Tetraspanin 7 promotes osteosarcoma cell invasion and metastasis by inducing EMT and activating the FAK-Src-Ras-ERK1/2 signaling pathway.

BACKGROUND: Tetraspanins are members of the 4-transmembrane protein superfamily (TM4SF) that function by recruiting many cell surface receptors and signaling proteins into tetraspanin-enriched microdomains (TEMs) that play vital roles in the regulation of key cellular processes including adhesion, motility, and proliferation. Tetraspanin7 (Tspan7) is a member of this superfamily that plays documented roles in hippocampal neurogenesis, synaptic transmission, and malignant transformation in certain tumor types. How Tspan7 influences the onset or progression of osteosarcoma (OS), however, remains to be defined. Herein, this study aimed to explore the relationship between Tspan7 and the malignant progression of OS, and its underlying mechanism of action. METHODS: In this study, the levels of Tspan7 expression in human OS cell lines were evaluated via qRT-PCR and western blotting. The effect of Tspan7 on proliferation was examined using CCK-8 and colony formation assays, while metastatic role of Tspan7 was assessed by functional assays both in vitro and in vivo. In addition, mass spectrometry and co-immunoprecipitation were performed to verify the interaction between Tspan7 and ß1 integrin, and western blotting was used to explore the mechanisms of Tspan7 in OS progresses. RESULTS: We found that Tspan7 is highly expressed in primary OS tumors and OS cell lines. Downregulation of Tspan7 significantly suppressed OS growth, metastasis, and attenuated epithelial-mesenchymal transition (EMT), while its overexpression had the opposite effects in vitro. Furthermore, it exhibited reduced OS pulmonary metastases in Tspan7-deleted mice comparing control mice in vivo. Additionally, we proved that Tspan7 interacted with ß1 integrin to facilitate OS metastasis through the activation of integrin-mediated downstream FAK-Src-Ras-ERK1/2 signaling pathway. CONCLUSION: In summary, this study demonstrates for the first time that Tspan7 promotes OS metastasis via interacting with ß1 integrin and activating the FAK-Src-Ras-ERK1/2 pathway, which could provide rationale for a new therapeutic strategy for OS.

CDC42 Regulates Cell Proliferation and Apoptosis in Bladder Cancer via the IQGAP3-Mediated Ras/ERK Pathway.

Bladder cancer (BC) is the most common malignant tumour of the urinary system. The current conventional treatments for BC have certain limitations. It is very urgent and necessary to find new treatment strategies for BC. Our study elucidated the underlying regulatory mechanisms of cell division control protein 42 homologue (CDC42) to regulate the development of BC. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence and immunohistochemistry were used to assess the expression of CDC42 and IQ motif-containing GTPase-activating protein 3 (IQGAP3) in BC tissues and BC cells. We induced the knockdown or overexpression by transfecting sh-CDC42 or oe-IQGAP3 into BC cells. In addition, cell proliferation and apoptosis were evaluated by cell counting kit-8 and flow cytometry assays, respectively. Moreover, proteins involved in the rat sarcoma (Ras)/extracellular regulated protein kinase (ERK) pathway were determined by Western blot. The expression of CDC42 and IQGAP3 was markedly upregulated in both BC tissues and BC cells. CDC42 silencing downregulated the expression of IQGAP3 and suppressed the Ras/ERK pathway. In addition, CDC42 silencing markedly promoted apoptosis and inhibited proliferation in BC cells. Further experiments showed that overexpression of IQGAP3 dramatically abolished the bioeffects mediated by CDC42 silencing on the proliferation and apoptosis of BC cells. All our results suggested that CDC42 promoted the Ras/ERK pathway by regulating IQGAP3, thus enhancing cell proliferation and suppressing cell apoptosis in BC cells and ultimately participating in the pathogenesis of BC.

FRA-1 suppresses apoptosis of Helicobacter pylori infected MGC-803 cells.

Previous research has demonstrated a correlation between elevated expression of Fos-related antigen 1 (FRA-1) and malignancies. Nevertheless, the role of FRA-1 in Helicobacter pylori infected gastric cancer cells remains vague. Our study aims to investigate whether FRA-1 plays a role in the apoptosis of MGC-803 induced by H. pylori and possible mechanisms. MGC-803 cells were used in vitro to establish a cell model of H. pylori infection. After stimulation with H. pylori, the expression of FRA-1 was increased in MGC-803 cells. H. pylori infection promoted the apoptosis of MGC-803 cells, and led to cell cycle arrest and increased oxidative stress levels. Furthermore, the knockdown of FRA-1 reinforced these changes. H. pylori decreased the expression of Bcl2, Caspase3 and Caspase9, while increased the level of BAX, Cleaved-Caspase3 and Cleaved-Caspase9; in addition, it led to the decrease of major proteins in Ras/Erk and PI3K/AKT signaling pathway. As expected, these changes were augmented by FRA-1 knockdown. Our results demonstrated that high expression of FRA-1 induced by H. pylori suppresses apoptosis in MGC-803 cells which may be regulated by oxidative stress and cycle arrest through caspase family, Ras/Erk and PI3K/AKT signaling pathway.

Dominant-negative antagonists of the Ras-ERK pathway: DA-Raf and its related proteins generated by alternative splicing of Raf.

The Ras-ERK pathway regulates a variety of cellular and physiological responses, including cell proliferation, differentiation, morphogenesis during animal development, and homeostasis in adults. Deregulated activation of this pathway leads to cellular transformation and tumorigenesis as well as RASopathies. Several negative regulators of this pathway have been documented. Each of these proteins acts at particular points of the pathway, and they exert specific cellular and physiological functions. Among them, DA-Raf1 (DA-Raf), which is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain, antagonizes the Ras-ERK pathway in a dominant-negative manner. DA-Raf induces apoptosis, skeletal myocyte differentiation, lung alveolarization, and fulfills tumor suppressor functions by interfering with the Ras-ERK pathway. After the findings of DA-Raf, several kinase-domain-truncated splicing variants of Raf proteins have also been reported. The family of these truncated proteins represents the concept that alternative splicing can generate antagonistic proteins to their full-length counterparts.

RSK1 vs. RSK2 Inhibitory Activity of the Marine ß-Carboline Alkaloid Manzamine A: A Biochemical, Cervical Cancer Protein Expression, and Computational Study.

Manzamines are complex polycyclic marine-derived ß-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia, and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, SIX1, and vacuolar ATPases. We hypothesized that additional, yet undiscovered molecular targets might be associated with Manzamine A's (MZA) reported pharmacological properties. We report here, for the first time, that MZA selectively inhibited a 90 kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases, while in vitro RSK kinase assays demonstrated a 10-fold selectivity in the potency of MZA against RSK1 versus RSK2. The effect of MZA on inhibiting cellular RSK1 and RSK2 protein expression was validated in SiHa and CaSki human cervical carcinoma cell lines. MZA's differential binding and selectivity toward the two isoforms was also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2-MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes, including growth, survival, and proliferation. Consequently, our findings have led us to hypothesize that MZA and the currently known manzamine-type alkaloids isolated from several sponge genera may have novel pharmacological properties with unique molecular targets, and MZA provides a new tool for chemical-biology studies involving RSK1.

[Yishen Tonglong Decoction inhibits the epithelial-mesenchymal transition and Ras/ERK signaling pathway in human prostate cancer DU-145 cells].

Objective: To observe the effect of Yishen Tonglong Decoction (YTD) on the epithelial-mesenchymal transition (EMT) and Ras/ERK signaling pathway in human PCa DU-145 cells and explore its action mechanism. METHODS: We treated human PCa DU-145 cells with normal plasma (the blank control) or plasma containing 5% (low-dose), 10% (medium-dose) and 15% (high-dose) YTD. After intervention, we examined the proliferation of the DU-145 cells in different groups with CCK-8 and their apoptosis by Annexin V/PI double staining. We detected the cell cycle by PI assay, the invasion and migration of the cells using the Transwell chamber and scratch test, and the expressions of the proteins and genes related to the EMT and Ras/ERK signaling pathways in the cells by Western blot and RT-PCR. RESULTS: Compared with the blank control group, high-, medium- and low-dose YTD significantly inhibited the proliferation of the PCa DU-145 cells, decreased their adherence and growth (P < 0.05, P < 0.01), promoted their apoptosis (P < 0.01), regulated their cell cycles (P < 0.05, P < 0.01), and reduced their in vitro invasion and migration abilities (P < 0.05), all in a dose-dependent manner. The results of Western blot and RT-PCR revealed down-regulated protein and mRNA expressions of N-cadherin, zinc finger transcription factor (Snail), Ras, p-ERK1/2 and ERK1/2, but up-regulated protein and mRNA expressions of E-cadherin in the PCa DU-145 cells treated with YTD (P < 0.05, P < 0.01). CONCLUSIONS: Yishen Tonglong Decoction can effectively inhibit the proliferation, promote the apoptosis, regulate the cell cycle and suppress the invasion and migration abilities and EMT process of human PCa DU-145 cells. The mechanism of Yishen Tonglong Decoction acting on PCa may be associated with its inhibitory effect on the EMT process and expression of the Ras/ERK signaling pathway in PCa cells./.

VPS33B negatively modulated by nicotine functions as a tumor suppressor in colorectal cancer.

The biological role of vacuolar protein sorting 33B (VPS33B) has not been examined in colorectal cancer (CRC). We report that VPS33B was downregulated in dextran sulfate sodium/azoxymethane (DSS/AOM) -induced CRC mice models and nicotine-treated CRC cells via the PI3K/AKT/c-Jun pathway. Reduced VPS33B is an unfavorable factor promoting poor prognosis in human CRC patients. VPS33B overexpression suppressed CRC proliferation, intrahepatic metastasis and chemoresistance of cisplatin (DDP) in vivo and in vitro through modulating the epidermal growth factor receptor (EGFR)/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and the downstream cell cycle or EMT-related factors. Furthermore, NESG1 as a newly identified tumor suppressor interacted with VPS33B via colocalization in the cytoplasm, and it was stimulated by VPS33B through the downregulation of RAS/ERK/c-Jun-mediated transcription. NESG1 also activated VPS33B expression via the RAS/ERK/c-Jun pathway. Suppression of NESG1 increased cell growth, migration and invasion via the reversion of the VPS33B-modulating signal in VPS33B-overexpressed cells. Taken together, VPS33B as a tumor suppressor is easily dysregulated by chemical carcinogens and it interacts with NESG1 to modulate the EGFR/RAS/ERK/c-Myc/p53/miR-133a-3p feedback loop and thus suppress the malignant phenotype of CRC.

DA-Raf, a dominant-negative regulator of the Ras-ERK pathway, is essential for skeletal myocyte differentiation including myoblast fusion and apoptosis.

Ras-activated ERK pathway (Raf-MEK-ERK phosphorylation cascade) regulates a variety of cellular responses including cell proliferation, differentiation, survival, and apoptosis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner. Here we show that DA-Raf plays essential roles in skeletal myocyte differentiation including myoblast fusion and in apoptosis, which are suppressed by the Ras-ERK pathway. Expression of DA-Raf was highly induced in C2C12 skeletal myocytes in a low serum concentration of differentiation condition and in NIH3T3 fibroblasts under a serum starvation apoptosis-inducing condition. Stable knockdown of DA-Raf resulted in suppression of muscle-specific gene expression, myoblast fusion, and apoptosis. In contrast, exogenous overexpression of DA-Raf prominently caused apoptosis. DA-Raf induces apoptosis by preventing ERK-RSK-mediated inhibitory phosphorylation of Bad. Although it has been reported that apoptosis triggers myoblast fusion, DA-Raf-induced apoptosis was not involved in myoblast fusion in C2C12 cells. These results imply that suppression of the Ras-ERK pathway by DA-Raf is essential for both myocyte differentiation including myoblast fusion and apoptosis but that apoptosis is not a prerequisite for myoblast fusion.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis.

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent-offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10-11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

CDK-11-Cyclin L is required for gametogenesis and fertility in C. elegans.

CDK11, a member of the cyclin-dependent kinase family, has been implicated in a diverse array of functions including transcription, RNA processing, sister chromatid cohesion, spindle assembly, centriole duplication and apoptosis. Despite its involvement in many essential functions, little is known about the requirements for CDK11 and its partner Cyclin L in a developing multicellular organism. Here we investigate the function of CDK11 and Cyclin L during development of the nematode Caenorhabditis elegans. Worms express two CDK11 proteins encoded by distinct loci: CDK-11.1 is essential for normal male and female fertility and is broadly expressed in the nuclei of somatic and germ line cells, while CDK-11.2 is nonessential and is enriched in hermaphrodite germ line nuclei beginning in mid pachytene. Hermaphrodites lacking CDK-11.1 develop normally but possess fewer mature sperm and oocytes and do not fully activate the RAS-ERK pathway that is required for oocyte production in response to environmental cues. Most of the sperm and eggs that are produced in cdk-11.1 null animals appear to complete development normally but fail to engage in sperm-oocyte signaling suggesting that CDK-11.1 is needed at multiple points in gametogenesis. Finally, we find that CDK-11.1 and CDK-11.2 function redundantly during embryonic and postembryonic development and likely do so in association with Cyclin L. Our results thus define multiple requirements for CDK-11-Cyclin L during animal development.

Carcinogenic role of K-Ras-ERK1/2 signaling in bladder cancer via inhibition of H1.2 phosphorylation at T146.

It has been reported that Ras-ERK signaling regulated tumor suppressive genes via epigenetic mechanisms. Herein, we set out to investigate the correlation between K-Ras-ERK1/2 signaling and H1.2 phosphorylation, to provide a better understanding of K-Ras-ERK signaling in cancer. A plasmid for expression of mutated K-Ras was transfected into human bladder carcinoma HT1197 cells. Western blot was carried out for testing the expression changes of ERK1/2 and H1.2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, soft-agar colony formation assay, and transwell assay were used to test the effects of H1.2 phosphorylation at T146 (H1.2 T146ph ) on HT1197 cells growth and migration. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and chromatin immunoprecipitation (ChIP) were performed to test whether H1.2 T146ph regulated K-Ras-ERK1/2 downstream genes. Furthermore, how K-Ras-ERK1/2 regulated H1.2 T146ph expression was studied. We found that the ERK1/2 was activated when K-Ras was mutated, and H1.2 T146ph expression was significantly downregulated by K-Ras mutation. H1.2 T146E for mimicking H1.2 T146ph significantly attenuated K-Ras mutation induced increases in HT1197 cells viability, colony formation, and relative migration. Besides, H1.2 T146ph regulated the transcription of K-Ras-ERK1/2 downstream genes, including NT5E, GDF15, CARD16, CYR61, IGFBP3, and WNT16B. Furthermore, K-Ras-ERK1/2 signaling inhibited H1.2 phosphorylation at T146 through degradation of DNA-PK, and the degraded DNA-PK by K-Ras-ERK1/2 possibly via modulation of MDM2. In conclusion, the activation of K-Ras-ERK1/2 signaling will repress the phosphorylation of H1.2 at T146, and thereby, promoted the growth and migration of bladder cancer cells. K-Ras-ERK1/2 signaling repressed H1.2 phosphorylation possibly by MDM2-mediated degradation of DNA-PK.

K-ras-ERK1/2 down-regulates H2A.XY142ph through WSTF to promote the progress of gastric cancer.

BACKGROUND: Histone H2AX phosphorylation at the site of Tyr-142 can participates in multiple biological progressions, which is including DNA repair. Ras pathway is closely involved in human cancers. Our study investigated the effects of Ras pathway via regulating H2AX.Y142ph. METHODS: Gastric cancer cell line SNU-16 and MKN1 cells were transfected with Ras for G12D and T35S site mutation. The phosphorylation of H2A.XY142 and ERK1/2, WSTF and MDM2 was detected by western blot. Cell viability, cell colonies and migration was analyzed by MTT assay, soft-agar colony formation assay, and Transwell assay, respectively. The expression of Ras pathway related downstream factors, EYA3 and WSTF was detected by qRT-PCR. The relationship between Ras and downstream factors were detected by ChIP. The cell cycle progression was measured by flow cytometry. RESULTS: RasG12D/T35V transection decreased the phosphorylation of H2A.XY142 and activated phosphorylation of ERK-1/2. H2A.XY142 inhibited cell viability, colonies and migration. H2A.XY142ph altered the expression of Ras downstream factors. CHIP assay revealed that RasG12D/T35V could bind to the promoters of these Ras pathway downstream factors. Silence of EYA3 increased H2A.XY142ph and inhibited cell viability, migration and percent cells in S stage. Furthermore, silence of EYA3 also changed the downstream factors expression. WSTF and H2A.XY142ph revealed the similar trend and MDM2 on the opposite. CONCLUSION: Ras/ERK signal pathway decreased H2A.XY142ph and promoted cell growth and metastasis. This Ras regulation process was down-regulated by the cascade of MDM2-WSTF-EYA3 to decrease H2A.XY142ph in SNU-16 cells.

DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, is a putative tumor suppressor.

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf-MEK-ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis.

Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons.

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP+) treatment (a model of Parkinson's disease). We show that MPP+-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12 cells (NPC12 cells) and rat cerebellar granule neurons (CGNs). Following MPP+ treatment, we found production of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12 cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP+ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP+-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP+-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP+-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.

Acquisition of anoikis resistance promotes alterations in the Ras/ERK and PI3K/Akt signaling pathways and matrix remodeling in endothelial cells.

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis resistance is a critical mechanism in tumor metastasis. Cancer cells deregulate and adapt their metabolism to survive in the absence of adhesion, spreading metastases to distant organs. These adaptations include abnormal regulation of growth factor receptors activating prosurvival signaling pathways, such as the Ras/ERK and PI3K/Akt pathways, and extracellular matrix remodeling, leading to metastasis by an increase of invasiveness and inhibiting anoikis. This study investigates the possible involvement of ECM components and signaling pathways in the regulation of resistance to anoikis in endothelial cells (EC). Endothelial cells submitted to stressful conditions by blocking adhesion to substrate (anoikis resistance) display an up-regulation of Ras/ERK and PI3k/Akt pathways by high expression of Ras, ERK, PI3K (p110α) and Akt (Thr 308). After ERK and PI3K inhibiting, all EC-derived cell lines studied showed lower growth, a decrease in invasive potential and a higher rate of apoptosis. Furthermore, anoikis-resistant cell lines display a decrease in the expression of fibronectin, collagen IV and hyaluronic acid and an increase in the expression of laminin, perlecan, αv, ß3, α5 and ß1 integrins subunits, hyaluronidades 1, 2 and 3 and metalloproteinases 2 and 9. These results indicate that the acquisition of anoikis resistance induced remodeling of the extracellular matrix and overexpression of the PI3K/Akt and Ras/ERK pathway components. Acquisition of resistance to anoikis is a potentially crucial step in endothelial cell transformation.

G-CSF improves CUMS-induced depressive behaviors through downregulating Ras/ERK/MAPK signaling pathway.

Neuronal plasticity in hippocampal neurons is closely related to memory, mood and behavior as well as in the development of depression. Granulocyte colony-stimulating factor (G-CSF) can promote neuronal plasticity and enhance motor skills. However, the function of G-CSF in depression remains poorly understood. In this study, we explored the biological role and potential molecular mechanism of G-CSF on depression-like behaviors. Our results showed that G-CSF was significantly downregulated in the hippocampus of chronic unexpected mild stress (CUMS) rats. Administration of G-CSF significantly reversed CUMS-induced depression-like behaviors in the open field test (OFT), sucrose preference test (SPT) and forced swimming test (FST). Moreover, G-CSF upregulated the expression of synaptic-associated proteins including polysialylated form of neural cell adhesion molecule (PSA-NCAM), synaptophysin (SYN), and postsynaptic density protein 95 (PSD-95) in the hippocampus and G-CSF significantly increased cell viability rate of hippocampal neurons in vitro. Further studies indicated that the renin-angiotensin system (Ras)/extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways was involved in the regulation of G-CSF on depressive-like behaviors and neuronal plasticity in CUMS rats. Taken together, our results showed that G-CSF improves depression-like behaviors via inhibiting Ras/ERK/MAPK signaling pathways. Our study suggests that G-CSF may be a promising therapeutic strategy for the treatment of depression.

Fgf8 signaling for development of the midbrain and hindbrain.

In this paper, we review how midbrain and hindbrain are specified. Otx2 and Gbx2 are expressed from the early phase of development, and their expression abuts at the midbrain hindbrain boundary (MHB), where Fgf8 expression is induced, and functions as an organizing molecule for the midbrain and hindbrain. Fgf8 induces En1 and Pax2 expression at the region where Otx2 is expressed to specify midbrain. Fgf8 activates Ras-ERK pathway to specify hindbrain. Downstream of ERK, Pea3 specifies isthmus (rhombomere 0, r0), and Irx2 may specify r1, where the cerebellum is formed.

RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis.

Neural cell fate specification is well understood in the embryonic cerebral cortex, where the proneural genes Neurog2 and Ascl1 are key cell fate determinants. What is less well understood is how cellular diversity is generated in brain tumors. Gliomas and glioneuronal tumors, which are often localized in the cerebrum, are both characterized by a neoplastic glial component, but glioneuronal tumors also have an intermixed neuronal component. A core abnormality in both tumor groups is overactive RAS/ERK signaling, a pro-proliferative signal whose contributions to cell differentiation in oncogenesis are largely unexplored. We found that RAS/ERK activation levels differ in two distinct human tumors associated with constitutively active BRAF. Pilocytic astrocytomas, which contain abnormal glial cells, have higher ERK activation levels than gangliogliomas, which contain abnormal neuronal and glial cells. Using in vivo gain of function and loss of function in the mouse embryonic neocortex, we found that RAS/ERK signals control a proneural genetic switch, inhibiting Neurog2 expression while inducing Ascl1, a competing lineage determinant. Furthermore, we found that RAS/ERK levels control Ascl1's fate specification properties in murine cortical progenitors--at higher RAS/ERK levels, Ascl1(+) progenitors are biased toward proliferative glial programs, initiating astrocytomas, while at moderate RAS/ERK levels, Ascl1 promotes GABAergic neuronal and less glial differentiation, generating glioneuronal tumors. Mechanistically, Ascl1 is phosphorylated by ERK, and ERK phosphoacceptor sites are necessary for Ascl1's GABAergic neuronal and gliogenic potential. RAS/ERK signaling thus acts as a rheostat to influence neural cell fate selection in both normal cortical development and gliomagenesis, controlling Neurog2-Ascl1 expression and Ascl1 function.

Sos1 regulates sustained TCR-mediated Erk activation.

The duration and/or the magnitude of Ras-Erk activation are known to be crucial for cell-fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl-releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T-cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T-cell activation.

Pea3 determines the isthmus region at the downstream of Fgf8-Ras-ERK signaling pathway.

It has been shown that strong Fgf8 signal activates Ras-ERK signaling pathway to determine metencephalon, which consists of rhombomere 1 (r1), where the cerebellum differentiates, and isthmus (r0). The present study was undertaken to check if Ets type transcription factor Pea3 functions downstream of Ras-ERK signaling to determine metencephalon. Pea3 misexpression resulted in repression of Otx2 expression in the mesencephalon, induction of Gbx2 and Fgf8 expression in the mesencephalon, and differentiation of the trochlear neurons in the posterior mesencephalon. Fate change of the tectum to the cerebellum did not occur. Repression of Pea3 function by misexpressing the chimeric molecule of Engrailed repressor domain EH1 and Pea3 (eh1-Pea3) resulted in induction of Otx2 expression in the metencephalon, repression of Gbx2 and Fgf8 expression in the metencephalon, and differentiation of the oculomotor neurons in the isthmus. It was concluded that Pea3 plays a pivotal role in determination of the isthmus (r0) property downstream of Fgf8-Ras-ERK signaling.