Semen Cuscutae-Fructus Lycii improves spermatogenic dysfunction by repairing the blood-testis barrier in rats according to in silico and in vitro methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae and Fructus Lycii (SC-FL) is a commonly used herbal pair for male infertility treatment. Studies have found that the mechanism of SC-FL treatment may be related to repairing the blood-testis barrier (BTB). The application of network pharmacology can be used to explore the correlation between medicines and diseases and predict the potential pharmacological mechanisms of SC-FL. AIM OF THE STUDY: This study aimed to explore the specific effects and mechanisms of SC-FL in repairing the BTB and initially revealed the mechanism of Chinese medicine treating male infertility through network pharmacology and animal experiments. MATERIALS AND METHODS: We searched databases using the network pharmacology method and performed mass spectrometry analysis. We analyzed and predicted the active ingredients, targets and key pathways of SC-FL in male infertility treatment. Then, we designed animal experiments to verify the results. Thirty-six Sprague-Dawley rats were randomly divided into the normal control group (NC group), spermatogenic dysfunction group (SD group) and SC-FL treatment group (SCFL group). Glucosides of Tripterygium wilfordii Hook. F (GTW) (40 mg/kg/d) was administered for 4 weeks to generate a spermatogenic dysfunction model. The rats in the SCFL group were given the SC-FL suspension (6 g/kg/d) daily. After 4 weeks of treatment, we detected the sperm quality of each group of rats and observed the cell morphology. Western blotting and qRT-PCR were used to detect the expression of BTB-related proteins in testicular tissues. RESULTS: 213 chemical ingredients of SC and FL were retrieved from the TCMSP database, and 54 effective chemical ingredients were obtained. Mass spectrometry analysis showed the above results were credible. Then, we identified 44 potential targets for the treatment of male infertility, and we plotted a network diagram of the interaction network between the core targets and a diagram of herbal medicine-active ingredient-target-disease interactions. The target genes were enriched according to biological functions, and 22 biological processes, 49 cellular components, 1487 molecular functions, and 122 signaling pathways were obtained. The results of the animal experiments showed that the sperm concentration and motility of the SCFL group were significantly improved compared with those of the SD group. Compared with those in the SD group, the structure and morphology of the Sertoli cells and seminiferous tubules of rats in the SCFL group improved, and the number of spermatogenic cells increased significantly. Western blotting and qRT-PCR results showed that compared with that in the SD group, the expression of p38 MAPK decreased significantly, and the expression of c-Jun, Occludin, ZO-1 and connexin 43 increased significantly in the SCFL group. CONCLUSION: We predicted that the active ingredients of SC-FL can treat male infertility by interacting with the core targets JUN, IL6, MAPK1, TP53, MYC, CCND1, AR, EGF, FOS, and MAPK8, and the possible mechanism is related to the MAPK signaling pathway. SC-FL can regulate the MAPK pathway and affect the expression of Occludin, ZO-1 and connexin 43 to repair damaged BTB and improve spermatogenic dysfunction induced by GTW, which may be one of the possible mechanisms.

Isoliquiritigenin Suppresses EMT-Induced Metastasis in Triple-Negative Breast Cancer through miR-200c/C-JUN/[Formula: see text]-Catenin.

Triple-negative breast cancer (TNBC) is the subtype of breast cancer with more aggressive growth and metastasis and without efficient therapies. Hence, it is worthwhile to search for potential effective drug candidates. According to our previous study, isoliquiritigenin (ISL) exerted a potent anticancer effect on breast cancer proliferation. Its effect on TNBC growth, metastasis and mechanism deserves further investigation. In this study, PCR array screened a significant increase of miR-200c in BT-549 and MDA-MB-231 cells after ISL treatment, and ISH exerted that miR-200c was expressed at a low level in breast cancer tissue of patients. We also found that ISL could up-regulate miR-200c, resulting in the inhibition of epithelial-mesenchymal transition. Meanwhile, ISL could inhibit metastasis and tumor growth in nude mice models through the increase of miR-200c. Further study displayed that ISL decreased c-Jun expression through the increase of miR-200c. Interestingly, we also detected that ISL might increase miR-200c expression through the demethylation of miR-200c promoter region. These findings indicated that ISL could be potentially developed as a novel drug candidate for TNBC in microRNA-based cancer therapies.

Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression.

Curcumae radix is the dry root of Curcuma longa L. (turmeric) that can be used either as a spice or traditional medicine. The aim of this study was to investigate the survival benefits and the anti-metastatic activity of curcumae radix extract (CRE) in MCF7 cells and in MMTV-PyMT transgenic mice-a mouse model of breast cancer metastasis. In vitro wound scratch assay revealed that CRE treatment inhibited cell motility and cell migration in a dose-dependent manner. To investigate the effect of CRE in breast cancer metastasis, MMTV-PyMT transgenic female virgin mice were used and randomly divided into two groups. For survival curve analysis, CRE was administered in a dose of 50 mg/kg to 8⁻20-week-old mice. Interestingly, CRE treatment significantly increased the median and prolonged survival of MMTV-PyMT mice. Furthermore, CRE treatment decreased tumor burden and inhibited cell proliferation in primary breast tumor, and also suppressed mammary tumor-derived lung metastasis. The size of the lung metastases substantially decreased in the CRE-treated group compared with the ones in the control group. Curcumae radix extract showed anti-metastatic activity through regulating the expression of metastasis markers including C-C Chemokine Receptor Type 7, Matrix Metalloproteinase 9 and the proto-oncogenes c-fos and c-jun. We demonstrated that these metastatic regulators were decreased when CCR7 expression was suppressed in MCF7 cells transfected with CCR7 siRNA. The results of this study show that curcumae radix exerts antitumor and anti-metastatic activities, and we suggest that curcumae radix might be a potential supplement for the treatment and prevention of breast cancer metastasis.

Heparin-binding epidermal growth factor-like growth factor downregulates expression of activator protein-1 transcription factor after intestinal ischemia-reperfusion injury.

The pathogenesis of necrotizing enterocolitis (NEC) is unknown. Ischemia and reperfusion (I/R) injury have been considered to be major contributing factors. More recent reports have noted that apoptosis is a significant and perhaps the principal contributor to cell death after I/R injury. Recent studies have revealed that activator protein 1 (AP-1) family proteins including c-Fos and c-Jun potentially induce either the proliferation or apoptosis of the cells in the brain, heart, kidney, and liver. c-Fos and c-Jun expression has also been reported to be upregulated in postischemic intestinal epithelial cells (IECs). Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a potent cytoprotective factor in various pathologic conditions and plays a pivotal role in mediating the earliest cellular responses to injury. This study aims to examine whether HB-EGF, a proven intestinal cytoprotective molecule, exerts its protective effects through modulation of AP-1 transcription factor after intestinal I/R injury. Thirty rats were randomly divided into the following 5 groups: (1) normal control group; (2) ischemia group; (3) I/R group; (4) ischemia group with HB-EGF (400 µg/kg), and (5) I/R group with HG-EGF (400 µg/kg). c-Fos and c-Jun messenger RNAs and protein levels were determined by real-time quantitative polymerase chain reaction (PCR) and Western analyses, respectively. Statistical analysis was performed using ANOVA with Dunn's test. The messenger RNA levels of the c-Fos and c-Jun increased after intestinal ischemia or the intestinal reperfusion phase. HB-EGF pretreatment significantly decreased c-Fos and c-Jun messenger RNAs. The expression of protein levels of c-Fos and c-Jun were correlation with the expression of messenger RNA level. HB-EGF intestinal cytoprotection is mediated, in part, by downregulation of the expression of AP-1 transcription factor after intestinal I/R injury.

DNA damage, apoptosis and C-myc, C-fos, and C-jun overexpression induced by selenium in rat hepatocytes.

OBJECTIVE: To study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes. METHODS: Sodium selenite at the doses of 5, 10, and 20 micromol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis (or comet assay). Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP nick end labelling) and flow cytometry. C-myc, c-fos, and c-jun expression in rat hepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method. RESULTS: At the doses of 5, 10, and 20 micromol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite (r=0.9501, P<0.01). Sodium selenite at the doses of 5, 10, and 20 micromol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates (%) of selenium-treated liver cells at the doses of 5, 10, and 20 micromol/kg were (3.72 +/- 1.76), (5.82 +/- 1.42), and (11.76 +/- 1.87) respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite (r=0.9897, P<0.01), these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P<0.01. CONCLUSION: Selenium at 5-20 micromol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

Inhibition of MAP kinases by crude extract and pure compound isolated from Commiphora mukul leads to down regulation of TNF-alpha, IL-1beta and IL-2.

The anti-inflammatory effect of the medicinal plant, Commiphora mukul gum was studied in peripheral blood mononuclear cells (PBMC). Bioassay-guided fractionation using conventional solvent extraction procedures, subsequent column fractionation, followed by monitoring specific activity in PBMC led to the isolation of a lead compound. Both crude ethyl acetate extract and the lead compound, thus isolated, showed inhibitory effect on proliferative response of PBMC in mitogenic lymphocyte proliferation and MLR assays. Further studies on inflammatory mediators such as IFN-gamma, IL-12, TNF-alpha, IL-1beta and NO showed down regulation, whereas no inhibition was observed in the case of anti-inflammatory cytokine IL-10. Immunoblot analysis revealed the inhibitory effect of crude ethyl acetate extract on phosphorylation of all the three mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 MAPK. In contrast treatment with pure compound showed no inhibitory effect on ERK. c-fos and c-jun mRNA levels were also reduced in PMA stimulated cells on treatment with crude extract and pure compound. This reduction in c-fos and c-jun levels, when taken together with inhibition of MAPK activation, provides a possible mechanism by which both crude ethyl acetate extract and purified compound isolated from C. mukul exert its action.

Soybean isoflavones inhibit estrogen-stimulated gene expression in mouse uteri.

This study was performed to examine the inhibitory effects of soybean isoflavones on estrogen-stimulated gene expression of the uteri in ovarectomized mice. Especially when compared with the inhibitory effect of genistein and daidzein as aglycosides described in our previous report, subcutaneous administration of the glycoside genistin significantly decreased the levels of estradiol-17beta (E2)-induced expressions of c-jun, interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha mRNAs (p < 0.005, p < 0.05 and p < 0.05, respectively) and seemingly proteins in the mice uteri, whereas the glycoside daidzin weakly inhibited E2-stimulated expressions of c-fos and IL-1alpha. Both genistin and daidzin seemed to have a weaker inhibitory effect than that of genistein and daidzein on the expression of estrogen-stimulated genes. It is suggested that those glycosides are naturally derived and generally absorbed from plant foods and might prevent E2-related endometrial carcinogenesis.

The colon mitosis-inhibitor pyroglutamyl-histidyl-glycine alters expression of immediate-early cancer-related genes in HT-29 cells.

BACKGROUND: The intestinal mitosis-inhibiting peptide pyroglu-His-GlyOH (pEHG), inhibits normal intestinal epithelial cells and the human colon adenocarcinoma cell line HT-29 and increases the expression of c-fos (1). In this study, we investigated the mechanisms of the growth-inhibiting effects of pEHG. MATERIALS AND METHODS: cDNA expression array was hybridized with cDNA from HT-29 cells exposed to pEHG or control. The results were confirmed with Northern blot or real-time PCR. RESULTS: pEHG(1 nM) provoked a significant increase in the expression of the early growth response protein 1 (egr-1) after an incubation of 20 minutes, while c-fos was confirmed up-regulated by the same treatment. We further studied the expression of fosB, c-jun and junB, in the AP-1 complex. fosB was up-regulated 20-fold, but only minor effects on jun variants were observed. CONCLUSION: pEHG stimulates the gene expression of some immediate-early transcription factors involved in cell proliferation.

Dietary lipids alter the effect of steroids on transport of glucose after intestinal resection: Part II. Signalling of the response.

BACKGROUND/PURPOSE: Glucocorticosteroids alter the function of the intestine. Budesonide (Bud) increases the jejunal D-glucose uptake, and this effect is prevented through a polyunsaturated fatty acid (PUFA) diet. This study was undertaken to assess the possible signalling effect of budesonide, prednisone (Pred), or dexamethasone (Dex) in animals with a 50% intestinal resection and fed chow or a diet enriched with saturated (SFA) or polyunsaturated fatty acids. METHODS: Northern blots were performed. RESULTS: Steroids reduced the jejunal but not the ileal expression of proglucagon. Ornithine decarboxylase (ODC) expression was reduced in the jejunum. CONCLUSIONS: c-jun, ODC, and proglucagon may be involved in the adaptive response that occurs with steroids and variations in dietary lipids after intestinal resection.

Involvement of adrenoceptors in the angiotensin II-induced expression of inducible transcription factors in the rat forebrain and hypothalamus.

Angiotensin II (Ang II) acts as a neuromodulator/neurotransmitter in specific brain nuclei involved in the regulation of blood pressure and volume homeostasis. It also induces a highly differentiated transcription factor expression in these nuclei. We investigated whether adrenoceptors, which modulate other central actions of angiotensin II like the vasopressin release, also play a role in the AT1 receptor-mediated expression of the transcription factors (TF) c-Fos, c-Jun and Krox-24 in the rat brain. Ang II, injected intracerebroventricularly, induced the expression of c-Fos, c-Jun and Krox-24 in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Pretreatment with the alpha 1-adrenoceptor antagonist, prazosin, significantly inhibited the Ang II-induced transcription factor expression in the SON and PVN. The alpha 2-adrenoceptor antagonist, yohimbine, also reduced Ang II-stimulated transcription factors significantly in both nuclei. This inhibition was mainly localized in vasopressinergic magnocellular neurons in both nuclei. The beta-adrenoceptor antagonist, propranolol, did not influence the Ang II-induced expression of TF. Our results show that both, Ang II-induced vasopressin release and transcription factor expression, involve the same neuronal connections in the brain, implicating that the signal transduction pathways leading to the two different effects are at least to a certain degree convergent.

Photoprotective effect of black tea extracts against UVB-induced phototoxicity in skin.

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.

Advantage of double labeled in situ hybridization for detecting the effects of glucocorticoids on the mRNAs of protooncogenes and neural peptides (TRH) in cultured hypothalamic neurons.

In order to study the effect of glucocorticoids on thyrotropin-releasing hormone (TRH) and protooncogenes, we describe a double labeled in situ hybridization method to explore this issue. The development of non-isotopic in situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology [C.L.E. Moine, E. Normand, B. Bloch, Use of non-radioactive probes for mRNA detection by in situ hybridization: interests and applications in the central nervous system, Cell. Mol. Biol. 41 (1995) 917-923]. These methods involve the anatomic localization of labeled RNA or DNA molecules which hybridize with complementary target RNA or DNA sequences in the cell. With regard to gene expression, in situ hybridization allows the study of specific mRNA levels and the distribution between various cell types. It also allows the comparison of mRNA levels at various stages of development. Double labeled in situ hybridization is able to detect the colocalization of two different mRNAs simultaneously. Accordingly, this approach is utilized for specific studies involving the expression and distribution of TRH mRNA and the protooncogenes, c-fos/c-jun, in cultured rat hypothalamic neurons [L. G. Luo, I.M.D. Jackson, Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons, Brain Research 791 (1998) 56-62]. Our protocol for double labeled in situ hybridization reflects a modification of a number of original protocols developed by others [H. Breitschopf, G. Suchanek, R.M. Gould, D.R. Colman, H. Lassmann, In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain, Acta Neurropathol. 84 (1992) 581-587; S. McQuaid, J. McMahon, G.M. Allan, A comparison of digoxigenin and biotin labeled DNA and RNA probes for in situ hybridization, Biotech. Histochem. 70 (1995) 147-154; E. Hrabovszky, M.E. Vrontakis, S.L. Petersen, Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats, J. Histochem. Cytochem. 43 (1995) 363-370]. This technique can be readily applied to various studies of cellular gene expression in the mammalian nervous system involving other neural peptides and transcription factors.

Effects of prolactin on expression of the mRNAs encoding the immediate early genes zif/268 (NGF1-A), nur/77 (NGF1-B), c-fos and c-jun in the hypothalamus.

Prolactin (PRL) exerts a short-loop negative feedback effect on hypothalamic neurons which control its secretion from the anterior pituitary gland. The purpose of this study was to identify the location of hypothalamic neurons which respond to acute PRL exposure. Increasing evidence indicates that excitation of neurons often results in the rapid transcription of immediate early genes (IEGs). In the present study, quantitative in situ hybridization histochemistry (ISHH) was used to visualize the induction of mRNAs for four different IEGs: zif/268 (NGF1-A), nur/77 (NGF1-B), c-fos and c-jun. Three groups of male rats were compared: unmanipulated controls, rats injected s.c. with 2.4 mg ovine PRL (oPRL) suspended in polyvinylpyrrolidone (PVP), and PVP-injected controls. Animals were decapitated 0, 0.5, 1, 2, 3 or 4 h following injection. In all rats, the four probes labeled cells within the cortex, particularly the cingulate and piriform cortices, the hippocampus and the striatum. In the arcuate nucleus, there was a modest increase in the average number of cells/animal which expressed zif/268 mRNA following the injection of PVP and oPRL at all times studied. The average area of grains/cell representing zif/268 message also increased following the injection stimulus. The number of neurons expressing nur/77 mRNA was greater in PRL-treated rats compared with PVP-treated controls 0.5 and 1 h following injection. Nur/77-labeled neurons were co-extensive with the tuberoinfundibular dopaminergic (TIDA) neurons. The data suggest that cells located within the arcuate nucleus are involved in mediating PRL autofeedback on the brain.

c-fos is a positive regulator of carcinogen enhancement of adenovirus transformation.

The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)

MK-801 inhibits the induction of immediate early genes in cerebral cortex, thalamus, and hippocampus, but not in substantia nigra following middle cerebral artery occlusion.

Middle cerebral artery (MCA) occlusion in rats induced c-fos and junB mRNA 4h later in all ipsilateral cortex outside the MCA distribution and in many subcortical structures: medial striatum; most of thalamus including medial and lateral geniculate nuclei: substantia nigra; and hippocampus. The N-methyl-D-aspartate (NMDA) antagonist, MK-801 (4 mg/kg, i.p.) inhibited c-fos and junB mRNA induction in the cortex, striatum, thalamus, and hippocampus but not in the substantia nigra. These data show that c-fos and junB mRNA induction in cortex, striatum, thalamus, hippocampus involves the activation of NMDA receptors whereas different receptors must be implicated in the induction in substantia nigra.

A single administration of ethanol simultaneously increases c-fos mRNA and reduces c-jun mRNA in the hypothalamus and hippocampus.

We have previously demonstrated that a single administration of ethanol induces the expression of c-fos mRNA in the hypothalamic paraventricular nucleus (PVN). However, Fos protein must interact with a member of the Jun family to form functional heterodimers. To determine whether ethanol may have differential effects on c-fos and c-jun expression, we injected male rats acclimated to a 25 degrees C environment with ethanol (3 g/kg b.wt.) or saline. Using in situ hybridization histochemistry with oligonucleotide probes, we found that ethanol increased c-fos mRNA in the PVN, but decreased c-jun mRNA both in the PVN and in hippocampus. Considering that ethanol produces hypothermia and that the PVN contains neurons activated during hypothermia, we evaluated the effect of cold on c-fos and c-jun mRNA. Both cold and ethanol increased c-fos mRNA, and the effects were additive. However, c-jun mRNA levels in both PVN and hippocampus were unaffected by temperature. Finally, c-jun mRNA levels in the hippocampus were significantly reduced by chronic ethanol exposure, and this trend was also observed in the PVN. These findings demonstrate that a single injection of ethanol has opposite effects on the expression of nuclear transcription factors which interact to regulate gene expression in the nervous system.

Protooncogene junB as a target for activin actions.

Activin, a member of the transforming growth factor-beta family of peptides, is implicated in the regulation of cell growth and differentiation in a variety of biological systems. We have sought to identify immediate early genes whose altered expression may provide a common nuclear event involved in activin-regulated phenotypic changes in many cell types. In both human K562 myelogenous leukemia and rat PC12 pheochromocytoma cells, activin treatment caused transient transcription-dependent and protein synthesis-independent increases of junB messenger RNA (mRNA) within 1 h, whereas neither c-jun nor c-fos mRNA were inducible. In K562 cells, this selective junB mRNA induction was synergistically augmented by treatment with 12-O-tetradecanoyl phorbol-13-acetate but not affected by forskolin. Furthermore, in PC12 cells, the up-regulation of junB mRNA by activin was observable even after high-dose treatment with 12-O-tetradecanoyl phorbol-13-acetate for 48 h, indicating that junB mRNA expression by activin is independent of both A- and C-kinases. Our report suggests that induction of this ubiquitous gene product may be a critical event shared by a set of activin-responsive tissues.