the kainic acid-treated group. GCEE treatment against kainic acid significantly increased the levels of glutathione in the cortex and hippocampus, and decreased the levels of formation of reactive oxygen species when compared to kainic acid-treated synaptosomes., Conclusions: The increased levels of glutathione and the reduced levels of reactive oxygen species formation lead us to conclude that GCEE may be beneficial as a potential antioxidant against neurodegenerative processes where excitotoxicity is involved.

Corticotropin-releasing factor (CRF) overexpressing (OE) mice are a genetic model that exhibits features of chronic stress. We investigated whether the adaptive feeding response to a hypocaloric challenge induced by food deprivation is impaired under conditions of chronic CRF overproduction. Food intake response to a 16-h overnight fast and ip injection of gut hormones regulating food intake were compared in CRF-OE and wild type (WT) littermate mice along with brain Fos expression, circulating ghrelin levels, and gastric emptying of a nonnutrient meal. CRF-OE mice injected ip with saline showed a 47 and 44% reduction of 30-min and 4-h cumulative food intake response to an overnight fast, respectively, compared with WT. However, the 30-min food intake decrease induced by ip cholecystokinin (3 microg/kg) and increase by ghrelin (300 microg/kg) were similar in CRF-OE and WT mice. Overnight fasting increased the plasma total ghrelin to similar levels in CRF-OE and WT mice, although CRF-OE mice had a 2-fold reduction of nonfasting ghrelin levels. The number of Fos-immunoreactive cells induced by fasting in the arcuate nucleus was reduced by 5.9-fold in CRF-OE compared with WT mice whereas no significant changes were observed in other hypothalamic nuclei. In contrast, fasted CRF-OE mice displayed a 5.6-fold increase in Fos-immunoreactive cell number in the dorsal motor nucleus of the vagus nerve and a 34% increase in 20-min gastric emptying. These findings indicate that sustained overproduction of hypothalamic CRF in mice interferes with fasting-induced activation of arcuate nucleus neurons and the related hyperphagic response.

Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes.

Neurons producing melanocortin receptor agonist, alpha-MSH derived from proopiomelanocortin, and antagonist, agouti-related protein, are known to be sensitive to metabolic stress such as food deprivation and glucoprivation. However, how these neurons respond to emotional/psychological stress remained to be elucidated. We report here that acute emotional stressors, i.e. restraint and forced swim, evoked mRNA expression of c-fos, a neuronal activation marker, in a high percentage of proopiomelanocortin neurons (up to 53% for restraint stress and 62% for forced swim), with marked variations along the rostro-caudal axis of the arcuate nucleus. In contrast, only a small population of agouti-related protein neurons in this brain region was activated. These neuronal activation patterns were correlated with behavioral reactions. Both stressors suppressed feeding and induced anxiety-like behavior in the elevated plus-maze test, as reflected by a reduction in the percentage of entries and time spent in the open arms. Central pretreatment with SHU9119, a melanocortin receptor antagonist, dose dependently attenuated the anorectic and anxiogenic effects elicited by acute restraint or forced swim. These results indicate that the melancortinergic pathway can be rapidly recruited by acute emotional stress, and that activation of melanocortin signaling is involved in mediating stress-induced anorexia and anxiety.

Our recent study suggested that cyclic compressive loading may promote chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures through the transforming growth factor (TGF)-beta signaling pathway. It has been shown that the activating protein 1 (AP-1) (Jun-Fos) complex mediated autoinduction of TGF-beta1 and its binding activity was essential for promoting chondrogenesis of mesenchymal cells, whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c-Fos, c-Jun, and TGF-beta type I and II receptors) and induction of their corresponding proteins in agarose culture of rabbit BM-MSCs subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were made by suspending BM-MSCs in 2% agarose gel (10(7) cells/ml) for cyclic, unconfined compression tests performed in a custom-made bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, c-Jun, and both TGF-beta receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c-Fos was detected only in the samples subjected to1-hour dynamic compressive loading. These findings suggest that the TGF-beta signal transduction and activities of AP-1 and Sox9 are involved in the early stage of BM-MSC chondrogenesis promoted by dynamic compressive loading.

Transcription factors, chromatin components and chromatin modification activities are involved in many diseases including cancer. However, the means by which alterations in these factors influence the epigenotype of specific cell types is poorly understood. One problem that limits progress is that regulatory regions of eukaryotic genes sometimes extend over large regions of DNA. To improve chromatin structure-function analysis over such large regions, we have developed an automated, relatively simple procedure that uses magnetic beads and a capillary sequencer for ligation-mediated-PCR (LM-PCR). We show that the procedure can be used for the rapid examination of chromatin fine-structure, nucleosome positioning as well as changes in transcription factor binding-site occupancy during cellular differentiation.

GnRH analog (GnRHa) and TGF-beta act directly on leiomyoma/myometrial smooth muscle cells (LSMCs and MSMCs) regulating diverse activities resulting in leiomyoma growth and regression. Because GnRH and TGF-beta receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of c-fos and c-jun, result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression, whose products are known to influence extracellular matrix turnover, which is critical in leiomyoma growth and GnRHa-induced regression. We found that GnRHa and TGF-beta in a dose- and time-dependent manner increased the level of phosphorylated ERK1/2 (pERK1/2) in LSMCs and MSMCs. GnRHa and TGF-beta increased ERK1/2 nuclear accumulation resulting in differential regulation of c-fos and c-jun mRNA expression via downstream signaling from MAPK kinase (MEK)1/2, because pretreatment with U0126, a synthetic inhibitor of MEK1/2, abolished basal and GnRHa- and TGF-beta-induced pERK1/2 and the expression of c-fos and c-jun. LSMCs and MSMCs also express fibronectin, type I collagen, and PAI-1 mRNA, and GnRHa and TGF-beta altered their expression in a cell-specific manner through MEK1/2. We concluded that GnRHa and TGF-beta acting through a MAPK/ERK pathway and transcriptional activation of c-fos/c-jun results in differential regulation of specific genes whose products may in part influence the outcome of leiomyoma growth and regression.

Background and Methods: The majority of human endometrial (>80%), ovarian (>80%) and breast (>50%) cancers express GnRH receptors. Their spontaneous and epidermal growth-factor-induced proliferation is dose- and time-dependently reduced by treatment with GnRH and its agonists. In this study, we demonstrate that the GnRH agonist triptorelin inhibits estradiol (E2)-induced cancer cell proliferation., Results: The proliferation of quiescent estrogen receptor alpha (ER alpha)-/ER beta-positive, but not of ER alpha-negative/ER beta-positive endometrial, ovarian and breast cancer cell lines, was significantly stimulated (P<0.001) (ANOVA) after treatment with E2 (10(-8) M). This effect was time- and dose-dependently antagonized by simultaneous treatment with triptorelin. The inhibitory effect was maximal at 10(-5) M concentration of triptorelin (P<0.001). In addition, we could show that, in ER alpha-/ER beta-positive cell lines, E2 induces activation of serum response element (SRE) and expression of the immediate early-response gene c-fos. These effects were blocked by triptorelin (P<0.001). E2-induced activation of estrogen-response element (ERE) was not affected by triptorelin., Conclusions: The transcriptional activation of SRE by E2 is due to ER alpha activation of the mitogen-activated protein kinase (MAPK) pathway. This pathway is impeded by GnRH, resulting in a reduction of E2-induced SRE activation and, in consequence, a reduction of E2-induced c-fos expression. This causes downregulation of E2-induced cancer cell proliferation.

Little is known about the impact of circulating lipids on brain processes. Building on evidence that chronic fat consumption stimulates hypothalamic peptides in close association with elevated triglycerides (TG), this study examined whether an acute rise in TG levels induced by fat emulsion can affect these hypothalamic systems. In normal weight rats, ip injection of Intralipid (20%, 5 ml) during the first 4 h after injection produced a robust increase in TG levels and nonesterified fatty acids, but had no impact on glucose, insulin, or leptin levels. This was accompanied by a marked increase in the expression of particular orexigenic peptides, galanin, orexins, and the opioid, enkephalin, which are known to be positively related to fat ingestion. This effect, similarly induced by 4 h of high fat diet consumption, was detected in the paraventricular nucleus (PVN) for galanin, in the perifornical hypothalamus (PFH) for orexins, and in the PVN, PFH, as well as the arcuate nucleus (ARC) for enkephalin. It was not seen, however, for neuropeptide Y and agouti-related protein localized in the ARC, which are unaffected or reduced by dietary fat. This site specificity was confirmed by c-Fos immunostaining, a marker of neuronal activity, which was increased by Intralipid in the PVN and PFH, but not in the ARC, and was detected in 20% of orexin-expressing neurons in the PFH. These findings suggest that circulating lipids, through different mechanisms, may stimulate hypothalamic neurons, which synthesize specific feeding stimulatory peptides that possibly contribute to hyperphagia during consumption of a fat-rich diet.

We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT-PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed.

Short-term assays for carcinogenicity testing of chemicals that use transgenic mice designed to have altered expression of genes mechanistically relevant to carcinogenesis are attractive alternatives to two-year dosing studies in rodents. The models that have been the received the greatest level of performance evaluation include p53(+/-), rasH2, Xpa/p53(+/-), and Tg.AC mice. For use of these models in a regulatory setting to evaluate the carcinogenic potential of pharmaceuticals, it is important to establish an assurance of assay specificity and positive predictivity based on studies using drugs with a wide spectrum of pharmacologic activity. For this purpose, 99 noncarcinogenic drugs were prioritized based on their activity in an in vitro induction assay correlative with a positive response in the Tg.AC assay (induction of the gadd153 promoter in HepG2 cells). Activities in two assays less predictive of Tg.AC activity (induction of c-fos and zeta-globin gene promoters) were also measured. Nine percent of the screened drugs induced the gadd153 promoter by at least fourfold. Several criteria were used to select candidates for subsequent in vivo testing in the Tg.AC assay: (1) sufficient drug solubility in appropriate skin paint vehicles to elicit systemic toxicity, (2) the level of induction of the gadd153 promoter by the drug, (3) the in vitro potency of the drug, and (4) the cost of the drug required for a 6-month study. Based on these criteria, amiloride, dipyridamole, and pyrimethamine were selected from 99 rodent noncarcinogens in a drug database for testing the specificity of the Tg.AC assay.

5-Fluoro-2'-deoxyuridine (FUdR) inhibits thymidylate synthase (TS). The inhibition of TS causes an imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools which subsequently induced cell death. We investigated the molecular mechanisms of cell death after treated with FUdR in F28-7 strain (which induced necrosis) and F28-7-A strain (mutant of F28-7 strain which induced apoptosis). After treated with FUdR, we observed different size of DNA fragmentations. F28-7 strain induced DNA cleavaged into 100-200 kbp fragments and F28-7-A strain induced DNA cleavaged into oligonucleosomal sized fragments. In F28-7 strain, FUdR induced the increased mRNA level of c-jun, c-fos and c-myc genes, caspase-3 like protease activity and the changes of mitochondrial membrane potential which were greater and earlier than those of F28-7-A strain. On the other hand, F28-7-A strain induced release of cytochrome c from mitochondrial, but not F28-7 strain. Furthermore, caspase-5 inhibitor was strongly inhibited the cell death of F28-7 strain. We suggest that it is concerned with intensity of intracellular signals in the cell death of F28-7 strain and F28-7-A strain.

Identifying genes upregulated in lead-resistant cells should give insight into lead toxicity and cellular protective mechanisms and may also result in identification of proteins that may be useful as biomarkers. Glial cells are thought to protect neurons against heavy metals. Rat glioma C6 cells share many properties of normal glial cells. To identify and analyze genes upregulated in a lead-resistant variant, PbR11, suppression subtractive hybridization (SSH) between mRNAs of wild-type and PbR11 cells was performed. Sequencing and database searches identified three genes, thrombospondin-1, heparin sulfate 6-sulfotransferase, and neuropilin-1, which play important roles in angiogenesis and axon growth during development. Two genes, HSP90 and UBA3, are involved in the ubiquitin-proteosome system. One gene was identified as that of a rat endogenous retrovirus and another, 2C9, is a transcript expressed in fos-transformed cells. PbR11 also overexpresses c-fos. Expression of these genes and effects of short-term lead exposure (24 h, up to 600 microM) on their expression in C6 cells was examined. The rat endogenous retrovirus and 2C9 are expressed only in PbR11 cells, and show no expression, either constitutive or lead-induced, in wild-type C6 cells. HSP90 is expressed at low level constitutively in C6 cells, but can be induced in a dose-dependent manner by lead. In contrast, thrombospondin-1 is repressed in a dose-dependent manner by lead. The other genes (HS6ST, neuropilin, and UBA3) show low constitutive expression and are neither upregulated nor downregulated by exposure to lead. We suggest that neuropilin-1, heparin sulfate 6-sulfotransferase, and thrombospondin-1 may be important targets for lead-induced developmental neurotoxicity.

PTH and PTH-related protein (PTHrP) are key mediators of skeletal development and homeostasis through their activation of the PTH-1 receptor. Previous studies have found that several AP-1 family members are regulated by PTH, such as c-fos, fra-1, and c-jun. There are numerous genes in the bone microenvironment that contain AP-1 sites, and different Fos family members are reported to have opposing transcriptional activities at AP-1 sites. The purpose of this study was to identify the effects of PTH on expression of the AP-1 protein complex member, fra-2, to extend our understanding of transcriptional regulators of PTH action. PTH induction of fra-2 messenger RNA (mRNA) levels in MC3T3-E1 preosteoblastic cells was maximal with 0.1 microM PTH (1-34). The expression in vitro was greatest 1 h after treatment and was present with N-terminal PTH but not PTH (7-34) or (53-84). Cycloheximide treatment induced fra-2 expression, and actinomycin D inhibited basal and PTHrP-induced expression. AP-1 protein in nuclear extracts of MC3T3-E1 cells was increased with PTH treatment at 3 h and consisted of high levels of Fra-2 protein, as evidenced by a supershift in an electrophoretic mobility shift assay and Western blot analysis. Up-regulation of steady-state fra-2 mRNA was also noted in vivo, where injection of PTH (1-34) (20 microgram) resulted in a more-than-7-fold maximal increase in fra-2 mRNA expression in the calvaria of mice, after 1 h of treatment. These data add to the transcriptional mediators induced by PTH and suggest that the interplay of AP-1 family members will provide insight into regulatory pathways of PTH and PTHrP for their anabolic and catabolic actions in bone.

The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor transduces signals after binding IGF-II or M6P-bearing growth factors. We hypothesized that this receptor relays paracrine signals between Sertoli cells and spermatogonia in the basal compartment of the seminiferous epithelium. For these studies spermatogonia were isolated from 8-day-old mice with purity >95% and viability >85% after overnight culture. The IGF-II/M6P receptors were present on the surface of spermatogonia, as detected by indirect immunofluorescence. We determined that both IGF-II and M6P-glycoproteins in Sertoli cell conditioned medium (SCM) modulate gene expression in isolated spermatogonia. The IGF-II produced dose-dependent increases in both rRNA and c-fos mRNA. These effects were mediated specifically by IGF-II/M6P receptors, as shown by studies using IGF-II analogues that are specific agonists for either IGF-I or IGF-II receptors. The SCM treatment also induced dose-dependent increases in rRNA levels, and M6P competition showed that this response required interaction with IGF-II/M6P receptors. The M6P-glycoproteins isolated from SCM by IGF-II/M6P receptor affinity chromatography increased spermatogonial rRNA levels at much lower concentrations than required by SCM treatment, providing further evidence for the paracrine activity of Sertoli M6P-glycoproteins. These results demonstrate that Sertoli cells secrete paracrine factors that modulate spermatogonial gene expression after interacting with cell-surface IGF-II/M6P receptors.

Human estrogen receptor-alpha messenger RNA (hERalpha mRNA) has a relatively short half-life, which was determined to be approximately 5 h in MCF-7 cell line after actinomycin D treatment. The 3'-untranslated region (3'UTR) of hERalpha mRNA was previously shown to completely down-regulate chloramphenicol acetyltransferase activity when present at the 3'-end of chloramphenicol acetyltransferase transcripts, suggesting a destabilizing function of the hERalpha 3'UTR sequence. Chimeric genes composed of a serum-inducible Fos promoter, GH-coding sequences, and different segments of the hERalpha complementary DNA 3'UTR sequence were used to confirm this hypothesis and to localize the RNA region responsible for the destabilizing effect. The presence of the complete hERalpha 3'UTR reduced the half-life of the reporter mRNA from more than 24 to 3 h. When the hERalpha 3'UTR was subdivided into four fragments (UTR1-4), one fragment, UTR2, retained the most ability to down-regulate the reporter mRNA (t1/2 = 4 h). A stretch of four AUUUA motifs within UTR2 was shown not to mediate mRNA destabilization. In contrast, further subdivision of the UTR2 into three parts (UTR2a-c) resulted in the loss of the destabilizing activity. Finally, recombination of two UTR2 subfragments (UTR2a and -b) partially restored this function, indicating a cooperative role among the three UTR2a-c subfragments in the process that leads to destabilization of the hERalpha transcript.

In order to elucidate the molecular mechanisms whereby ovarian hormones, and particularly estrogens, modulate fat cell metabolism, we investigated the effects of estradiol administration on c-fos and c-jun expressions in fat cells from ovariectomized (OVX) rats. Estradiol treatment resulted in a rapid increase in c-fos and c-jun messenger RNA (mRNA) and protein levels (about 2-fold). These effects of estradiol on c-fos and c-jun mRNAs were blocked by actinomycin D but not by cycloheximide treatment, suggesting that estradiol modulates c-fos and c-jun transcription. Moreover, the estradiol-induction of both transcripts was partially suppressed by the estrogen-receptor antagonist ICI 182,780. In contrast, progesterone administration did not affect c-fos and c-jun mRNA levels indicating a hormonal specificity of estrogen action. However, an antagonism of estradiol-induction of both genes was observed after progesterone treatment. In addition, the estradiol-induced changes in c-fos and c-jun mRNA expressions could not be observed in castrated males suggesting a gender-specific effect of estradiol. Finally, in OVX rats, estradiol treatment stimulated the specific AP-1 DNA binding activity (about 5-fold) in adipocyte nuclear extracts as assessed by electrophoretic mobility shift assays. These results suggest that some of the estrogen effects in fat cells from female rats are mediated through induction of the AP-1 complex expression and consequently through modulation of the AP-1 dependent gene expression in adipocytes.

When murine peritoneal macrophages were stimulated for 30 min with arachidonic acid, the growth-associated immediate early gene c-fos was induced in a concentration-dependent manner as assessed by Northern blot analysis. The arachidonic acid-induced c-fos mRNA expression was inhibited by a cyclooxygenase inhibitor, indomethacin, but not by a lipoxygenase inhibitor, nordihydroguaiaretic acid. Macrophages produced prostaglandin (PG) E(2) from arachidonic acid as determined by an enzyme immunoassay. Northern blot analysis revealed the expression of PGE receptor EP2 and EP4 subtypes, but not EP1 and EP3 in murine macrophages. PGE(2) brought about a marked elevation of cAMP, and c-fos mRNA expression was increased by PGE(2) and dibutyryl cAMP in these cells. These results suggest that arachidonic acid is transformed to PGE(2), which then binds to EP2 and EP4 receptors to increase intracellular cAMP and c-fos mRNA expression. Furthermore, the induction of c-fos by arachidonic acid, PGE(2), and cAMP was suppressed by pretreatment with interleukin (IL)-4. We also showed that the tyrosine phosphorylation of a Janus kinase, JAK3, is enhanced by IL-4 treatment, suggesting that the PGE(2)-mediated c-fos mRNA induction is inhibited by IL-4 through the tyrosine phosphorylation of JAK3.

Regulation of the activator protein-1 (AP-1) complex is very intricate because it involves phosphorylation state, protein-protein, and protein-DNA interactions. In these studies, the regulation of AP-1 activity, with emphasis on c-fos and c-jun regulation, was investigated using cannabinol (CBN) in primary mouse splenocytes in vitro. Cannabinoid compounds exhibit immunosuppressive actions that are putatively mediated through Gi-protein coupled receptors that negatively regulate adenylate cyclase. However, recent studies suggest that cannabinoids modulate other signaling cascades. Indeed, we demonstrate that CBN inhibited binding to AP-1-containing sites from the interleukin-2 promoter. This inhibition of binding was, in part, due to decreased nuclear expression of c-fos and c-jun. We further determined that the effects of CBN were due to posttranslational modifications of these phosphoproteins and showed that CBN inhibited the activation of ERK MAP kinases. Thus, cannabinoid-induced immunosuppression involves disruption of the ERK signaling cascade.

During the preovulatory and estradiol-progesterone-induced GnRH-LH surge, a subpopulation of GnRH neurons transiently expresses the transcription factor c-fos, which is a useful marker of cell activation. To further characterize this subpopulation of GnRH neurons, multiple immunohistochemical procedures were applied to visualize GnRH, c-Fos, KA2, GluR5, GluR6, and GluR7 receptor subunits during different phases of the estrogen-progesterone-induced LH surge. The results show that the LH surge begins at 1400 h and peaks at 1600 h before returning to baseline late in the evening. At 1400 h, about 50% of the GnRH neurons contained c-Fos, and this percentage remained high at 65% at 1600 and 2000 h. During the surge, 50% of the c-Fos-positive GnRH neurons contained KA2 receptor subunit protein at 1400 h, 65% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 1600 h, and 50% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 2000 h. As KA2 subunits require other kainate-preferring subunits to form functional receptor channels, we examined GnRH neurons for the presence of GluR5, GluR6, and GluR7 messenger RNA (mRNA) and protein. The results show that the KA2-containing GnRH neurons also contain GluR5 receptor subunit mRNA and protein, and that these GnRH neurons are c-Fos positive during the steroid-induced LH surge. To determine whether administration of kainate is sufficient to induce c-Fos in GnRH neurons, steroid-primed animals received iv injections of subseizure-inducing amounts of kainic acid and were processed for immunohistochemistry and in situ hybridization. The results show that kainic acid causes a significant increase in circulating LH; however, it does not induce c-Fos in GnRH neurons, nor does it cause an increase in GnRH mRNA. Together, the results suggest that a large subset of GnRH neurons expresses KA2 as well as GluR5 receptor subunits, which would allow the formation of functional glutamate receptor channels, and that this subset of GnRH neurons is activated during the steroid-induced LH surge.

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including the initiation of labor and milk ejection. OTR messenger RNA levels are highly regulated, reaching the greatest concentration in the uterus at the end of gestation, and in the mammary gland during lactation. Factors directly effecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by characterizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region between -85 and -65 that was required for both basal and serum-induced expression in a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OTRs. This DNA region contains an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif. The specific Ets factor binding to the OTR promoter was identified, by electrophoretic mobility immunoshift assays, to be GABP alpha/beta. Co-transfection of a -85 OTR/luciferase construct with vectors expressing GABP alpha and GABP beta1 had only a modest effect on expression, but cotransfection with GABP alpha/beta- with c-Fos/c-Jun-expressing plasmids resulted in an increase of almost 10-fold in luciferase activity. Mutation of either the GABP- or CRE-like binding sites obliterated the induction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We showed the requirement for GABP alpha/beta and c-Fos/c-Jun in endogenous OTR gene expression, using oligonucleotide GABP and AP-1 binding decoys to inhibit serum-induced increases in 125I-labeled OT antagonist binding to Hs578T cells. Our work is the first characterization of the proximal promoter region of the human OTR gene, and it sets the stage for studying regulation of OTR expression in breast cells.

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.

Several extracellular stimuli mediated by G protein-coupled receptors activate c-fos promoter. Recently, we and other groups have demonstrated that signals from G protein-coupled receptors stimulate mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The activation of these three MAPKs is mediated in part by the G protein betagamma subunit (Gbetagamma). In this study, we characterized the signals from Gbetagamma to c-fos promoter using transient transfection of c-fos luciferase into human embryonal kidney 293 cells. Activation of m2 muscarinic acetylcholine receptor and overexpression of Gbetagamma, but not constitutively active Galphai2, stimulated c-fos promoter activity. The c-fos promoter activation by m2 receptor and Gbetagamma was inhibited by beta-adrenergic receptor kinase C-terminal peptide (betaARKct), which functions as a Gbetagamma antagonist. MEK1 inhibitor PD98059 and kinase-deficient mutant of JNK kinase, but not p38 MAPK inhibitor SB203580, attenuated the m2 receptor- and Gbetagamma-induced c-fos promoter activation. Activated mutants of Ras and Rho stimulated the c-fos promoter activity, and the dominant negative mutants of Ras and Rho inhibited the c-fos promoter activation by m2 receptor and Gbetagamma. Moreover, c-fos promoter activation by m2 receptor, Gbetagamma, and active Rho, but not active Ras, was inhibited by botulinum C3 toxin. These data indicated that both Ras- and Rho-dependent signaling pathways are essential for c-fos promoter activation mediated by Gbetagamma.

The postnatal development of ocular dominance columns (ODCs) in monkey visual cortex provides an exquisite model for studying mechanisms of experience-guided neuronal plasticity. While the presence of columns at birth in Old World monkeys is now well established, it remains unclear whether cortical neurons at this early stage are capable of modulating gene expression in response to changing sensory conditions. Using a set of monocular deprivation and stimulation protocols, we examined activity-driven expression of the immediate-early genes (IEGs) c-fos and zif268 during the critical period of development. We observed well-delineated patterns of ODCs produced by sensory regulation of both IEGs throughout the critical period, starting as early as the first postnatal day. The expression levels are similar in layers II/II, IVC and VI throughout development, with no selective decline in the thalamorecepient layer (layer IVC) of adult monkeys. A narrow strip of non-columnar c-Fos expression was observed at the border of layers IVC and V. Our results show that neurons in monkey visual cortex are equipped at birth with the molecular machinery for coupling sensory inputs to active genomic responses and that this responsivity extends throughout the critical period. The findings are discussed within the context of a possible role for IEGs in sensory-driven cortical plasticity during development.

Background: Epidemiologic studies have demonstrated that individuals who eat more fruits and vegetables and/or have high levels of serum beta-carotene have a lower risk of cancer, especially lung cancer. However, recent human intervention studies using beta-carotene supplements have shown an increase in the risk of lung cancer among smokers and asbestos workers. In this study, we used an animal model system to evaluate the hazard associated with a combination of high-dose beta-carotene supplementation and tobacco smoking., Methods: Ferrets were given a beta-carotene supplement, exposed to cigarette smoke, or both for 6 months. Cell proliferation and squamous metaplasia in lung tissue were assessed by examination of proliferating-cell nuclear antigen expression and histopathologic examination, respectively. beta-Carotene and retinoid concentrations in lung tissue and plasma samples were analyzed by high-performance liquid chromatography. Expression of genes for retinoic acid receptors (RARs) and activator protein-1 (encoded by the c-Jun and c-Fos genes) in lung tissue specimens was examined by western blotting., Results: A strong proliferative response in lung tissue and squamous metaplasia was observed in all beta-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared with control groups, all three treatment groups had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited 18%-73% reductions in RARbeta gene expression; however, RARalpha and RARgamma gene expression was not reduced. Ferrets given a beta-carotene supplement and exposed to tobacco smoke had threefold to fourfold elevated expression of the c-Jun and c-Fos genes., Conclusions: Diminished retinoid signaling, resulting from the suppression of RARbeta gene expression and overexpression of activator protein-1, could be a mechanism to enhance lung tumorigenesis after high-dose beta-carotene supplementation and exposure to tobacco smoke.

To examine whether adrenomedullin (AM), a novel vasodilator peptide, acts as a growth modulator in the vasculature, the effects of AM on protein tyrosine phosphorylation, mitogen-activated protein kinase (MAPK) activation, protooncogene expression, DNA synthesis, and cell proliferation were studied in cultured rat vascular smooth muscle cells (VSMC). AM and calcitonin gene-related peptide (CGRP), although weaker than AM, stimulated DNA synthesis and cell proliferation of quiescent VSMC, whose effects were inhibited by a CGRP receptor antagonist, CGRP-(8-37). AM induced a rapid increase in MAPK activity, followed by the expression of the immediate early protooncogene c-fos. AM-induced MAPK activation and cell proliferation were completely blocked by protein tyrosine kinase inhibitors (genistein and ST638). Moreover, AM rapidly induced tyrosine phosphorylation of several proteins (approximately 120, approximately 90, and approximately 50 kDa) and transiently increased association of a tyrosine-phosphorylated protein (approximately 120 kDa) and Shc with the glutathione-S-transferase-Grb2 fusion protein. A MAPK kinase inhibitor (PD98059) also reduced the AM-induced MAPK activation, c-fos messenger RNA expression, and cell proliferation. Although AM has been shown to induce vasodilation through cAMP production in VSMC, a cAMP antagonist (Rp-cAMP-thionate) and a protein kinase A inhibitor (KT5720) failed to block AM-induced MAPK activation and DNA synthesis. Moreover, 8-bromo-cAMP and forskolin did not affect the MAPK activity. AM had no effect on either the intracellular Ca2+ concentration or inositol 1,4,5-trisphosphate formation. In addition, a protein kinase C inhibitor (GF109203X) did not inhibit the AM-induced MAPK activation. These data suggest that in addition to its vasodilatory effect through the cAMP-dependent pathway, AM exerts its mitogenic activity via protein tyrosine kinase-mediated MAPK activation in quiescent rat VSMC.

The cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family plays a major role in the expression of skeletal-specific genes and skeletal tissue development. We analyzed the changes of the amount, degree of phosphorylation and binding activity of the CREB/ATF family in the course of development of the murine calvarial osteoblastic cell line MC3T3-E1 as an in vitro model system of bone formation. The amount of CREB in the whole-cell extract detectable by Western blot analysis was high through all stages of development and maximal in the proliferation stage. The degree of phosphorylation estimated with anti-phosphorylated CREB antibody changed greatly and reached high levels in the proliferation stage and early mineralization stage. The ratio of phosphorylated CREB to total CREB in the CREB-CRE complex was also examined by gel shift assay. Although the binding to the consensus/CRE probe reached almost equally high levels in the proliferation stage and early mineralization stage, the relative level of phosphorylated CREB in the CREB-CRE complex was different in these two stages. In the early mineralization stage, most CREB bound to consensus/CRE was phosphorylated, while both phosphorylated and unphosphorylated CREB were bound to consensus/CRE in the proliferation stage. ATF-1 was also detected as a minor component bound to the consensus/CRE probe. The alteration of the binding of CREB to consensus/CRE over the course of osteoblast development supports the hypothesis that CREB may regulate the expression of genes defining the developmental sequence of MC3T3-E1 cells.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.

PTH and PTH-related protein (PTHrP) bind to the PTH-1 (PTH/PTHrP) receptor and produce anabolic and catabolic effects in bone. To investigate postreceptor mechanisms of action, MC3T3-E1 cells were induced to differentiate to optimize PTH-1 receptor expression, and differentiated MC3T3-E1 cells were treated with varying doses of PTH (1-34) for 1 h. Northern blot analysis revealed a dose-dependent stimulation of steady state c-fos messenger RNA (mRNA), with measurable expression at doses as low as 1 pM PTH. The time course of c-fos mRNA induction was rapid, with peak levels detected at 30-45 min. Increased steady state c-fos mRNA was due to increased transcription of the c-fos gene as demonstrated by nuclear run-on assays and was dependent on the temporal differentiation state of the MC3T3-E1 cells. Stimulation of c-fos mRNA was induced exclusively by N-terminal PTH and PTHrP (which is also responsible for cAMP activation), and did not occur with PTH (7-34), (53-84), or PTHrP (107-139). The effects of PTH (1-34) on c-fos stimulation were dependent on intracellular cAMP. Forskolin [a guanine-nucleotide-binding protein (G(alpha)) agonist] stimulated c-fos mRNA, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (a cAMP antagonist), 1,9 dideoxyforskolin (a cAMP independent analog of forskolin), and phorbol 12-myristate 13-acetate (a protein kinase C activator) did not. Furthermore, THFA inhibited the ability of PTH (1-34) to stimulate c-fos mRNA in a time-dependent manner. These findings indicate that c-fos is transcriptionally regulated by PTH (1-34) in osteoblastic cells, and that cAMP is a mediator of PTH-stimulated c-fos induction. Several known bone-associated proteins contain DNA binding sites in their promoter regions that recognize c-fos in conjunction with c-jun (AP-1 sites). Consequently, the induction of c-fos by PTH (1-34) in osteoblastic cells may be a sensitive indicator of PTH effects in vitro and in vivo, and provide valuable information regarding mechanisms of PTH action in bone.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.

To shed light on the molecular mechanisms involved in the pathogenesis of uterine leiomyomas, transcript levels of the immediate early genes c-fos, c-myc, and c-jun and of the estrogen receptor (ER) and progesterone receptor (PR) were determined in tissue samples of human myometrium and leiomyoma. The messenger RNA (mRNA) content was analyzed by RT-PCR. mRNAs for c-fos, c-myc, c-jun, ER, and PR were detected in all 18 samples of leiomyoma and corresponding myometrial tissue collected in this study. Interestingly, in contrast to healthy tissues, we found a distinct and significant reduction of c-fos mRNA in the tumor. These data were substantiated by the finding of lowered c-Fos protein levels in leiomyomas tissues. Moreover, transcripts of c-jun and c-myc were less abundant in most of the leiomyomas than in the myometrium. This different expression of the protooncogenes in leiomyomas and myometrium was independent of the phase of the menstrual cycle in which samples were collected. In contrast to the reduced transcript levels observed for the immediate early genes, the ER and PR mRNA contents of the leiomyomas and myometrium did not differ. These results were confirmed by immunohistochemical studies for ER and PR protein. In conclusion, our data show that the deregulated expression of protooncogenes, especially of c-fos, is linked to the pathogenesis of leiomyomas. Confirmation of a potential role of downregulated c-fos levels for the benign character of these tumors requires further investigation. Additionally, the findings suggest that sex steroids do not influence the different expression patterns of c-fos, c-myc, and c-jun in leiomyomas, as compared with myometrium.

The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-gamma induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-gamma increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-gamma, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3-6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription.

We have previously demonstrated antagonistic interactions between the major signal transduction pathways in human thyroid follicles: TSH acting via protein kinase A (PKA) attenuated phorbol ester [acting via protein kinase C (PKC)] as well as epidermal growth factor (EGF)-protein tyrosine kinase (PTK)-mediated cell proliferation, whereas the PKC and PTK pathways inhibited PKA-mediated cell differentiation. In view of the key role played by the protooncogenes c-jun and c-fos in the cascade of events leading to cell proliferation and differentiation, we examined whether the antagonism we observed between the pathways could be related to changes in the expression of these genes. The experimental model used was the same in vitro system as that used in the above study on cell growth and differentiation: thyroid follicles of human origin cultured in suspension under serum-free conditions. Both EGF (1-50 ng/mL) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 10(-11)-10(-7) mol/L) dose and time dependently stimulated c-jun and c-fos messenger ribonucleic acid (mRNA) expression. The c-jun and c-fos mRNA stimulation elicited by TPA was reduced by the PKC inhibitors, chelerythrine and staurosporine, and could not be mimicked by 4alpha-phorbol 12,13-didecanoate (a phorbol ester that fails to activate PKC), whereas the stimulation induced by EGF was diminished by the PTK inhibitor, genistein. This indicates a PKC- and PTK-mediated pathway triggered by TPA and EGF, respectively. TSH induced an increase in c-jun and c-fos mRNA, which, though significant, was small compared to that elicited by TPA or EGF. Addition of TSH (0.1-0.5 mU/mL), however, to either TPA or EGF dose dependently inhibited the c-jun and c-fos mRNA elicited by these agents. The repressive action of TSH on the effects of TPA and EGF mRNA were mimicked by forskolin and 8-bromo-cAMP, suggesting that the TSH inhibitory action is PKA mediated. The TSH inhibitory action seems to require de novo protein synthesis, as it was abrogated in the presence of cycloheximide. In conclusion, the present study provides novel data on c-jun and c-fos gene expression and their modulation by the major signal transduction pathways operating in human thyrocytes. Moreover, using the same serum-free system of human thyroid follicles cultured with the same agents and at the same doses as in our previous study on cell growth and differentiation, we found the TSH/PKA pathway to inhibit PKC- and EGF/tyrosine kinase-induced c-jun and c-fos mRNA, i.e. antagonistic effects parallel to those previously observed measuring cell proliferation. The findings suggest an association between human thyroid cell proliferation and c-jun and c-fos gene expression.

The effect of prostaglandin E2 (PGE2) on osteoclast (OC) differentiation is unclear, either stimulator or inhibitor, depending on the in vitro system used. This probably reflects indirect mechanisms through intermediate cells. We have investigated the direct effect of PGE2 on human OC differentiation from cord blood monocytes (CBMs) in the absence of stromal cells. Macrophages and multinucleated cells (MNCs) resembling OCs form in cultures of CBMs stimulated by 1,25-dihydroxyvitamin D3. In the present study, CBMs were cultured for 3 weeks, as previously described, in the presence or absence of PGE2. The number of MNCs was significantly reduced in the presence of PGE2 as was the proliferation of cultured CBMs, assessed on day 7. Immunohistochemistry was performed to evaluate macrophage markers (CD11b and CD14) and OC marker (beta3-chain). PGE2 significantly increased the numbers of CD11b-positive and CD14-positive cells, whereas the number of beta3-chain-positive cells was significantly decreased. beta3-Chain, c-fos, and human calcitonin receptor (h-CTR) messenger RNA (mRNA) expressions were evaluated by reverse transcription-PCR with RNA extracted from cultured CBMs. In the presence of PGE2, expression of beta3-chain and c-fos mRNA was reduced from the first week of culture. h-CTR mRNA expression was also reduced, and only the h-CTR1 isoform was detected in the presence of PGE2. In addition, when PGE2 was added only during the last week of culture, when no CBM proliferation occurred, the number of CD11b- and beta3-positive cells was unchanged compared to that in the control culture, as were the proportion of MNCs, the fusion index, and the expression of c-fos mRNA. In conclusion, our results suggest that PGE2 has an inhibitory effect on human OC differentiation from CBMs, possibly by reducing precursor proliferation in these cultures. We also hypothesize that PGE2 may reduce OC differentiation by increasing the proportion of precursor cells that differentiate into macrophages. In addition, this may be the result of inhibition of the c-fos expression in CBMs.

Activation of steroid hormone receptors by steroid hormones alters both the physiology and behavior of animals. Steroid hormone receptors (e.g., progestin receptors) can also be activated in the absence of steroid hormones by pharmacological treatment with neurotransmitters or neuropeptides. However, it is not known if progesterone-independent activation of brain progestin receptors occurs under natural, physiological, conditions. We report that increases in reproductive behavior and brain immediate early gene expression in female rats induced by mating stimuli can be blocked by prior treatment with progesterone antagonists in the absence of circulating progesterone. This suggests that progestin receptors are activated in a progesterone-independent manner by a physiologically relevant stimulus in female rats, thus implicating a novel pathway by which mating stimuli and other environmental influences could activate steroid receptors to influence neuronal response and behavior.

The present study was carried out to investigate how hormonal changes caused by chronic alcohol exposure of rats during the late period of gestation are coordinated with neuroendocrine functions of the fetal thymus, namely thymic expression of CRH and POMC genes. Alcohol consumption by pregnant dams led to a 5-fold elevation of plasma corticosterone (CORT) levels and significantly decreased fetal CORT levels. This generally inverse correlation between maternal and fetal CORT was absent in alcohol-consuming dams and their male fetuses on day 19 of gestation. These day 19 fetuses also had an attenuated plasma testosterone surge that occurred in the male control (pair-fed) fetus on day 19 of embryonic life. Furthermore, fetal alcohol exposure (FAE) resulted in a significant increase in thymic CRH and a decrease in thymic POMC expression in the male fetuses only, specifically on embryonic day 19. Thus, the strong positive correlation between CRH and POMC gene expression in the thymus of pair-fed male and female FAE fetuses was abolished in the FAE males. However, regardless of embryonic age or treatment, a strong positive correlation between thymic POMC gene expression and plasma testosterone levels in the male fetuses was detected. These data suggest that the sexually dimorphic effect of FAE on the fetal thymic POMC and CRH expression in males is driven by testosterone and may be related, therefore, to the presence of alcohol at the time of the prenatal testosterone surge in the male fetuses.

cAMP-responsive-element (CRE)-binding factors interaction with nucleosomal DNA has been investigated in vitro on the human c-fos promoter. Analysis of nucleosome reconstitution of this promoter shows a preferential nucleosome positioning on the proximal promoter sequences, including the CRE centered at -60 relative to the start site of transcription. CRE-binding protein (CREB) and modulator protein (CREM) are unable to interact with their recognition site incorporated in a nucleosome. However, competition between transcription factor binding and nucleosome assembly allows CREM binding and induces important modifications in the nucleosomal structure suggesting the displacement of nucleosomes. These findings imply that binding of transcription factors to the CRE prior to cAMP induction might be required to prevent the incorporation of this element in a nucleosome.

Employing immunohistochemistry and in situ hybridization, we studied the temporal and cell type specific localization of c-Fos and c-Jun proteins and the corresponding messenger RNAs (mRNAs) elicited by a single 17beta-estradiol (E2) injection in the uteri of castrated adult mice. Cellular expression of mRNAs was in parallel with the synthesis of proteins within 1 h. E2 stimulated the c-fos expression rapidly and transiently in the epithelium and vascular endothelium. A second small peak of c-Fos protein and c-fos mRNA expression occurred around 11-13 h in the epithelium. No detectable amount of c-fos transcript and protein was present throughout the time course (0-24 h) in the stromal and myometrial cells. E2 treatment caused differential c-jun expression in all uterine cell types. In the epithelium, c-jun mRNA and protein expression was decreased during 1-6 h post injection, and thereafter returned showing small peak around 11-13 h. Induction of c-Jun protein and c-jun mRNA was evident in the stromal and myometrial cells at 2-3 h, and then the expression gradually decreased and returned to nearly control level by 24 h. E2 treatment induced rapid and transient activation of c-jun in the vascular endothelium. Present results suggest that transient increase of c-Fos and decrease of c-Jun protein at the early phase and coexpression of these proteins at the late phase contribute the proliferation of endometrial epithelium in mature mice. Furthermore, c-Fos and c-Jun expression in the vascular endothelium at the early phase may participate in the uterine imbibition.

The ability to create artificial menstrual cycles in the rhesus monkey provides a model for studies on the regulation of genes and gene networks by estradiol or progesterone (P) in the primate endometrium. This model allowed us to create both a normal level of secretory phase P or an inadequate level of secretory phase P, i.e. endometria that cannot support implantation. The objective of our present study focused on PCR analyses of genes for several factors that are believed to be important in the proper maturation of the endometrium. Complementary DNA (cDNA) populations were prepared from endometria harvested on day 13 (peak E level), days 21-23 of an adequate secretory phase (PcDNA) and days 21-23 of an inadequate secretory phase (IcDNA). Although placental protein 14, leukemia inhibitory factor and 17-beta hydroxysteroid dehydrogenase displayed highly upregulated levels in PcDNA (P-activated genes), there was little or no up-regulation in IcDNA. Transforming growth factor-beta 2 and its receptor and insulin growth factor-I and its receptor were up-regulated in PcDNA, whereas little or no expression was observed in IcDNA. Regulators of the cell cycle and transcription, such as retinoblastoma, c-fos, and c-jun genes, were also greatly underexpressed in IcDNA compared with PcDNA. Interestingly, one gene that we studied, keratinocyte growth factor, that was up-regulated by P (peak E levels vs. PcDNA) was more highly expressed in IcDNA. This latter result suggests that low levels of circulating P are sufficient for expression of this gene, whereas high sustained P levels result in an autologous down-regulation. These data show that the regulation of genes that may play pivotal roles in endometrial maturation are differentially expressed in IcDNA vs. PcDNA and may, in part, characterize improper endometrial maturation.

We have identified cDNAs encoding Mel-N1, the mouse homologue of a human nervous system-specific RNA-binding protein, Hel-N1. Two major mRNA transcripts of Mel-N1 were detected predominantly in the adult mouse brain by Northern blot analysis. To gain insight into the RNA binding specificity of Mel-N1, we performed iterative in vitro RNA selection. The resulting in vitro selected RNAs were found to contain AU-rich sequences as well as a GAAA motif in the majority of clones. By means of in vitro binding assays we demonstrate that this GAAA sequence appears to significantly affect the Mel-N1 RNA-binding efficiency. Our studies further reveal that Mel-N1 can bind to its own 3' untranslated region (3'UTR) as well as to the c-fos 3'UTR, and is localized predominantly in the cytoplasmic region in cells, suggesting that posttranscriptional autoregulation of Mel-N1 gene expression occurs in vivo.

EWS-FLI-1 is a chimeric protein produced in most Ewing's sarcomas. It results from the fusion of the N-terminal-encoding region of the EWS gene to the C-terminal DNA-binding domain (the ETS domain) encoded by the FLI-1 ets family gene. Both EWS-FLI-1 and FLI-1 proteins function as transcription factors that bind specifically to ets sequences (the ets boxes) present in promoter elements. EWS- FLI-1 is a powerful transforming protein, whereas FLI-1 is not. In a search for potential DNA binding sites for these two proteins, we have tested their ability to recognize the serum responsive element (SRE) in the c-fos promoter. This cis element contains an ets box which can be occupied by members of the ETS protein family which do not bind DNA autonomously but form a ternary complex with a second protein, p67SRF (serum responsive factor). We demonstrate here that EWS-FLI-1, but not FLI-1, is able to form a ternary complex on the c-fos SRE. Using a GST pull-down assay, we show that both FLI-1 and EWS-FLI-1 interact in vitro with SRF in the absence of DNA. In electromobility shift assays, EWS-FLI-1 binding to the SRE is detectable in the absence of SRF whereas the binding of FLI-1 is not, suggesting that the interaction with DNA is the step which limits ternary complex formation by FLI-1. Deletion of the N-terminal portion of FLI-1 resulted in a protein which behaved as EWS-FLI-1, suggesting the existence of an N- terminal inhibitory domain in the normal protein. Taken together, our data indicate that there are intrinsic differences in the binding of EWS-FLI-1 and FLI-1 proteins to distinct ets sequences.

The p53 tumour suppressor protein is a potent transcription factor which plays a central role in the defence of cells against DNA damage and the propagation of malignant clones. We have previously shown that phosphorylation of serine 386 in mouse p53 by the growth- associated protein kinase, casein kinase II (CKII), plays an important role in the ability of p53 to block the proliferation of drug-resistant colonies. In this paper we show that blocking phosphorylation of serine 386 through an alanine substitution, or placing a constitutive negative charge at this position in the form of aspartate, had no significant influence on p53-dependent transcriptional activation of a promoter containing 13 copies of a p53 consensus binding sequence, or of the p21WAF1 promoter which is a natural target for p53. In contrast, the alanine mutant showed a weak reduction in the ability of p53 to repress expression from the c-fos promoter, which is a target for p53-dependent repression in vivo. Strikingly, when the repression of the SV40 early promoter was examined, a reduction in the repression capacity of up to 5-fold was observed. Moreover, repression of the SV40 promoter could be partially restored by aspartic acid substitution at the phosphorylation site. These data indicate that phosphorylation at a specific C-terminal site can selectively regulate p53-dependent repression, but not transactivation.