Induction Of C-fos mRNA Research Articles

Evidence for a lack of phasic inhibitory properties of habituated stressors on HPA axis responses in rats

This experiment tested the hypothesis that habituation to repeated stressor exposures is produced by phasic inhibitory influence on the neural circuitry that normally drives the paraventricular nucleus of the hypothalamus and subsequently the adrenocortical hormone response to psychological stress. Such a process would be expected to lower the acute response to a novel stressor when experienced concurrently with a habituated stressor. Rats were exposed to restraint or no stress conditions for 14 consecutive days. On the 15th day, the rats were exposed to the control condition (no stress), acute restraint, loud noise, or restraint and loud noise concurrently. Blood was taken and assayed for ACTH and corticosterone and brains were collected to examine c-fos messenger RNA expression in several brain areas. As predicted, the rats that received the same (homotypic) stressor repeatedly and again on the test day displayed low levels of ACTH and corticosterone, similar to the control conditions (i.e., showed habituation). All rats that received a single novel stressor on the test day, regardless of prior stress history, exhibited high levels of ACTH and corticosterone. The rats that received two novel stressors also displayed high levels of ACTH and corticosterone, but little evidence of additivity was observed. Importantly, when a novel stressor was concurrently given with a habituated stressor on the test day, no reduction of HPA axis response was observed when compared to previously habituated rats given only the novel stressor on the test day. In general, c-fos mRNA induction in several stress responsive brain areas followed the same patterns as the ACTH and corticosterone data. These data suggest that habituation of the adrenocortical hormone response to psychological stressors is not mediated by phasic inhibition of the effector system.

A food predictive cue must be attributed with incentive salience for it to induce c-fos mRNA expression in cortico-striatal-thalamic brain regions

A food predictive cue must be attributed with incentive salience for it to induce c-fos mRNA expression in cortico-striatal-thalamic brain regions

Repeated administration of methamphetamine blocked cholecystokinin-octapeptide injection-induced c-fos mRNA expression without change in capsaicin-induced junD mRNA expression in rat cerebellum

In the cerebellum, there are numerous cholecystokinin (CCK-8)-containing fibers. Since systemic CCK-8 injection-induced anxiety (psychological stress) activates the locus coeruleus cells that send mossy fiber inputs to the cerebellum, we examined whether systemic CCK-8 injections activate the rat and mouse cerebellum. First, injections of CCK-8 were found to induce c-fos mRNA expression in a vague patchy pattern that is different from single methamphetamine-induced Zebrin band-like c-fos mRNA expression, suggesting that the CCK-8 activating mossy fibers induce gene expression differently from the dopamine-containing mossy fibers in the ventral tegmental area. Second, since CCK-8 facilitates neural activity of dopamine in the midbrain, we examined whether repeated methamphetamine administration that induced behavioral sensitization had similar effects on the cerebellar CCK system. Repeated administration of methamphetamine suppressed the CCK-8-induced c-fos mRNA expression in the rat cerebellum. Third, capsaicin injections (physical stress) into a hind limb of the rat increased junD mRNA expression with no effect on c-fos mRNA expression, and repeated methamphetamine injections had no effect on the capsaicin-induced expression of junD mRNA. Fourth, either single injection of methamphetamine or CCK-8 to mice increased c-fos mRNA expression in the locus coeruleus, and so noradrenalin, but not dopamine, might interact with CCK-8-activating system. However, we considered the possibility unlikely. Thus, we conclude that repeated methamphetamine administration though dopamine selectively inhibits the c-fos mRNA expression after CCK-8 injection in the cerebellum.

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.

Quercetin Potentiates UVB-Induced c-Fos Expression: Implications for Its Use as a Chemopreventive Agent

Quercetin (Qu) is currently being investigated as a chemopreventive agent for several cancers, including nonmelanoma skin cancer induced by UV light. We previously reported that Qu degradation has important consequences on signaling and cell biology. In the current study, we report that Qu induces c-Fos mRNA and protein expression through activation of p38 and cAMP-responsive element binding protein (CREB), and Qu potentiates UVB-induced c-Fos expression. Inclusion of ascorbic acid (AA) in cell culture medium stabilizes Qu and completely prevents both Qu- and UVB-induced p38 and CREB activation, leading to a blockade of c-fos gene expression through reduced CREB/cAMP-responsive element binding. AA stabilizes c-Fos mRNA, increasing steady-state levels even when c-fos gene expression is suppressed, but this has no effect on c-Fos protein levels in either mock- or UVB-irradiated cells. We report that Qu blocks mammalian target of rapamycin signaling and inhibits c-Fos protein expression directly through this mechanism because cotreatment with Qu and AA resulted in the complete suppression of UVB-induced c-Fos protein expression even in the presence of significantly increased mRNA levels. We further confirmed that this was not due to increased protein turnover because inhibition of proteasome activity with MG-132 did not raise c-Fos protein levels in Qu+AA-treated cells. Together, these data indicate that although Qu has been reported to have some beneficial properties as a chemopreventive agent, it is also capable of inducing c-fos expression, a cellular event important for the promotion phase of tumor development, if it is not stabilized.

Real Time Monitoring of Endogenous Messenger RNA Using Linear Antisense Probe

In eukaryotic cells mRNA plays a key role in gene regulation. However, the function of mRNA is not fully understood because direct analysis of endogenous mRNAs in living cells has been difficult. We developed a method for the observation of endogenous mRNA in living cells using two fluorescently labeled linear antisense 2′O-methyl RNA oligonucleotides. When those two antisense probes, each is labeled with different fluorescent dyes, are hybridized to an adjacent sequence of the target mRNA, the distance between two fluorophores becomes close and FRET occurs.Here we applied linear antisense probes to the real time monitoring of endogenous mRNA, which will be useful in understanding the function of mRNA as well as the intracellular localization. First, two kinds of linear antisense probes were microinjected into the cytoplasm of living COS7 cells and the FRET signal from cells was recorded over time to examine the kinetics of the hybridization reaction with c-fos mRNA. The hybridization reaction of linear antisense probes proceeded quickly and time constants of linear antisense probe was estimated to be less than one minute. When using Molecular Beacon, the conventional probe for endogenous mRNAs, it took more than one hour to complete the hybridization. Next, the induction of c-fos mRNA in the cytoplasm of COS7 cells was investigated in real time using linear antisense probes. As a result, the elevation of c-fos mRNA expressed in the cytoplasm was observed within one hour after the stimulation with PMA (phorbol 12-myristate 13-acetate). In conclusion, we showed the linear antisense probes are advantageous in monitoring of mRNAs due to their prominent kinetics in hybridizing with target mRNAs in living cells.

The immediate early genes, c-fos, c-jun and AP-1, are early markers of platinum analogue toxicity in human proximal tubular cell primary cultures

These studies tested the hypothesis that c-fos, c-jun and AP-1 are early markers of platinum analogue-induced proximal tubule nephrotoxicity in primary rat proximal tubule (RPT) and human proximal tubule (HPT) cell cultures. The order of platinum analogue toxicity was cisplatin > transplatin > carboplatin in RPT and HPT cultures. Following a 2-h platinum analogue treatment, c-fos protein expression correlated with toxicity. Maximal c-fos protein levels were observed at 8-h (RPT) and 4-h (HPT) post-platinum analogue treatment. c-jun and AP-1 protein levels were maximal 4-h and 8-h, respectively, post cisplatin treatment in HPT cultures. In contrast, c-jun and AP-1 protein were not detected in RPT cultures. c-fos and c-jun mRNA levels were maximal at 60 and 120-min in RPT cell cultures, respectively, whilst c-fos and c-jun mRNA levels were maximal at 120-min in HPT cultures. Differences between HPT and RPT responses to cisplatin reveal inter-species differences associated with induction of c-fos and c-jun mRNA and protein, which in turn form the functional AP-1 complex prior to the onset of cellular toxicity. These studies highlight the utility of HPT cultures as an in vitro model system, and the potential of c-fos and c-jun as early markers of nephrotoxicity to screen therapeutic lead compounds.

Hypothalamic Injection of Oxyntomodulin Suppresses Circulating Ghrelin-Like Immunoreactivity

Ghrelin is a gastric peptide that regulates appetite and GH secretion. Circulating ghrelin levels are elevated by fasting and suppressed postprandially. However, the mechanisms regulating circulating ghrelin levels are unclear. Oxyntomodulin is an anorexic peptide hormone released from L cells in the gut. We investigated the effects of intracerebroventricular (icv) administration of oxyntomodulin on circulating ghrelin levels. The icv administration of 1, 3, or 10 nmol oxyntomodulin reduced circulating acylated and total (acylated and des-acylated) ghrelin 60 min after icv injection. Administration of 1 nmol oxyntomodulin directly into the arcuate nucleus of the hypothalamus significantly reduced total and acylated ghrelin levels, and administration of 3 nmol oxyntomodulin into the lateral ventricle induced c-fos mRNA expression in arcuate nucleus neurons expressing the glucagon-like peptide-1 (GLP-1) receptor. In a final study, the reduction in total ghrelin observed after icv injection of 3 nmol oxyntomodulin was blocked by coadministration of the GLP-1 receptor antagonist exendin (9-39). These studies suggest oxyntomodulin reduces peripheral ghrelin levels via GLP-1 receptor-dependent hypothalamic pathways. Postprandial release of anorexic gut hormones may thus act centrally to contribute to the postprandial reduction in circulating ghrelin.

Behavioral effects and pattern of brain c-fos mRNA induced by 2,5-dihydro-2,4,5-trimethylthiazoline, a component of fox feces odor in GAD67-GFP knock-in C57BL/6 mice

Predator odors, which are non-intrusive and naturalistic stressors of high ethological relevance, were used to study the neurobiology of innate fear in rodents. The present study investigates behavioral effects and the induction of c-fos mRNA in adult male predator naive mice caused by acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of the fox feces odor. On the behavioral level, TMT potently increased unconditioned freezing and decreased non-defensive grooming behavior. With quantitative real time PCR we established a strong TMT-induced activation in the bed nucleus of the stria terminalis (BNST) (eight-fold increase, p < 0.016) and in the ventral olfactory bulb (two-fold increase, p < 0.036). In contrast, no significant TMT-induced c-fos induction could be observed in the dorsal olfactory bulb or in the amygdala. Our results display robust fear responses of GAD67-GFP knock-in mice exposed to TMT and suggest that the ventral olfactory bulb and the BNST are strongly activated during the elicitation of fear through predator odor in these transgenic mice.

Signal transduction and functional selectivity of F15599, a preferential post‐synaptic 5‐HT1A receptor agonist

Activation of post-synaptic 5-HT(1A) receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT(1A) receptor agonist. F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. F15599 was highly selective for 5-HT(1A) receptors in binding experiments and in [(35)S]-GTPgammaS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT(1A) receptors. In cell lines expressing h5-HT(1A) receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT(1A) receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [(35)S]-GTPgammaS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated G(alphai) than G(alphao) activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT(1A) receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT(1A) receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT(1A) receptors in vivo almost as potently as F13714. F15599 showed a distinctive activation profiles for 5-HT(1A) receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT(1A) receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition.

Signal transduction and gene expression in cultured accessory olfactory bulb neurons

Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate α1 and α2 adrenergic receptors in combination with N-methyl- d-aspartic acid and tested expression of the immediate-early gene (IEG) c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, mitogen-activated protein kinase (MAPK) and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by protein kinase C and MAPK.

Serotonin Hyperinnervation Abolishes Seizure Susceptibility inOtx2Conditional Mutant Mice

The homeobox-containing transcription factor Otx2 is crucially involved in fate determination of midbrain neurons. Mutant mice, in which Otx2 was conditionally inactivated by a Cre recombinase expressed under the transcriptional control of the Engrailed1 (En1) gene (En1(cre/+); Otx2(flox/flox)), show a reduced number of dopaminergic neurons and an increased number of serotonergic neurons in the ventral midbrain. Despite these developmental anatomical alterations, En1(cre/+); Otx2(flox/flox) adult mice display normal motor function. Here, we further investigated the neurological consequences of Otx2 inactivation in adult En1(cre/+); Otx2(flox/flox) mice. Adult En1(cre/+); Otx2(flox/flox) mice showed increased serotonin (5-HT) levels in the pons, ventral midbrain, hippocampus (CA3 subfield), and cerebral cortex, as indicated by HPLC and immunohistochemistry. Conversely, SERT (5-HT transporter) levels were decreased in conditional mutant brains. As a consequence of this increased 5-HT hyperinnervation, En1(cre/+); Otx2(flox/flox) mice were resistant to generalized seizures induced by the glutamate agonist kainic acid (KA). Indeed, prolonged pretreatment of En1(cre/+); Otx2(flox/flox) mice with the 5-HT synthesis inhibitor para-chlorophenylalanine (pCPA) restored brain 5-HT content to control levels, fully reestablishing KA seizure susceptibility. Accordingly, c-fos mRNA induction after KA was restricted to the hippocampus in En1(cre/+); Otx2(flox/flox) mice, whereas a widespread c-fos mRNA labeling was observed throughout the brain of En1(cre/+); Otx2(flox/flox) mice pretreated with pCPA. These results clearly show that increased brain 5-HT levels are responsible for seizure resistance in En1(cre/+); Otx2(flox/flox) mice and confirm the important role of 5-HT in the control of seizure spread.

ΔFosB Mediates Epigenetic Desensitization of the c-fosGene After Chronic Amphetamine Exposure

The molecular mechanisms underlying the transition from recreational drug use to chronic addiction remain poorly understood. One molecule implicated in this process is DeltaFosB, a transcription factor that accumulates in striatum after repeated drug exposure and mediates sensitized behavioral responses to psychostimulants and other drugs of abuse. The downstream transcriptional mechanisms by which DeltaFosB regulates drug-induced behaviors are incompletely understood. We reported previously the chromatin remodeling mechanisms by which DeltaFosB activates the expression of certain genes; however, the mechanisms underlying DeltaFosB-mediated gene repression remain unknown. Here, we identify c-fos, an immediate early gene rapidly induced in striatum after acute psychostimulant exposure, as a novel downstream target that is repressed chronically by DeltaFosB. We show that accumulation of DeltaFosB in striatum after chronic amphetamine treatment desensitizes c-fos mRNA induction to a subsequent drug dose. DeltaFosB desensitizes c-fos expression by recruiting histone deacetylase 1 (HDAC1) to the c-fos gene promoter, which, in turn, deacetylates surrounding histones and attenuates gene activity. Accordingly, local knock-out of HDAC1 in striatum abolishes amphetamine-induced desensitization of the c-fos gene. In concert, chronic amphetamine increases histone H3 methylation on the c-fos promoter, a chromatin modification also known to repress gene activity, as well as expression levels of the H3 histone methyltransferase, KMT1A (lysine methyltransferase 1A, formerly SUV39H1). This study reveals a novel epigenetic pathway through which DeltaFosB mediates distinct transcriptional programs that may ultimately alter behavioral plasticity to chronic amphetamine exposure.

Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Food Intake in Mice Through Activation of the Hypothalamic Melanocortin System

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), exert anorexigenic activities. While alpha-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

The role of the posterior medial bed nucleus of the stria terminalis in modulating hypothalamic–pituitary–adrenocortical axis responsiveness to acute and chronic stress

The bed nucleus of the stria terminalis (BST) plays a prominent role in brain integration of acute responses to stressful stimuli. This study tests the hypothesis that the BST plays a complementary role in regulation of physiological changes associated with chronic stress exposure. Male Sprague-Dawley rats received bilateral ibotenate lesions or sham lesions of the posterior medial region of the BST (BSTpm), an area known to be involved in inhibition of HPA axis responses to acute stress. Chronic stress was induced by 14-day exposure to twice daily stressors in an unpredictable sequence (chronic variable stress, CVS). In the morning after the end of CVS, stressed and non-stressed controls were exposed to a novel restraint stress challenge. As previously documented, CVS caused adrenal hypertrophy, thymic involution, and attenuated body weight gain. None of these endpoints were affected by BSTpm lesions. Chronic stress exposure facilitated plasma corticosterone responses to the novel restraint stress and elevated CRH mRNA. Lesions of the BSTpm increased novel stressor-induced plasma ACTH and corticosterone secretion and enhanced c-fos mRNA induction in the paraventricular nucleus of the hypothalamus (PVN). In addition, lesion of the BSTpm resulted in an additive increase in CVS-induced facilitation of corticosterone responses and PVN CRH expression. Collectively these data confirm that the BSTpm markedly inhibits HPA responses to acute stress, but do not strongly support an additional role for this region in limiting HPA axis responses to chronic drive. The data further suggest that acute versus chronic stress integration are subserved by different brain circuitry.

Tenascin-C induction by cyclic strain requires integrin-linked kinase

Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition. In ILK wild-type but not ILK −/− fibroblasts, cyclic strain-induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MAL, a transcriptional co-activator that links actin assembly to gene expression. These findings support a role for RhoA in ILK-mediated mechanotransduction. Rescue of ILK −/− fibroblasts by expression of wild-type ILK restored these responses to cyclic strain. Mechanosensation is not entirely abolished in ILK −/− fibroblasts, since cyclic strain activated Erk-1/2 and PKB/Akt, and induced c-fos mRNA in these cells. Conversely, lysophosphatidic acid stimulated RhoA and induced both c-fos and tenascin-C mRNA in ILK −/− cells. Thus, the signaling pathways controlling tenascin-C expression are functional in the absence of ILK, but are not triggered by cyclic strain. Our results indicate that ILK is selectively required for the induction of specific genes by mechanical stimulation via RhoA-mediated pathways.

Histamine enhances the production of human β-defensin-2 in human keratinocytes

The anti-microbial peptide human beta-defensin-2 (hBD-2), produced by epidermal keratinocytes, plays pivotal roles in anti-microbial defense, inflammatory dermatoses, and wound repair. hBD-2 induces histamine release from mast cells. We examined the in vitro effects of histamine on hBD-2 production in normal human keratinocytes. Histamine enhanced TNF-alpha- or IFN-gamma-induced hBD-2 secretion and mRNA expression. Histamine alone enhanced transcriptional activities of NF-kappaB and activator protein-1 (AP-1) and potentiated TNF-alpha-induced NF-kappaB and AP-1 activities or IFN-gamma-induced NF-kappaB and STAT1 activities. Antisense oligonucleotides against NF-kappaB components p50 and p65, AP-1 components c-Jun and c-Fos, or H1 antagonist pyrilamine suppressed hBD-2 production induced by histamine plus TNF-alpha or IFN-gamma. Antisense oligonucleotide against STAT1 only suppressed hBD-2 production induced by histamine plus IFN-gamma. Histamine induced serine phosphorylation of inhibitory NF-kappaBalpha (IkappaBalpha) alone or together with TNF-alpha or IFN-gamma. Histamine induced c-Fos mRNA expression alone or together with TNF-alpha, whereas it did not further increase c-Jun mRNA levels enhanced by TNF-alpha. Histamine induced serine phosphorylation of STAT1 alone or together with IFN-gamma, whereas it did not further enhance IFN-gamma-induced tyrosine phosphorylation of STAT1. The histamine-induced serine phosphorylation of STAT1 was suppressed by MAPKK (MEK) inhibitor PD98059. These results suggest that histamine stimulates H1 receptor and potentiates TNF-alpha- or IFN-gamma-induced hBD-2 production dependent on NF-kappaB, AP-1, or STAT1 in human keratinocytes. Histamine may potentiate anti-microbial defense, skin inflammation, and wound repair via the induction of hBD-2.

Rhythmic Dependent Light Induction of Gonadotrophin‐Releasing Hormone‐I Expression and Activation of Dopaminergic Neurones within the Premammillary Nucleus of the Turkey Hypothalamus

Our previous studies using turkey hens have demonstrated that c-fos mRNA (a marker of neuronal activation) is expressed in gonadotrophin-releasing hormone-I (GnRH-I), vasoactive intestinal peptide (VIP) and dopamine (DA) neurones following electrical stimulation in the preoptic area. DA has been shown to have both stimulatory and inhibitory effects on the GnRH-I/luteinising hormone (LH), follicle-stimulating hormone (FSH) and VIP/prolactin (PRL) systems. To identify the DA neurones that mediate the stimulatory influences of photoperiod on the reproductive system, we examined c-fos mRNA induction in DA, GnRH-I, and VIP neurones in the turkey hypothalamus using a dark-interruption experimental design. A 30-min light period was provided to short day (6L : 18D) photosensitive turkeys at times when birds were responsive to light (14 h after first light) and at times when birds were unresponsive to light (8 h and 20 h after first light). The only area where DA neurones were activated when the birds were provided with light was in the nucleus premammillaris (PMM). The number of activated DA neurones was significantly greater when light was provided at 14 h (during the photoinducible phase) than at 8 h or 20 h. At 14 h, there was also an increase in the number of GnRH-I neurones activated in the area of the nucleus commissura pallii (nCPa), as well as an up-regulation of GnRH-I mRNA expression. No expression of c-fos mRNA was observed in VIP neurones in the nucleus infundibularis or up-regulation of VIP mRNA expression in any of the experimental light treatments. These results are the first evidence to demonstrate a relationship between the dopaminergic system in the PMM and the GnRH-I system in the nCPa during the photoinduction of avian reproductive activity.

Inducible cAMP early repressor splice variants ICER I and IIγ both repress transcription of c‐fos and chromogranin A

Inducible cAMP early repressor (ICER) splice variants are generated upon activation of an alternative, intronic promoter within the CREM gene. ICER is proposed to downregulate both its own expression, and the expression of other genes, containing cAMP-responsive promoter elements. To examine the biological function of the two ICER splice variants, I and IIgamma, in comparable cellular systems, we generated HEK 293 cell variants with controllable overexpression of either ICER I or IIgamma. These two splice variants contain two different variants of DNA binding domains. Overexpression of either ICER I or IIgamma strongly represses CRE-driven reportergene transcription but not AP1- or NFkappaB-driven transcription. Thus, high specificity is maintained even at ICER overexpression. We here show that both ICER I and IIgamma repress Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated c-fos mRNA induction with similar efficiency, indicating that both splice variants play an important role in modulating PACAP-mediated transcriptional activation of the c-fos gene. ICER I and IIgamma also repress cAMP-mediated activation of chromogranin A (CgA), indicating that these splice variants may function as negative feedback regulators in CgA synthesis. The proliferation rate was not altered in cells overexpressing ICER I or IIgamma. Thus, in the epithelial cells HEK 293, ICER I and IIgamma splice variants seem to exert similar biological function.

Bed nucleus of the stria terminalis subregions differentially regulate hypothalamic-pituitary-adrenal axis activity: implications for the integration of limbic inputs.

Limbic and cortical neurocircuits profoundly influence hypothalamic-pituitary-adrenal (HPA) axis responses to stress yet have little or no direct projections to the hypothalamic paraventricular nucleus (PVN). Numerous lines of evidence suggest that the bed nucleus of the stria terminalis (BST) is well positioned to relay limbic information to the PVN. The BST comprises multiple anatomically distinct nuclei, of which some are known to receive direct limbic and/or cortical input and to heavily innervate the PVN. Our studies test the hypothesis that subregions of the BST differentially regulate HPA axis responses to acute stress. Male Sprague Dawley rats received bilateral ibotenate lesions, targeting either the principal nucleus in the posterior BST or the dorsomedial/fusiform nuclei in the anteroventral BST. Posterior BST lesions elevated plasma ACTH and corticosterone in response to acute restraint stress, increased stress-induced PVN c-fos mRNA, and elevated PVN corticotropin-releasing hormone (CRH) and parvocellular arginine vasopressin (AVP) mRNA expression relative to sham-lesion animals. In contrast, anterior BST lesions attenuated the plasma corticosterone response and decreased c-fos mRNA induction in the PVN but did not affect CRH and parvocellular AVP mRNA expression in the PVN. These data suggest that posterior BST nuclei are involved in inhibition of the HPA axis, whereas the anteroventral BST nuclei are involved in HPA axis excitation. The results indicate that the BST contains functional subdomains that play different roles in integrating and processing limbic information in response to stress and further suggest that excitatory as well as inhibitory limbic information is funneled through these important cell groups.