Long Noncoding RNA Fos Downstream Transcript Is Developmentally Dispensable but Vital for Shaping the Poststroke Functional Outcome.

Background and Purpose: Stroke induces the expression of several long noncoding RNAs in the brain. However, their functional significance in poststroke outcome is poorly understood. We recently observed that a brain-specific long noncoding RNA called Fos downstream transcript (FosDT) is induced rapidly in the rodent brain following focal ischemia. Using FosDT knockout rats, we presently evaluated the role of FosDT in poststroke brain damage. Methods: FosDT knockout rats were generated using CRISPR-Cas9 genome editing on a Sprague-Dawley background. Male and female FosDT−/− and FosDT+/+ cohorts were subjected to transient middle cerebral artery occlusion. Postischemic sensorimotor deficits were evaluated between days 1 and 7 and lesion volume on day 7 of reperfusion. The developmental expression profile of FosDT was determined with real-time polymerase chain reaction and mechanistic implications of FosDT in the ischemic brain were conducted with RNA-sequencing analysis and immunostaining of pathological markers. Results: FosDT expression is developmentally regulated, with the adult cerebral cortex showing significantly higher FosDT expression than neonates. FosDT−/− rats did not show any anomalies in growth and development, fertility, brain cytoarchitecture, and cerebral vasculature. However, when subjected to transient focal ischemia, FosDT−/− rats of both sexes showed enhanced sensorimotor recovery and reduced brain damage. RNA-sequencing analysis showed that improved poststroke functional outcome in FosDT−/− rats is partially associated with curtailed induction of inflammatory genes, reduced apoptosis, mitochondrial dysfunction, and oxidative stress. Conclusions: Our study shows that FosDT is developmentally dispensable, mechanistically important, and a functionally promising target to reduce ischemic brain damage and facilitate neurological recovery.

Circuit-specific hippocampal ΔFosB underlies resilience to stress-induced social avoidance.

Chronic stress is a key risk factor for mood disorders like depression, but the stress-induced changes in brain circuit function and gene expression underlying depression symptoms are not completely understood, hindering development of novel treatments. Because of its projections to brain regions regulating reward and anxiety, the ventral hippocampus is uniquely poised to translate the experience of stress into altered brain function and pathological mood, though the cellular and molecular mechanisms of this process are not fully understood. Here, we use a novel method of circuit-specific gene editing to show that the transcription factor ΔFosB drives projection-specific activity of ventral hippocampus glutamatergic neurons causing behaviorally diverse responses to stress. We establish molecular, cellular, and circuit-level mechanisms for depression- and anxiety-like behavior in response to stress and use circuit-specific gene expression profiling to uncover novel downstream targets as potential sites of therapeutic intervention in depression.

Fra1 Controls Rheumatoid Factor Autoantibody Production by Bone Marrow Plasma Cells and the Development of Autoimmune Bone Loss.

Next to proinflammatory cytokines, autoimmunity has been identified as a key trigger for osteoclast activation and bone loss. IgG-rheumatoid factor (IgG-RF) immune complexes, which are present in patients with rheumatoid arthritis, were shown to boost osteoclast differentiation. To date, the regulation of IgG-RF production in the absence of inflammatory triggers is unknown. Herein, we describe Fra1 as a key checkpoint that controls IgG-RF production by plasma cells and regulates autoimmune-mediated bone loss. Fra1 deficiency in B cells (Fra1ΔBcell ) led to increased IgG1-producing bone marrow plasma cells, enhanced IgG-RF production, and increased bone loss associated with elevated osteoclast numbers after immunization. The effect of IgG-RF on osteoclasts in vitro and on osteoclasts associated with bone loss in vivo was dependent on FcγR, especially FcγR3. Furthermore, immunization of WT mice with T-cell-dependent antigens induced a significant and robust decrease in Fra1 expression in bone marrow B cells, which was followed by increased IgG1 production and the induction of osteoclast-mediated bone loss. Overall, these data identify Fra1 as a key mediator of IgG-RF production and autoimmune-mediated bone loss. © 2019 American Society for Bone and Mineral Research.

Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts.

Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone-resorbing capacity on c-Fos-deficient Sh3bp2KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c-Fos-deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research.

FOSL1 Inhibits Type I Interferon Responses to Malaria and Viral Infections by Blocking TBK1 and TRAF3/TRIF Interactions.

Innate immune response plays a critical role in controlling invading pathogens, but such an immune response must be tightly regulated. Insufficient or overactivated immune responses may lead to harmful or even fatal consequences. To dissect the complex host-parasite interactions and the molecular mechanisms underlying innate immune responses to infections, here we investigate the role of FOS-like antigen 1 (FOSL1) in regulating the host type I interferon (IFN-I) response to malaria parasite and viral infections. FOSL1 is known as a component of a transcription factor but was recently implicated in regulating the IFN-I response to malaria parasite infection. Here we show that FOSL1 can act as a negative regulator of IFN-I signaling. Upon stimulation with poly(I:C), malaria parasite-infected red blood cells (iRBCs), or vesicular stomatitis virus (VSV), FOSL1 "translocated" from the nucleus to the cytoplasm, where it inhibited the interactions between TNF receptor-associated factor 3 (TRAF3), TIR domain-containing adapter inducing IFN-ß (TRIF), and Tank-binding kinase 1 (TBK1) via impairing K63-linked polyubiquitination of TRAF3 and TRIF. Importantly, FOSL1 knockout chimeric mice had lower levels of malaria parasitemia or VSV titers in peripheral blood and decreased mortality compared with wild-type (WT) mice. Thus, our findings have identified a new role for FOSL1 in negatively regulating the host IFN-I response to malaria and viral infections and have identified a potential drug target for controlling malaria and other diseases. IMPORTANCE: Infections of pathogens can trigger vigorous host immune responses, including activation and production of type I interferon (IFN-I). In this study, we investigated the role of FOSL1, a molecule previously known as a transcription factor, in negatively regulating IFN-I responses to malaria and viral infections. We showed that FOSL1 was upregulated and translocated into the cytoplasm of cells after stimulation for IFN-I production. FOSL1 could affect TRAF3 and TRIF ubiquitination and consequently impaired the association of TRAF3, TRIF, and TBK1, leading to inhibition of IFN-I signaling. In vivo experiments with FOSL1 knockout chimeric mice further validated the negative role of FOSL1 in IFN-I production and antimicrobial responses. This report reveals a new functional role for FOSL1 in IFN-I signaling and dissects the mechanism by which FOSL1 regulates IFN-I responses to malaria and viral infections, which can be explored as a potential drug target for disease control and management.

c-Jun Is Required for Nuclear Factor-κB-Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages.

Previously, we have reported that Fos-related antigen-1 (Fra-1) transcription factor promotes LPS-induced acute lung injury and mortality, and that LPS-induced Fra-1 expression in the lung occurs predominantly in alveolar macrophages. Nuclear factor-κB (NF-κB) and c-Jun transcription factors play key roles in modulating inflammatory and immune responses induced by infectious and non-infectious insults. Here, we report that NF-κB and c-Jun coregulate Fra-1 induction by LPS in alveolar macrophages and that this regulation occurs through both the NF-κB and the extracellular signal-regulated protein kinase (ERK) signaling pathways. Transient transfections with Fra-1 promoter-reporter constructs and inhibitor studies revealed that the transcriptional activation of Fra-1 by LPS in alveolar macrophages is mediated by NF-κB and ERK1/2 signaling. Importantly, chromatin immunoprecipitation assays revealed the recruitment of c-Jun and NF-κB to the endogenous Fra-1 promoter after LPS stimulation. We found that inhibition of ERK1/2 signaling reduced LPS-stimulated c-Jun and NF-κB recruitment to the promoter. Likewise, NF-κB inhibitor blocked LPS-induced NF-κB and c-Jun binding to the promoter. ERK1/2 inhibition had no effect on c-Jun activation but suppressed LPS-stimulated NF-κB phosphorylation. Finally, functional assays showed reduced levels of LPS-stimulated NF-κB regulated proinflammatory IL-1ß and macrophage inflammatory protein-1α expression and increased antiinflammatory IL-10 expression in lung alveolar macrophages of Fra-1-null mice in vivo. Thus, our studies indicate that NF-κB and c-Jun coregulate LPS-induced Fra-1 transcription and that Fra-1 selectively modulates LPS-stimulated inflammatory cytokine expression in lung alveolar macrophages during inflammatory lung injury.

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen.

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-D-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ∆FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ∆FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-∆FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.

Osteocyte-directed bone demineralization along canaliculi.

The mammalian skeleton stores calcium and phosphate ions in bone matrix. Osteocytes in osteocyte lacunae extend numerous dendrites into canaliculi less than a micron in diameter and which are distributed throughout bone matrix. Although osteoclasts are the primary bone-resorbing cells, osteocytes also reportedly dissolve hydroxyapatite at peri-lacunar bone matrix. However, robust three-dimensional evidence for peri-canalicular bone mineral dissolution has been lacking. Here we applied a previously reported Talbot-defocus multiscan tomography method for synchrotron X-ray microscopy and analyzed the degree of bone mineralization in mouse cortical bone around the lacuno-canalicular network, which is connected both to blood vessels and the peri- and endosteum. We detected cylindrical low mineral density regions spreading around canaliculi derived from a subset of osteocytes. Transmission electron microscopy revealed both intact and demineralized bone matrix around the canaliculus. Peri-canalicular low mineral density regions were also observed in osteopetrotic mice lacking osteoclasts, indicating that osteoclasts are dispensable for peri-canalicular demineralization. These data suggest demineralization can occur from within bone through the canalicular system, and that peri-canalicular demineralization occurs not uniformly but directed by individual osteocytes. Blockade of peri-canalicular demineralization may be a therapeutic strategy to increase bone mass and quality.

Therapy-induced tumour secretomes promote resistance and tumour progression.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

Expression profiling of genes regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary fibrosis.

BACKGROUND: The Fra-1/AP-1 transcription factor regulates the expression of genes controlling various processes including migration, invasion, and survival as well as extracellular remodeling. We recently demonstrated that loss of Fra-1 leads to exacerbated bleomycin-induced pulmonary fibrosis, accompanied by enhanced expression of various inflammatory and fibrotic genes. To better understand the molecular mechanisms by which Fra-1 confers protection during bleomycin-induced lung injury, genome-wide mRNA expression profiling was performed. RESULTS: We found that Fra-1 regulates gene expression programs that include: 1) several cytokines and chemokines involved in inflammation, 2) several genes involved in the extracellular remodeling and cell adhesion, and 3) several genes involved in programmed cell death. CONCLUSION: Loss of Fra-1 leads to the enhanced expression of genes regulating inflammation and immune responses and decreased the expression of genes involved in apoptosis, suggesting that this transcription factor distinctly modulates early pro-fibrotic cellular responses.

fosB-null mice display impaired adult hippocampal neurogenesis and spontaneous epilepsy with depressive behavior.

Patients with epilepsy are at high risk for major depression relative to the general population, and both disorders are associated with changes in adult hippocampal neurogenesis, although the mechanisms underlying disease onset remain unknown. The expression of fosB, an immediate early gene encoding FosB and ΔFosB/Δ2ΔFosB by alternative splicing and translation initiation, is known to be induced in neural progenitor cells within the subventricular zone of the lateral ventricles and subgranular zone of the hippocampus, following transient forebrain ischemia in the rat brain. Moreover, adenovirus-mediated expression of fosB gene products can promote neural stem cell proliferation. We recently found that fosB-null mice show increased depressive behavior, suggesting impaired neurogenesis in fosB-null mice. In the current study, we analyzed neurogenesis in the hippocampal dentate gyrus of fosB-null and fosB(d/d) mice that express ΔFosB/Δ2ΔFosB but not FosB, in comparison with wild-type mice, alongside neuropathology, behaviors, and gene expression profiles. fosB-null but not fosB(d/d) mice displayed impaired neurogenesis in the adult hippocampus and spontaneous epilepsy. Microarray analysis revealed that genes related to neurogenesis, depression, and epilepsy were altered in the hippocampus of fosB-null mice. Thus, we conclude that the fosB-null mouse is the first animal model to provide a genetic and molecular basis for the comorbidity between depression and epilepsy with abnormal neurogenesis, all of which are caused by loss of a single gene, fosB.

Fra-1/AP-1 transcription factor negatively regulates pulmonary fibrosis in vivo.

The Fra-1/AP-1 transcription factor plays a key role in tumor epithelial cell progression; however, its role in pathogenic lung fibrosis remains unclear. In the present study, using a genetic approach (Fra-1 deficient mice), we have demonstrated a novel regulatory (protective) role for Fra-1 in lung fibrosis. We found greater levels of progressive interstitial fibrosis, characterized by increased levels of inflammation, collagen accumulation, and profibrotic and fibrotic gene expression in the lungs of Fra-1(Δ/Δ) mice than in those of Fra-1(+/+) mice following bleomycin treatment. Fra-1 knockdown in human lung epithelial cells caused the upregulation of mesenchymal marker N-cadherin, concomitant with a downregulation of the epithelial phenotype marker E-cadherin, under basal conditions and in response to bleomycin and TGF-ß1. Furthermore, Fra-1 knockdown caused an enhanced expression of type 1 collagen and the downregulation of collagenase (MMP-1 and MMP-13) gene expression in human lung epithelial cells. Collectively, our findings demonstrate that Fra-1 mediates anti-fibrotic effects in the lung through the modulation of proinflammatory, profibrotic and fibrotic gene expression, and suggests that the Fra-1 transcription factor may be a potential target for pulmonary fibrosis, a progressive disorder with poor prognosis and treatment.

Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17.

Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α-converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs.

The restructuring of dopamine receptor subtype gene transcripts in c-fos KO mice.

Although c-Fos protein is one of the principal molecules in intracellular signaling, c-fos gene disruption is associated with alterations in neuronal functions that do not correspond to its importance in function. The aim of the study was to evaluate the changes of dopaminergic system together with acetylcholinesterase (AChE) in c-fos disruption (KO). KO male mice showed an increase in D1-like receptor (279% of WT) and D2-like receptor (345% of WT) binding sites in the cortex. On the gene expression level (assessed by real-time PCR), lower quantities of D1R-mRNA (0.64) and D5R-mRNA (0.6) were found in females when compared to males in the frontal cortex, higher D2R-mRNA in the parietal (1.43) and temporal (2.64) cortex and lower AChE-mRNA (0.67). On the contrary, female striatum contained higher level of D2R-mRNA (1.62) and AChE-mRNA (1.57) but lower level of D3R-mRNA (0.73). Hypothalamic D1R-mRNA, D2R-mRNA and D4R-mRNA were higher in females (1.38, 1.63, and 1.68, respectively). Disruption of c-fos increased selectively D5R-mRNA (1.31) in male parietal cortex, D2R-mRNA (1.72) in male temporal cortex, and cerebellar D2R-mRNA in both males (1.43) and females (1.42), respectively. In females, we found rather decrease in DR-mRNA. Multiple correlations in mRNA quantities (in WT mice) were found, which changed considerably upon c-fos KO. Main interactions in WT were inter-regional, CNS of KO underwent an extensive restructuring comprising intraregional interactions in the frontal cortex, hypothalamus, and cerebellum. These changes in DR (between others) could be considered as one of the adaptive mechanisms in c-fos KO mice.

Oxidant-induced cell death and Nrf2-dependent antioxidative response are controlled by Fra-1/AP-1.

AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1(Δ/Δ)) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H(2)O(2)- and diquat-induced cell death, compared to their wild-type (Fra-1(+/+)) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1(Δ/Δ) cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1(Δ/Δ) cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses.

The transmembrane adaptor protein, linker for activation of T cells (LAT), regulates RANKL-induced osteoclast differentiation.

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein we investigate the role of the transmembrane adaptor proteins in RANKL-induced osteoclastogenesis. LAT positively regulates osteoclast differentiation and is up-regulated by RANKL via c-Fos and NFATc1, whereas LAB and LIME act as negative modulators of osteoclastogenesis. In addition, silencing of LAT by RNA interference or overexpression of a LAT dominant negative in bone marrow-derived macrophage cells attenuates RANKL-induced osteoclast formation. Furthermore, LAT is involved in RANKL-induced PLC(γ) activation and NFATc1 induction. Thus, our data suggest that LAT acts as a positive regulator of RANKL-induced osteoclastogenesis.

Impaired vibration of auditory ossicles in osteopetrotic mice.

In the middle ear, a chain of three tiny bones (ie, malleus, incus, and stapes) vibrates to transmit sound from the tympanic membrane to the inner ear. Little is known about whether and how bone-resorbing osteoclasts play a role in the vibration of auditory ossicles. We analyzed hearing function and morphological features of auditory ossicles in osteopetrotic mice, which lack osteoclasts because of the deficiency of either cytokine RANKL or transcription factor c-Fos. The auditory brainstem response showed that mice of both genotypes experienced hearing loss, and laser Doppler vibrometry revealed that the malleus behind the tympanic membrane failed to vibrate. Histological analysis and X-ray tomographic microscopy using synchrotron radiation showed that auditory ossicles in osteopetrotic mice were thicker and more cartilaginous than those in control mice. Most interestingly, the malleal processus brevis touched the medial wall of the tympanic cavity in osteopetrotic mice, which was also the case for c-Src kinase-deficient mice (with normal numbers of nonresorbing osteoclasts). Osteopetrotic mice showed a smaller volume of the tympanic cavity but had larger auditory ossicles compared with controls. These data suggest that osteoclastic bone resorption is required for thinning of auditory ossicles and enlargement of the tympanic cavity so that auditory ossicles vibrate freely.

FosB null mutant mice show enhanced methamphetamine neurotoxicity: potential involvement of FosB in intracellular feedback signaling and astroglial function.

Previous studies show that (1) two members of fos family transcription factors, c-Fos and FosB, are induced in frontal brain regions by methamphetamine; (2) null mutation of c-Fos exacerbates methamphetamine-induced neurotoxicity; and (3) null mutation of FosB enhances behavioral responses to cocaine. Here we sought a role of FosB in responses to methamphetamine by studying FosB null mutant (-/-) mice. After a 10 mg/kg methamphetamine injection, FosB(-/-) mice were more prone to self-injury. Concomitantly, the intracellular feedback regulators of Sprouty and Rad-Gem-Kir (RGK) family transcripts had lower expression profiles in the frontoparietal cortex and striatum of the FosB(-/-) mice. Three days after administration of four 10 mg/kg methamphetamine injections, the frontoparietal cortex and striatum of FosB(-/-) mice contained more degenerated neurons as determined by Fluoro-Jade B staining. The abundance of the small neutral amino acids, serine, alanine, and glycine, was lower and/or was poorly induced after methamphetamine administration in the frontoparietal cortex and striatum of FosB(-/-) mice. In addition, methamphetamine-treated FosB(-/-) frontoparietal and piriform cortices showed more extravasation of immunoglobulin, which is indicative of blood-brain barrier dysfunction. Methamphetamine-induced hyperthermia, brain dopamine content, and loss of tyrosine hydroxylase immunoreactivity in the striatum, however, were not different between genotypes. These data indicate that FosB is involved in thermoregulation-independent protective functions against methamphetamine neurotoxicity in postsynaptic neurons. Our findings suggest two possible mechanisms of FosB-mediated neuroprotection: one is induction of negative feedback regulation within postsynaptic neurons through Sprouty and RGK. Another is supporting astroglial function such as maintenance of the blood-brain barrier, and metabolism of serine and glycine, which are important glial modulators of nerve cells.

Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1.

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra-1-deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra-1 transgenic osteoblasts, suggesting a role for Fra-1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi-dependent regulation of MGP. In MC3T3-E1 cells and primary calvaria-derived osteoblasts, Pi increased MGP and Fra-1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi-stimulated MGP and Fra-1 expression, indicating that ERK1/2 is required for Pi-dependent regulation of MGP and Fra-1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra-1 interacts with the MGP promoter in response to Pi in MC3T3-E1 cells. Finally, we found that in fra-1 knockdown MC3T3-E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra-1-deficient mice failed to exhibit a Pi-dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2-Fra-1 pathway.

C-Fos elimination compensates for disabled-2 requirement in mouse extraembryonic endoderm development.

Disabled-2 (Dab2) is expressed in primitive endoderm cells as they are differentiating from the inner cell mass and dab2 deficiency in mice results in lethality at E5.5-E6.5 due to the disorganization of the endoderm layers. Here we show that Dab2 suppresses c-Fos expression in endoderm cells. A morphological normal primitive endoderm layer was observed in putative E5.5 dab2 (-/-):c-fos (-/-) embryos, indicating that the primitive endoderm defect due to the loss of Dab2 is rescued by deletion of the c-fos gene. The lethality of the double knockout embryos was delayed until E9.5-E10.5 and the defective embryos failed to undergo organogenesis. We conclude that Dab2 plays a role in epithelial organization by suppression of c-Fos expression and suggest that unsuppressed c-Fos can lead to disruption of primitive endoderm epithelial organization, yet an additional dab2 function is required for later organogenesis.