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The proenkephalin gene is a well-studied model of transcription factor-target gene interaction in the nervous system and has been proposed as a regulatory target of the protein product of the immediate-early gene c-fos. This regulatory mechanism has been proposed, in part, because the cAMP response element 2 (CRE-2) site, the key DNA regulatory element within the proenkephalin second-messenger-inducible enhancer, avidly binds AP-1 proteins, including Fos, in vitro. However, we observe a dissociation in the time course of activation of c-fos and proenkephalin mRNA in rat striatum after administration of the dopamine D2 receptor antagonist haloperidol. This result prompted us to investigate the composition of protein complexes in striatal nuclear extracts that bind to the CRE-2 site. Even though our striatal nuclear extracts had substantial basal and haloperidol-inducible AP-1-binding activities that contained Fos, we could not detect Fos in complexes bound to the CRE-2 element. Instead, as determined by antibody supershift analysis, we detect CRE-binding protein (CREB)-like proteins binding to CRE-2 in both basal and haloperidol-stimulated conditions. Finally, we show that haloperidol induces CREB protein phosphorylation in striatum.
Amphetamine is a psychostimulant drug of abuse that can produce long-lived changes in behavior including sensitization and dependence. The neural substrates of these drug effects remain unknown, but based on their prolonged time course, we hypothesize that they involve drug-induced alterations in gene expression. It has recently been demonstrated that amphetamine regulates the expression of several genes, including c-fos, via dopamine D1 receptors in rat striatum. Here we report that amphetamine induces phosphorylation of transcription factor cAMP response element binding protein (CREB) in rat striatum in vivo and that dopamine D1 receptor stimulation induces phosphorylation of CREB within specific complexes bound to cAMP regulatory elements. In addition, we show by antisense injection that CREB is necessary for c-fos induction by amphetamine in vivo. Since CREB has been implicated in the activation of a number of immediate-early genes as well as several neuropeptide genes, CREB phosphorylation may be an important early nuclear event mediating long-term consequences of amphetamine administration.
We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.
Haloperidol has been shown to induce rapid and transient expression of c-fos messenger RNA and Fos protein in striatal neurons via dopamine D2 receptors. Regulation of the c-fos gene by cyclic AMP and Ca2+ has been shown to be dependent on a DNA regulatory element within its promoter that binds the constitutively expressed transcription factor cyclic AMP response element binding protein. Cyclic AMP response element binding protein binds to an oligonucleotide containing the calcium/cyclic AMP response element of the c-fos promoter sequence in striatal cell extracts; the amount of binding is not regulated by haloperidol treatment. We have previously shown that haloperidol induces cyclic AMP response element binding protein phosphorylation in the striatum. Here we show by intrastriatal injection of antisense oligonucleotides that haloperidol-induced Fos expression is dependent on cyclic AMP response element binding protein. Intrastriatal injections of phosphorothioate oligonucleotides, in antisense orientation to cyclic AMP response element binding protein messenger RNA, reduce levels of cyclic AMP response element binding protein and completely prevent haloperidol-mediated induction of Fos. Oligonucleotides in sense orientation have no such effect. We observed a markedly different time course of the Fos protein inhibition by cyclic AMP response element binding protein antisense oligonucleotides compared to c-fos antisense oligonucleotides. This most likely reflects the different half-lives of c-fos and cyclic AMP response element binding protein messenger RNA and proteins. Neither cyclic AMP response element binding protein nor c-fos antisense oligonucleotide injection reduced c-Jun immunostaining in the striatum. We conclude that haloperidol induces Fos via transcription factor cyclic AMP response element binding protein.
Monitoring expression of c-fos and other immediate-early genes has proven a useful method for determining potential sites of action of antipsychotic drugs. Most studies of the effects of antipsychotic drugs on immediate-early gene expression have focused on the basal ganglia and allied cortical regions. We now report that clozapine administration markedly increases both the number of cells expressing Fos protein-like immunoreactivity and the amount of Fos protein in the thalamic paraventricular nucleus, but not the contiguous mediodorsal thalamic nucleus. Comparable doses of several dopamine D2-like antagonists, including raclopride, sulpiride, remoxipride and haloperidol, did not induce Fos expression in the paraventricular nucleus. However, loxapine and very high doses of haloperidol resulted in a small but significant increase in paraventricular nucleus Fos expression. The dopamine D1 receptor antagonist SCH23390 did not induce Fos in the paraventricular nucleus or alter the magnitude of the clozapine-elicited increase in Fos expression. The serotonergic 5-hydroxytryptamine2a/2c antagonist ritanserin, alone or in combination with sulpiride, did not increase Fos expression in the paraventricular nucleus. Similarly, the 5-hydroxytryptamine2:D2 antagonist risperidone did not change the amount of Fos protein in the paraventricular nucleus. Neither the alpha 1 adrenergic antagonist prazosin nor the muscarinic cholinergic antagonist scopolamine mimicked the effect of clozapine. The key placement of the paraventricular nucleus as an interface between the reticular formation and forebrain dopamine systems suggests that this thalamic nucleus may be an important part of an extended neural network subserving certain actions of antipsychotic drugs.
Fifteen archival human osteosarcoma specimens were examined by in situ hybridization for the expression of human and mouse transforming growth factor-beta (isoforms 1, 2, and 3), c-fos, and metalloproteinase (stromelysin-3 and matrilysin). Osteosarcoma subtypes were confirmed by review of patients' radiographs, histopathology, and age at diagnosis. The outcome and method of treatment were documented. The subtypes of osteosarcoma consisted of nine conventional osteosarcomas and two each of fibroblastic, telangiectatic, and post-radiation osteosarcomas. Each specimen was histologically examined under light microscopy, and then adjacent paraffin sections were assayed with sense and anti-sense RNA probes by in situ hybridization. The probes localized to the neoplastic cells, confirming the methodology of the technique. Human transforming growth factor-beta 1 had the most uniform binding affinity to the osteosarcomas examined and was more specific in binding than mouse transforming growth factor-beta 1. Specific mRNA encoding for the transforming growth factor-beta s, c-fos, and metalloproteinases are detectable in patterns within osteosarcoma cells, and collectively, their expression parallels the different histopathologic subtypes. The less differentiated subtypes (telangiectatic and post-radiation osteosarcomas) expressed the fewest molecular markers. Osteosarcoma is a heterogeneous tumor. Differential expression of matrilysin in osteosarcoma is the first reported detection of metalloproteinase activity in human skeletal sarcoma.
Stress-induced alterations in expression of c-fos protein (Fos) in mesencephalic dopamine (DA) neurons of the rat were examined in order to discern which midbrain DA neurons are metabolically activated by stress. Restraint stress for 30 min increased the number of DA neurons exhibiting Fos-like immunoreactivity in the ventral tegmental area (VTA), but not in the substantia nigra or retrorubral field. Stress elicited an increase in the number of DA neurons expressing Fos in specific nuclei within the VTA. Administration of the anxiogenic beta-carboline FG 7142 also increased the total number of VTA DA neurons expressing Fos protein, whereas pretreatment with an anxiolytic benzodiazepine (diazepam) partially prevented the stress-induced increase in Fos expression. Restraint stress for 30 min increased concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens and striatum, as well as in the prefrontal cortex. Retrograde tracer studies revealed that stress increased Fos protein expression in a distinct subset of DA neurons projecting to the prefrontal cortex. In contrast, Fos expression was not increased in any DA neurons projecting to the nucleus accumbens. The present data indicate that there are at least two functionally distinct DA systems embedded within the prefrontal cortex of the rat.
Polyoma virus (Py) causes neoplastic transformation in vitro and multiple tumors in vivo. The role played by large and middle T antigens (LT, MT) and their mechanisms of action are focused here. Py-transformed Balb-3T3 cells become independent of platelet-derived growth factor (PDGF) for growth. JE, c-fos, c-jun and c-myc are 'immediate early' genes induced in response to PDGF. To test whether these cellular genes play a role in malignant transformation by Py, we generated a number of transfectant cell lines overexpressing LT, MT or both. Characterization of these cell lines revealed that: (a) MT but not LT causes morphological transformation, ability to grow in agarose suspension; (b) cooperation between LT and MT is evident in vitro, however, high and simultaneous LT and MT expression does not warrant tumorigenic potential; (c) MT expression does not correlate with tumorigenic potential but alters the probability of eliciting tumors; (d) JE and c-myc (but not c-fos or c-jun) are constitutively expressed in MT transfectants. MT induction is followed by c-myc induction 1.5 h later. We conclude that some of the 'immediate-early' genes may play pivotal roles in Py transformation.