KDM2B/FBXL10 targets c-Fos for ubiquitylation and degradation in response to mitogenic stimulation.

KDM2B (also known as FBXL10) controls stem cell self-renewal, somatic cell reprogramming and senescence, and tumorigenesis. KDM2B contains multiple functional domains, including a JmjC domain that catalyzes H3K36 demethylation and a CxxC zinc-finger that recognizes CpG islands and recruits the polycomb repressive complex 1. Here, we report that KDM2B, via its F-box domain, functions as a subunit of the CUL1-RING ubiquitin ligase (CRL1/SCF(KDM2B)) complex. KDM2B targets c-Fos for polyubiquitylation and regulates c-Fos protein levels. Unlike the phosphorylation of other SCF (SKP1-CUL1-F-box)/CRL1 substrates that promotes substrates binding to F-box, epidermal growth factor (EGF)-induced c-Fos S374 phosphorylation dissociates c-Fos from KDM2B and stabilizes c-Fos protein. Non-phosphorylatable and phosphomimetic mutations at S374 result in c-Fos protein which cannot be induced by EGF or accumulates constitutively and lead to decreased or increased cell proliferation, respectively. Multiple tumor-derived KDM2B mutations impaired the function of KDM2B to target c-Fos degradation and to suppress cell proliferation. These results reveal a novel function of KDM2B in the negative regulation of cell proliferation by assembling an E3 ligase to targeting c-Fos protein degradation that is antagonized by mitogenic stimulations

Immunohistochemical C-FOS expression and autoradiography to study galnin/neuropeptide y Y1 receptor-receptor interactions in the amygdala

We have shown Galanin(GAL)/Neuropeptide Y Y1 receptor(Y1) interactions in the nucleus tractus solitarius and the arcuate nucleus. Since both peptides play an important role in mood disorders, the aim of this work was to study GAL/Y1 interactions in the amygdala(AMY), key nucleus for fear, mood, and motivation. We have combined the analysis of the expression of c-Fos immunoreactivity(c-Fos IR) with an autoradiographic study in the AMY. Groups of anaesthetized rats (n=4) received intracerebroventricular injections(icv) of GAL(3nmol) and the Y1 agonist Leu31-Pro34NPY(3nmol) alone or in combination, and were sacrificed 90 minutes after the injections. Immunohistochemical detection of c-Fos protein(1:5000) in AMY sections and stereological analysis were performed in: Basal(BA), lateral(LA), Central [lateral capsular subdivision(CeC), lateral intermediate subdivision(CeI), medial subdivision(CeM)] and the medial paracapsular intercalated(ITC) subnuclei of the AMY. For Autoradiography, rats (n=6) were sacrificed 15 minutes after icv injections of GAL(3nmol) and AMY sections were incubated with Y1 agonist [125I]-Leu31-Pro34-PPY (25 pM). Autoradiograms were analyzed using the NIH image analysis system. Student’s unpaired t- test and ANOVA followed by Newman-Keuls were used, respectively. We observed within the AMY that GAL increased c-Fos IR in ITC and CeC; the Y1 agonist induced both, an increase of c-Fos IR in BA and CeC and a decrease of c-Fos IR in LA and ITC. The coadministration of both peptides induced a specific effect in the ITC, significantly decreasing the c-Fos expression (P<0,05) induced by GAL or the Y1 agonist alone. Moreover, we observed that GAL significantly increased (p<0,05) the Y1 receptor agonist binding [I125]Leu31Pro34-PYY in the AMY. These results demonstrate an interaction between GAL and Y1 at the cellular and receptor level in the AMY and suggest that endogenous GAL and NPY system might interact to regulate emotional behaviours.Spanish CVI6476, TV3-Marató 090130/31/32 and Universidad de Málaga (Campus de Excelencia Internacional Andalucía Tech

ΔFosB Regulates Gene Expression and Cognitive Dysfunction in a Mouse Model of Alzheimer\u27s Disease.

Alzheimer\u27s disease (AD) is characterized by cognitive decline and 5- to 10-fold increased seizure incidence. How seizures contribute to cognitive decline in AD or other disorders is unclear. We show that spontaneous seizures increase expression of ΔFosB, a highly stable Fos-family transcription factor, in the hippocampus of an AD mouse model. ΔFosB suppressed expression of the immediate early gene c-Fos, which is critical for plasticity and cognition, by binding its promoter and triggering histone deacetylation. Acute histone deacetylase (HDAC) inhibition or inhibition of ΔFosB activity restored c-Fos induction and improved cognition in AD mice. Administration of seizure-inducing agents to nontransgenic mice also resulted in ΔFosB-mediated suppression of c-Fos, suggesting that this mechanism is not confined to AD mice. These results explain observations that c-Fos expression increases after acute neuronal activity but decreases with chronic activity. Moreover, these results indicate a general mechanism by which seizures contribute to persistent cognitive deficits, even during seizure-free periods

Multiple mechanisms of growth hormone-regulated gene transcription

Diverse physiological actions of growth hormone (GH) are mediated by changes in gene transcription. Transcription can be regulated at several levels, including post-translational modification of transcription factors, and formation of multiprotein complexes involving transcription factors, co-regulators and additional nuclear proteins; these serve as targets for regulation by hormones and signaling pathways. Evidence that GH regulates transcription at multiple levels is exemplified by analysis of the proto-oncogene c-fos. Among the GH-regulated transcription factors on c-fos, C/EBPbeta appears to be key, since depletion of C/EBPbeta by RNA interference blocks the stimulation of c-fos by GH. The phosphorylation state of C/EBPbeta and its ability to activate transcription are regulated by GH through MAPK and PI3K/Akt-mediated signaling cascades. The acetylation of C/EBPbeta also contributes to its ability to activate c-fos transcription. These and other post-translational modifications of C/EBPbeta appear to be integrated for regulation of transcription by GH. The formation of nuclear proteins into complexes associated with DNA-bound transcription factors is also regulated by GH. Both C/EBPbeta and the co-activator p300 are recruited to c-fos in response to GH, altering c-fos promoter activation. In addition, GH rapidly induces spatio-temporal re-localization of C/EBPbeta within the nucleus. Thus, GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins. Similar integration involving other transcription factors, including Stats, appears to be a feature of regulation by GH of other gene targets.Fil: Ceseña, Teresa I.. University of Michigan; Estados UnidosFil: Cui, Tracy Xiao. University of Michigan; Estados UnidosFil: Piwien Pilipuk, Graciela. Fundación Instituto Leloir; ArgentinaFil: Kaplani, Julianne. University of Michigan; Estados UnidosFil: Calinescu, Anda Alexandra. Michigan State University; Estados UnidosFil: Huo, Jeffrey S.. University of Michigan; Estados UnidosFil: Iñiguez Lluhí, Jorge A.. University of Michigan; Estados UnidosFil: Kwok, Roland. University of Michigan; Estados UnidosFil: Schwartz, Jessica. University of Michigan; Estados Unido

Medial prefrontal cortex neuronal activation and synaptic alterations after stress-induced reinstatement of palatable food seeking: a study using c-fos-GFP transgenic female rats

Relapse to maladaptive eating habits during dieting is often provoked by stress and there is evidence for a role of ovarian hormones in stress responses and feeding. We studied the role of these hormones in stress-induced reinstatement of food seeking and medial prefrontal cortex (mPFC) neuronal activation in c-fos-GFP transgenic female rats, which express GFP in strongly activated neurons. Food-restricted ovariectomized or sham-operated c-fos-GFP rats were trained to lever-press for palatable food pellets. Subsequently, lever-pressing was extinguished and reinstatement of food seeking and mPFC neuronal activation was assessed after injections of the pharmacological stressor yohimbine (0.5-2 mg/kg) or pellet priming (1-4 noncontingent pellets). Estrous cycle effects on reinstatement were also assessed in wild-type rats. Yohimbine- and pellet-priming-induced reinstatement was associated with Fos and GFP induction in mPFC; both reinstatement and neuronal activation were minimally affected by ovarian hormones in both c-fos-GFP and wild-type rats. c-fos-GFP transgenic rats were then used to assess glutamatergic synaptic alterations within activated GFP-positive and nonactivated GFP-negative mPFC neurons following yohimbine-induced reinstatement of food seeking. This reinstatement was associated with reduced AMPA receptor/NMDA receptor current ratios and increased paired-pulse facilitation in activated GFP-positive but not GFP-negative neurons. While ovarian hormones do not appear to play a role in stress-induced relapse of food seeking in our rat model, this reinstatement was associated with unique synaptic alterations in strongly activated mPFC neurons. Our paper introduces the c-fos-GFP transgenic rat as a new tool to study unique synaptic changes in activated neurons during behavio

Expression Of The Proto-oncogene C-fos Following Electrical Kindling In The Rat

Kindling is an example of permanent change in brain function that results from repeated elicitation of epileptiform neural activity. The molecular/genetic mechanisms by which kindling is produced and maintained are just beginning to be addressed. Recently two proto-oncogenes, c-fos and c-Ha-ras, have been identified as possible mediators of intracellular signal transduction and may mediate the kindling process. Additionally, c-fos, has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes that occur during kindling. The objective of the present study was to: (i) further investigate the enhancement of c-fos and c-Ha-ras levels following kindled seizures and (ii) investigate the role of c-fos in the plastic changes underlying kindling.;In this study, male hooded rats, were electrically kindled and the resulting c-fos and c-Ha-ras gene expression was quantified using Northern blot analysis. The results indicated that: (i) c-fos was constitutively expressed in forebrain and cerebellum, (ii) basal levels of c-fos were equivalent in naive and in fully kindled rats that has been seizure free for 3 weeks, (iii) an amygdala-pyriform kindled seizure massively and transiently increased c-fos levels, (iv) kindled seizure enhancement of c-fos was observed throughout forebrain and cerebellum, (v) enhancement of AD duration corresponded to enhanced c-fos levels, (vi) enhanced c-fos levels were observed in the amygdala-pyriform contralateral to the kindled site, and the enhancement did not depend on the occurrence of Ad in the contralateral amygdala-pyriform, (vii) electrical stimulations not resulting in AD increased c-fos levels, (viii) c-fos levels were increased by control stimulation, (ix) c-fos induction was partially refractory, (x) PTZ caused c-fos induction independent of a motor convulsion, and (xi) kindled seizures did not alter the expression of c-Ha-ras.;The most parsimonious explanation that accounts for the observations made in this study is that c-fos was expressed simply as a consequence of neural activity and not exclusively due to the specific neural activity (afterdischarge) required for kindling

Delayed c-Fos activation in human cells triggers XPF induction and an adaptive response to UVC-induced DNA damage and cytotoxicity

The oncoprotein c-Fos has been commonly found differently expressed in cancer cells. Our previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here, we demonstrate that in human diploid fibroblasts UV-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained upregulation of c-Fos goes along with transcriptional stimulation of the NER gene xpf, which harbors an AP-1 binding site in the promoter. Data gained on c-Fos knockdown and c-Fos overexpressing human cells provide evidence that c-Fos/AP-1 stimulates upregulation of XPF, thereby increasing the cellular repair capacity protecting from UVC-induced DNA damage. When these cells are pre-exposed to a low non-toxic UVC dose and challenged with a subsequent high dose of UVC irradiation, they show accelerated repair of UVC-induced DNA adducts and reduced cell kill. The data indicate a protective role of c-Fos induction by triggering an adaptive response pathway

The kinase MSK1 is required for induction of c-fos by lysophosphatidic acid in mouse embryonic stem cells

BACKGROUND: The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos. RESULTS: Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins. CONCLUSION: These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos

Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope

Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C

Sympathetic-correlated c-Fos expression in the neonatal rat spinal cord in vitro

An isolated thoracic spinal cord of the neonatal rat in vitro spontaneously generates sympathetic nerve discharge (SND) at ~25°C, but it fails in SND genesis at ≤ 10°C. Basal levels of the c-Fos expression in the spinal cords incubated at ≤ 10°C and ~25°C were compared to determine the anatomical substrates that might participate in SND genesis. Cells that exhibited c-Fos immunoreactivity were virtually absent in the spinal cords incubated at ≤ 10°C. However, in the spinal cords incubated at ~25°C, c-Fos-positive cells were found in the dorsal laminae, the white matter, lamina X, and the intermediolateral cell column (IML). Cell identities were verified by double labeling of c-Fos with neuron-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), or choline acetyltransferase (ChAT). The c-Fos-positive cells distributed in the white matter and lamina X were NeuN-negative or GFAP-positive and were glial cells. Endogenously active neurons showing c-Fos and NeuN double labeling were scattered in the dorsal laminae and concentrated in the IML. Double labeling of c-Fos and ChAT confirmed the presence of active sympathetic preganglionic neurons (SPNs) in the IML. Suppression of SND genesis by tetrodotoxin (TTX) or mecamylamine (MECA, nicotinic receptor blocker) almost abolished c-Fos expression in dorsal laminae, but only mildly affected c-Fos expression in the SPNs. Therefore, c-Fos expression in some SPNs does not require synaptic activation. Our results suggest that spinal SND genesis is initiated from some spontaneously active SPNs, which are capable of TTX- or MECA-resistant c-Fos expression