A single dose of methamphetamine leads to a long term reversal of the blunted dopamine D1 receptor-mediated neocortical c-fos responses in mice deficient for D2 and D3 receptors

Dopamine D(1) receptors play an essential role in the induction of expression of the immediate-early gene c-fos in response to pharmacological stimuli. In the forebrain of wild-type mice, administration of a D(1) receptor agonist leads to c-fos mRNA expression levels that are substantially higher than corresponding levels expressed after indirect stimulation of dopamine receptors with methamphetamine. In mice deficient for D(2) and D(3) receptors, c-fos mRNA levels expressed in response to D(1) agonist administration are significantly blunted. However, a single dose of methamphetamine (5 mg/kg) leads to a long lasting reversal of the blunted c-fos responses in these mutants. In the forebrain, this reversal is restricted to the neocortex. Moreover, methamphetamine also enhances c-fos expression levels in preadolescent wild-type mice that normally express low c-fos mRNA in response to D(1) agonist stimulation. Thus, a single dose of methamphetamine leads to a long term increase in D(1) receptor-dependent c-fos responses in brains with either low (preadolescent mice) or blunted (adult D(2) and D(3) mutant mice) c-fos expression levels. A similar long term reversal of the blunted c-fos responses is achieved with a single dose of a full D(1) agonist. These results indicate that the constitutive inactivation of D(2) and D(3) receptors leads to a decrease in agonist-promoted D(1) receptor activity that can be reversed by intermittent agonist stimulation.     

Use of c-Src and c-Fos knockout mice for the studies on the role of c-Src kinase signaling in the expression of toxicity of TCDD

Previously, we reported that several of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) were not fully expressed in c-src −/− and −/+ mice (1). In the current study, we studied the basic molecular mechanism of their differential responses. First, we could show that chemical inhibition of c-src kinase could produce practically the same phenomenon of reduced toxicity of TCDD in wild-type mice cotreated with geldanamycin, a specific chemical inhibitor known to suppress c-src kinase. Second, we established that the level of the Ah receptor associated with c-src kinase (2) is indeed low in c-src deficient mice as well as geldanamycin-treated mice. We could show, at the same time, that the effect of c-src deficiency on the toxicity of TCDD is very selective; that is, despite the reduction of many of its toxic signs, the enlargement of liver, induction of cytochrome P450, and other drug-metabolizing enzymes took place normally in those c-src–deficient mice. Apparently, induction of these detoxification enzymes are independent of c-src–mediated pathway. Based on the known signaling pathways of c-src, we tested c-fos–deficient mice and found that some of the c-src–dependant toxic signs of TCDD such as thymic atrophy and decrease in adipose tissue weight were also reduced in c-fos–deficient mice, indicating that these two toxic effects are likely to be mediated through both c-src and c-fos. However, other TCDD responses noticeable in c-fos–deficient mice; downregulation of receptors for epidermal growth factor (EGF), tumor necrosis factor (TNFα), and retinoic acid; and up-regulation of the T3 receptor. These findings clearly show that c-fos mediates only a part of c-src signaling pathway in transducing these specific toxic actions of TCDD as mediated by c-fos. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 263–274, 1998     

A new cAMP response element in the transcribed region of the human c-fos gene.

In NIH 3T3 cells the c-fos gene is induced rapidly and transiently by cAMP. As shown by the analysis of 3T3 cells stably transfected with promoter mutants of the human c-fos gene this induction does not depend on the dyad symmetry element (position -320 to -300), but involves at least two other non-related sites: an element located around position -60 resembling the cAMP response element of the fibronectin and somatostatin genes (which has been described before), and an element located between positions +18 and +38. Destruction of one or the other element in the c-fos gene reduces cAMP inducibility. The cAMP response of c-fos promoter CAT gene constructs also depends on these elements in transient transfection assays. When cloned in front of the albumin TATA box, both elements independently mediate cAMP inducibility. These elements do not bind the same protein as shown in gel retardation analyses, suggesting that two different cAMP inducible factors mediate the activation of the c-fos gene by cAMP.     

Aberrant neuronal connectivity in CHL1-deficient mice is associated with altered information processing-related immediate early gene expression

In humans, loss or alteration of the CHL1/CALL gene may contribute to mental impairment associated with the 3p-syndrome, caused by distal deletions of the short (p) arm of chromosome 3, and schizophrenia. Mice deficient for the Close Homologue of L1 (CHL1) show aberrant connectivity of hippocampal mossy fibers and olfactory sensory axons, suggesting participation of CHL1 in the establishment of neuronal networks. Furthermore, behavioral studies showed that CHL1-deficient mice react differently towards novel experimental environments. These data raise the hypothesis that processing of information, possibly novel versus familiar, may be altered in the absence of CHL1. To test this hypothesis, brain activities were investigated after presentation of a novel, familiar, or neutral gustatory stimulus using metabolic mapping with ((14)C)-2-deoxyglucose (2-DG) and analysis of mRNA expression of the immediate early genes (IEGs) c-fos and arg 3.1/arc by in situ hybridization. 2-DG labeling revealed only small differences between CHL1-deficient and wild-type littermate mice. In contrast, while the specific novelty-induced increase in c-fos expression was maintained in most of the brain areas analyzed, c-fos mRNA expression was similar after the novel and familiar taste in several brain areas of the CHL1-deficient mice. Furthermore, in these mutants, arg 3.1/arc expression was slightly reduced after the novel taste and increased after the familiar taste, leading to a similar arg 3.1/arc mRNA expression after both stimuli. Our results indicate that, in contrast to controls, CHL1-deficient mice might process novel and familiar information similarly and suggest that the altered neuronal connectivity in these mutants disturbs information processing at the molecular level.     

Activation of c-fos promoter by Gbetagamma-mediated signaling: involvement of Rho and c-Jun N-terminal kinase

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.     

Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.     

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells

The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.     

Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana

Leaf variegation has long been known as a recessive genetic trait in higher plants. Unlike albino mutants, leaf-variegated mutants are non-lethal and thus enable us to study a novel mechanism of plastid development and maintenance. Variegation results from a defect that makes chloroplast development unstable, since at least part of the tissues gives rise to normal chloroplasts. Despite the fact that leaf-variegated mutants have contributed to the findings of maternal inheritance or have been used as genetic markers, these mutations and the responsible loci have been poorly understood at the molecular level. A comprehensive study of the leaf-variegated mutants is possible in Arabidopsis, since such mutants have been known and the cloning can be at relative ease as a model plant. Here I summarize recent progress on characterization of the Arabidopsis leaf-variegated mutants. Detailed analysis of the responsible loci revealed that variegation is caused by a defect in various metabolic pathways related to organelle functions. Thus, studies on these genes provide us with novel redundant mechanisms by which heteroplasmic organelles such as plastids and mitochondria can survive from an environmental stress.     

Isolation and characterization of perithecial development mutants in neurospora

The isolation and characterization of mutants that block perithecial development in Neurospora crassa are described. Several classes of mutants have been isolated after UV mutagenesis, and those that block perithecial development when used as the female (protoperithecial) component of a cross have been further characterized. These mutants fall into 29 complementation groups. Twelve of the 33 mutants block development at the protoperithecial stage; no other clustering of block points is observed. Many of the mutants show an altered vegetative growth rate as well; in several mutants this lower growth rate cosegregates with the female sterile phenotype. Only one mutant also blocks development of the perithecium when used as the conidial parent. None of the mutants are temperature sensitive; two can be suppressed by growth on a complete crossing medium. There is no indication that the mutants are at or in the mating-type locus, nor are any of the mutants mating-type specific. Genetic mosaics have been formed using mixtures of mutant and marked wild-type nuclei; no mutants are cell autonomous by this criterion. The significance of these results in terms of "developmental" mutants isolated in other organisms and in relation to models of eukaryotic development is discussed.     

Nongenomic effects of estradiol on aggression under short day photoperiods

In several vertebrate species, the effects of estrogens on male aggressive behavior can be modulated by environmental cues. In song sparrows and rodents, estrogens modulate aggression in the nonbreeding season or winter-like short days, respectively. The behavioral effects of estrogens are rapid, which generally is considered indicative of nongenomic processes. The current study further examined the hypothesis that estradiol acts nongenomically under short days by utilizing a protein synthesis inhibitor, cycloheximide (CX). Mice were housed in either short or long day photoperiods, and treated with an aromatase inhibitor. One hour before resident–intruder testing mice were injected with either CX or saline vehicle, and 30 min later were treated orally with either cyclodextrin conjugated estradiol or vehicle. Under short days, mice treated with estradiol showed a rapid decrease in aggressive behavior, independent of CX administration. CX alone had no effect on aggression. These results show that protein synthesis is not required for the rapid effects of estradiol on aggression, strongly suggesting that these effects are mediated by nongenomic processes. We also showed that estradiol suppressed c-fos immunoreactivity in the caudal bed nucleus of the stria terminalis under short days. No effects of estradiol on behavior or c-fos expression were observed in mice housed under long days. Previously we had also demonstrated that cage bedding influenced the directional effects of estrogens on aggression. Here, we show that the phenomenon of rapid action of estradiol on aggression under short days is a robust result that generalizes to different bedding conditions.     

Genetic and Physiological Characteristics of a Slow-Growing Circadian Clock Mutant of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa with a period length of about 25.8 hours (4 hr longer than wild type) has been isolated after mutagenesis of the band strain. This mutant, called frq-5, segregates as a single nuclear gene, maps near the centromere on linkage group III, and is unlinked to four previously described clock mutants clustered on linkage group VII R (Feldman and Hoyle 1973, 1976). frq-5 differs from the other clock mutants in at least two other respects: (1) it is recessive in heterokaryons, and (2) it grows at about 60% the rate of the parent band strain on both minimal and complete media. Double mutants between frq-5 and each of the other clock mutants show additivity of period length--two long period mutants produce a double mutant whose period length is longer than either of the two single mutants, while a long and a short period double mutant has an intermediate period length. Although slow growth and long periodicity of frq-5 have segregated together among more than 300 progeny, slow growth per se is not responsible for the long period, since all the double mutants have the slow growth characteristic of frq-5, but have period lengths both shorter and longer than wild type.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein.

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.