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Chronic cerebral hypoperfusion (CCH)-mediated cognitive impairment is a serious problem worldwide. However, given its complexity, the underlying mechanisms by which CCH induces cognitive dysfunction remain unclear, resulting in a lack of effective treatments. In this study, we aimed to determine whether changes in the expression of RasGRF1, an important protein associated with cognition and synaptic plasticity, underlie the associated impairments in cognition after CCH. We found that RasGRF1 levels markedly decreased following CCH. Through prediction and validation studies, we observed that miRNA-323-3p was upregulated after CCH and could bind to the 3'-untranslated region of Rasgrf1 mRNA and regulate its expression in vitro . Moreover, the inhibition of miRNA-323-3p upregulated Rasgrf1 expression in the hippocampus after CCH, which was reversed by Rasgrf1 siRNA. This suggests that miRNA-323-3p is an important regulator of Rasgrf1. The Morris water maze and Y maze tests showed that miRNA-323-3p inhibition and Rasgrf1 upregulation improved spatial learning and memory, and electrophysiological measurements revealed deficits in long-term potentiation after CCH that were reversed by Rasgrf1 upregulation. Dendritic spine density and mature mushroom spine density were also improved after miRNA-323-3p inhibition and Rasgrf1 upregulation. Furthermore, Rasgrf1 upregulation by miRNA-323-3p inhibition improved dendritic spine density and mature mushroom spine density and ameliorated the deterioration of synapses and postsynaptic density. Overall, RasGRF1 regulation attenuated cognitive impairment, helped maintain structural and functional synaptic plasticity, and prevented synapse deterioration after CCH. These results suggest that Rasgrf1 downregulation by miRNA-323-3p plays an important role in cognitive impairment after CCH. Thus, RasGRF1 and miRNA-323-3p may represent potential therapeutic targets for cognitive impairment after CCH.

Objective: The aim of the study is to analyze the correlation between high myopia susceptibility and Ras protein-specific guanine nucleotide-releasing factor-1(RASGRF1) gene polymorphism among college students in Zhejiang., Methods: A stratified whole-group sampling method was used to select 218 cases of college students in Zhejiang who met the inclusion and exclusion criteria from January, 2019, to December, 2021, and they were divided into 77 cases (154 eyes) in the high myopia group and 141 cases (282 eyes) in the medium-low myopia group according to the degree of myopia, and 109 cases of college volunteers without myopia from the same period of medical examination in the region were included in the control group. The single nucleotide polymorphisms (SNPs) located in functional regions were selected by searching the literature and genetic databases, and the base sequences of rs939658, rs4778879, and rs8033417 loci were obtained by genotyping candidate SNPs using multiplex ligase detection reaction technique. The cardinality test was used to compare the differences in genotype frequency distribution of each locus of the RASGRF1 gene between the high myopia group and the low to moderate myopia group and the control group., Results: The genotype frequencies and allele frequencies of the RASGRF1 gene rs939658 locus in the high myopia group compared with the moderate-low myopia group and the control group were not statistically significant ( P > 0.05). The genotype frequencies and allele frequencies of the rs4778879 locus of the RASGRF1 gene were compared among the three groups, and the differences were not statistically significant ( P > 0.05). The genotype frequency and allele frequency of the rs8033417 locus of the RASGRF1 gene differed significantly among the three groups ( P < 0.05)., Conclusion: The polymorphism of the rs8033417 locus of the RASGRF1 gene was significantly correlated with the susceptibility of high myopia among college students in Zhejiang., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Jipan Yi et al.)

Menin is necessary for the formation of the menin/mixed lineage leukemia (MLL) complex and is recruited directly to chromatin. Menin is an important tumor suppressor in several cancer types, including lung cancer. Here, we investigated the role of MLL in menin-regulated lung tumorigenesis. Ablation of MLL suppressed KrasG12D-induced lung tumorigenesis in a genetically engineered mouse model. MLL deficiency decreased histone H3 lysine 4 trimethylation (H3K4me3) and subsequently suppressed expression of the Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1) gene. Rasgrf1 was essential for the GTP-bound active state of Kras and the activation of Kras downstream pathways as well as their cancer-promoting activities. MI-3, a small-molecule inhibitor targeting MLL, specifically inhibited the growth of Kras-mutated lung cancer cells in vitro and in vivo with minimal effect on wild-type Kras lung cancer growth. Together, these results demonstrate a novel tumor promoter function of MLL in mutant Kras-induced lung tumorigenesis and further indicate that specific blockade of the MLL-Rasgrf1 pathway may be a potential therapeutic strategy for the treatment of tumors containing Kras mutations., Significance: Activation of mutant Kras is dependent on MLL-mediated epigenetic regulation of Rasgrf1, conferring sensitivity to small-molecule inhibition of MLL in Kras-driven lung cancer., (©2022 American Association for Cancer Research.)

We have previously shown that high runner (HR) mice (from a line genetically selected for increased wheel-running behavior) have distinct, genetically based, neurobiological phenotypes as compared with non-selected control (C) mice. However, developmental programming effects during early life, including maternal care and parent-of-origin-dependent expression of imprinted genes, can also contribute to variation in physical activity. Here, we used cross-fostering to address two questions. First, do HR mice have altered DNA methylation profiles of imprinted genes in the brain compared to C mice? Second, does maternal upbringing further modify the DNA methylation status of these imprinted genes? To address these questions, we cross-fostered all offspring at birth to create four experimental groups: C pups to other C dams, HR pups to other HR dams, C pups to HR dams, and HR pups to C dams. Bisulfite sequencing of 16 imprinted genes in the cortex and hippocampus revealed that the HR line had altered DNA methylation patterns of the paternally imprinted genes, Rasgrf1 and Zdbf2, as compared with the C line. Both fostering between the HR and C lines and sex modified the DNA methylation profiles for the paternally expressed genes Mest, Peg3, Igf2, Snrpn, and Impact. Ig-DMR, a gene with multiple paternal and maternal imprinted clusters, was also affected by maternal upbringing and sex. Our results suggest that differential methylation patterns of imprinted genes in the brain could contribute to evolutionary increases in wheel-running behavior and are also dependent on maternal upbringing and sex., (© 2022. The Author(s).)

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. In this study, we clarified that a glucose response is involved in an inhibitory mechanism of proline utilization in yeast. Our genetic screen showed that strains with a loss-of-function mutation on the CDC25 gene can utilize proline even under fermentation conditions. Cdc25 is a regulator of the glucose response consisting of the Ras/cAMP-dependent protein kinase A (PKA) pathway. Moreover, we found that activation of the Ras/PKA pathway is necessary for the inhibitory mechanism of proline utilization. The present data revealed that crosstalk exists between the carbon and proline metabolisms. Our study could hold promise for the development of wine yeast strains that can efficiently assimilate proline during the fermentation processes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Purpose: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history., Experimental Design: We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers., Results: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo., Conclusions: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983., (©2022 American Association for Cancer Research.)

Spitz neoplasms, according to 2018 WHO Blue Book, are morphologically defined by spindled and/or epithelioid melanocytes and genetically by either HRAS mutations or kinase gene fusions. The terminology "spitzoid" refers to lesions with similar morphology but with alternate or undefined genetic anomalies. Herein, we present 3 melanocytic neoplasms with a spitzoid cytomorphology, variable nuclear atypia, and harboring undescribed fusions involving RASGRF1. Two cases presented as unpigmented papules on the heel of a 26-year-old female (case 1) and the forearm of a 13-year-old boy (case 2). They were classified as low-grade melanocytomas (WHO 2018). The third case appeared as a pigmented ulcer on the sole of a 72-year-old female (case 3) that displayed diagnostic features of an invasive melanoma (Breslow thickness 6 mm, Clark level V). A wide skin reexcision identified an epidermotropic metastasis, and sentinel lymph node biopsy displayed multiple subcapsular metastatic deposits. RNA sequencing revealed CD63::RASGRF1, EHBP1::RASGRF1, and ABCC2::RASGRF1 fusions in cases 1 to 3, respectively. They were confirmed by a RASGRF1 break-apart fluorescence in situ hybridization technique. Translocations of RASGRF1, a gene coding a guanine nucleotide exchange factor but not a kinase, have rarely been reported in tumors. While all these cases showed spitzoid cytomorphology, it is too early to tell if they are true Spitz neoplasms as currently defined., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Cells control the properties of the cytoplasm to ensure proper functioning of biochemical processes. Recent studies showed that cytoplasmic density varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here, we study how a cell cycle oscillator comprising cyclin-dependent kinase (Cdk1) responds to changes in cytoplasmic density by systematically diluting or concentrating cycling Xenopus egg extracts in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density: as low as 0.2× to more than 1.22× relative cytoplasmic density (RCD). A further dilution or concentration from these values arrested the system in a low or high steady state of Cdk1 activity, respectively. Interestingly, diluting an arrested cytoplasm of 1.22× RCD recovers oscillations at lower than 1× RCD. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of tuning, forming a hysteresis loop. We propose a mathematical model which recapitulates these observations and predicts that the Cdk1/Wee1/Cdc25 positive feedback loops do not contribute to the observed robustness, supported by experiments. Our system can be applied to study how cytoplasmic density affects other cellular processes., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Tanshinone IIA (Tan IIA) is reported to have neuroprotective effects to suppress cell apoptosis of cortical neurons induced by Aβ 25-35 through inhibiting oxidative stress. Nevertheless, few studies have investigated the effects of Tan IIA on depressive disorder. Here, we aimed to measure the effects of Tan IIA on chronic unpredictable mild stress (CUMS) induced mouse model and its underlying mechanism. For 28 days, mice were subjected to CUMS while Tan IIA was administered once daily at doses of 0, 1, 2.5, 5, or 10 mg/kg. CUMS exposure increased depressive-like behaviors, as indicated by increased immobility time in the forced swim and tail suspension tests, decreased sucrose preference in the sucrose preference test, and reduced exploratory behavior in the open field test. All of these behaviors were reversed dose-dependently by Tan IIA treatment. Oxidative stress was determined by measuring malondialdehyde, glutathione peroxidase, and superoxide dismutase activity and total antioxidant capacity. Levels of pro-inflammatory factors IL-1β and IL-18, cAMP response element binding protein and brain derived neurotrophic factor were detected by ELISA and western blot assay, respectively. The results showed that CUMS increased oxidative stress and pro-inflammatory factors and decreased levels of cAMP response element binding protein and brain-derived neurotrophic factor. Tan IIA treatment again reversed these effects. Importantly, RasGRF1 expression increased in CUMS-exposed mice but decreased after Tan IIA administration. Using RasGRF1 -/- mice to determine the role of RasGRF1 in mice exposed to CUMS, we found that knockdown of RasGRF1 reversed the effects of CUMS on mice, just like Tan IIA. These results indicate that Tan IIA may reverse depressive-like behaviors in CUMS-exposed mice by regulating RasGRF1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: We pursued genomic analysis of an exceptional responder with non-small cell lung cancer (NSCLC) through a multi-platform effort to discover novel oncogenic targets., Experimental Design: In this open-label, single-arm phase II study (NCT01829217), an enriched cohort of patients with advanced NSCLC was treated with the multi-kinase inhibitor sunitinib. The primary endpoint was objective response rate. Tissue was collected for multi-platform genomic analysis of responders, and a candidate oncogene was validated using in vitro models edited by CRISPR-Cas9., Results: Of 13 patients enrolled, 1 patient (8%), a never smoker, had a partial response lasting 33 months. Genomic analysis of the responder identified no oncogenic variant using multi-platform DNA analysis including hotspot allelotyping, massively parallel hybrid-capture next-generation sequencing, and whole-exome sequencing. However, bulk RNA-sequencing (RNA-seq) revealed a novel fusion, TMEM87A-RASGRF1 , with high overexpression of the fusion partners. RASGRF1 encodes a guanine exchange factor which activates RAS from GDP-RAS to GTP-RAS. Oncogenicity was demonstrated in NIH/3T3 models with intrinsic TMEM87A-RASGRF1 fusion. In addition, activation of MAPK was shown in PC9 models edited to express this fusion, although sensitivity to MAPK inhibition was seen without apparent sensitivity to sunitinib., Conclusions: Sunitinib exhibited limited activity in this enriched cohort of patients with advanced NSCLC. Nonetheless, we find that RNA-seq of exceptional responders represents a potentially underutilized opportunity to identify novel oncogenic targets including oncogenic activation of RASGRF1., (©2020 American Association for Cancer Research.)

Prostate cancer is a heterogeneous disease and optimum gene targeting treatment is often impermissible. We aim to determine the intratumoral genomic heterogeneity of prostate cancer and explore candidate genes for targeted therapy. Exome sequencing was performed on 37 samples from 16 patients with prostate cancer. Somatic variant analysis, copy number variant (CNV) analysis, clonal evolution analysis and variant spectrum analysis were used to study the intratumoral genomic heterogeneity and genetic characteristics of metastatic prostate cancer. Our study confirmed the high intratumoral genetic heterogeneity of prostate cancer in many aspects, including number of shared variants, tumor mutation burden (TMB), variant genes, CNV burden, weighted genome instability index (wGII), CNV profiles, clonal evolutionary process, variant spectrum and mutational signatures. Moreover, we identified several common genetic characteristics of prostate cancer. Alterations of DNA damage repair genes, RTK/RAS pathway associated gene RASGRF1 and autophagy gene EPG5 may be involved in tumorigenesis in prostate cancer. CNV burden and DNA damage repair (DDR) genes may be associated with metastasis of prostate cancer., (© 2020 UICC.)

RASGRF1 (GRF1) is a calcium-stimulated guanine-nucleotide exchange factor that activates RAS and RAC GTPases. In hippocampus neurons, it mediates the action of NMDA and calcium-permeable AMPA glutamate receptors on specific forms of synaptic plasticity, learning, and memory in both male and female mice. Recently, we showed GRF1 also regulates the HPA axis response to restraint stress, but only in female mice before puberty. In particular, we found that after 7 days of restraint stress (7DRS) (30 min/day) both elevated serum CORT levels and induction of an anxiolytic phenotype normally observed in early adolescent (EA) female mice are blocked in GRF1-knockout mice. In contrast, no effects were observed in EA male or adult females. Here, we show this phenotype is due, at least in part, to GRF1 loss in CRF cells of the paraventricular nucleus of the hypothalamus, as GRF1 knockout specifically in these cells suppressed 7DRS-induced elevation of serum CORT levels specifically in EA females, but only down to levels found in comparably stressed EA males. Nevertheless, it still completely blocked the 7DRS-induced anxiolytic phenotype observed in EA females. Interestingly, loss of GRF1 in CRF cells had no effect after only three restraint stress exposures, implying a role for GRF1 in 7DRS stress-induced plasticity of CRF cells that appears to be specific to EA female mice. Overall, these findings indicate that GRF1 in CRF cells makes a key contribution to the distinct response EA females display to repeated stress.

N-methyl-D-aspartate receptors (NMDARs) that contain the NR2A and NR2B subunits play a critical role in neuronal plasticity and dendritogenesis. Gain-and-loss-of function studies indicate that NR2B, but not NR2A, promotes dendritic branching. Accumulating evidence indicates that stimulation of NMDARs activates NADPH oxidase (NOX2), thereby generating superoxide. However, the molecular underpinnings of this process are not understood. RasGRF1, a guanine nucleotide exchange factor, is key for several forms of neuronal plasticity and interacts directly with the tail of NR2B. We investigated whether the NR2B-NMDAR/RasGRF1 pathway regulates the activity of NOX2 and whether superoxide production is required for dendritogenesis. We measured superoxide production in developing primary cultures of hippocampal neurons from 3 to 25 days in vitro (DIV) with the probe dihydroethidium (dHE). We found the highest dHE levels at early and intermediate developmental stages (3-15 DIV), when the NR2B-NMDAR expression is abundant. During these early/intermediate developmental stages, but not in mature neurons (>15 DIV), NMDAR activity is required for superoxide production. We also found that disrupting the NR2B-RasGRF1 interaction led to reduced dHE fluorescence intensity and moreover inhibited dendritic branching in hippocampal neurons. Together, our data indicate that superoxide production is induced by the NR2B-NMDARs/RasGRF1/NOX2 pathway and promotes dendritogenesis., (© 2019 Wiley Periodicals, Inc.)

Small GTPases regulate cytoskeletal dynamics, cell motility, and division under precise spatiotemporal control. Different small GTPases exhibit cross talks to exert feedback response or to act in concert during signal transduction. However, whether and how specific cytoskeletal components' feedback to upstream signaling factors remains largely elusive. Here, we report an intriguing finding that disruption of the Arp2/3-branched actin specifically reduces RhoA activity but upregulates its total protein abundance. We further dissect the mechanisms underlying these circumstances and identify the altered cortactin/p190RhoGAP interaction and weakened CCM2/Smurf1 binding to be involved in GTP-RhoA reduction and total RhoA increase, respectively. Moreover, we find that cytokinesis defects induced by Arp2/3 inhibition can be rescued by activating RhoA. Our study reveals an intricate feedback from the actin cytoskeleton to the small GTPase. Our work highlights the role of Arp2/3-branched actin in signal transduction aside from its function in serving as critical cytoskeletal components to maintain cell morphology and motility.

Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late-stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors, such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including Gpx4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

Ras/Raf/MEK/ERK (Ras-ERK) signaling has been implicated in the effects of drugs of abuse. Inhibitors of MEK1/2, the kinases upstream of ERK1/2, have been critical in defining the role of the Ras-ERK cascade in drug-dependent alterations in behavioral plasticity, but the Ras family of small GTPases has not been extensively examined in drug-related behaviors. We examined the role of Ras Guanine Nucleotide Releasing Factor 1 (RasGRF1) and 2 (RasGRF2), upstream regulators of the Ras-ERK signaling cascade, on cocaine self-administration (SA) in male mice. We first established a role for Ras-ERK signaling in cocaine SA, demonstrating that pERK1/2 is upregulated following SA in C57BL/6N mice in striatum. We then compared RasGRF1 and RasGRF2 KO mouse lines, demonstrating that cocaine SA in RasGRF2 KO mice was increased relative to WT controls, whereas RasGRF1 KO and WT mice did not differ. This effect in RasGRF2 mice is likely mediated by the Ras-ERK signaling pathway, as pERK1/2 upregulation following cocaine SA was absent in RasGRF2 KO mice. Interestingly, the lentiviral knockdown of RasGRF2 in the NAc had the opposite effect to that in RasGRF2 KO mice, reducing cocaine SA. We subsequently demonstrated that the MEK inhibitor PD325901 administered peripherally prior to cocaine SA increased cocaine intake, replicating the increase seen in RasGRF2 KO mice, whereas PD325901 administered into the NAc decreased cocaine intake, similar to the effect seen following lentiviral knockdown of RasGRF2. These data indicate a role for RasGRF2 in cocaine SA in mice that is ERK-dependent, and suggest a differential effect of global versus site-specific RasGRF2 inhibition. SIGNIFICANCE STATEMENT Exposure to drugs of abuse activates a variety of intracellular pathways, and following repeated exposure, persistent changes in these pathways contribute to drug dependence. Downstream components of the Ras-ERK signaling cascade are involved in the acute and chronic effects of drugs of abuse, but their upstream mediators have not been extensively characterized. Here we show, using a combination of molecular, pharmacological, and lentiviral techniques, that the guanine nucleotide exchange factor RasGRF2 mediates cocaine self-administration via an ERK-dependent mechanism, whereas RasGRF1 has no effect on responding for cocaine. These data indicate dissociative effects of mediators of Ras activity on cocaine reward and expand the understanding of the contribution of Ras-ERK signaling to drug-taking behavior., (Copyright © 2019 the authors.)

A decrease in the incidence of bovine mastitis, the costliest disease in the dairy industry, can be facilitated through genetic marker-assisted selective breeding programs. Identification of genomic variants associated with mastitis resistance is an ongoing endeavor for which genome-wide association studies (GWAS) using high-density arrays provide a valuable tool. We identified single nucleotide polymorphisms (SNPs) in Holstein dairy cattle associated with mastitis resistance in a GWAS by using a high-density SNP array. Mastitis-resistant (15) and mastitis-susceptible (28) phenotypic extremes were identified from 224 lactating dairy cows on commercial dairy farm located in Utah based on multiple criteria of mastitis resistance over an 8-month period. Twenty-seven quantitative trait loci (QTLs) for mastitis resistance were identified based on 117 SNPs suggestive of genome-wide significance for mastitis resistance (p ≤ 1 × 10 -4 ), including 10 novel QTLs. Seventeen QTLs overlapped previously reported QTLs of traits relevant to mastitis, including four QTLs for teat length. One QTL includes the RAS guanyl-releasing protein 1 gene (RASGRP1), a candidate gene for mastitis resistance. This GWAS identifies 117 candidate SNPs and 27 QTLs for mastitis resistance using a selective genotyping approach, including 10 novel QTLs. Based on overlap with previously identified QTLs, teat length appears to be an important trait in mastitis resistance. RASGRP1, overlapped by one QTL, is a candidate gene for mastitis resistance.

Background: Diabetic cardiomyopathy (DCM) is characterized by cardiac fibrosis and stiffness, which often develops into heart failure. This study investigated the role of Ras protein-specific guanine nucleotide releasing factor 1 (RasGRF1) in the development of DCM., Methods: Forty-eight mice were divided into four groups ( n = 12 per group): Group 1: Wild-type (WT) mice, Group 2: RasGRF1 deficiency (RasGRF1 -/- ) mice. Group 3: Streptozotocin (STZ)-induced diabetic WT mice, Group 4: STZ-induced diabetic RasGRF1 -/- mice. Myocardial functions were assessed by cardiac echography. Heart tissues from all of the mice were investigated for cardiac fibrosis, inflammation, and oxidative stress markers., Results: Worse impaired diastolic function with elevation serum interleukin (IL)-6 was found in the diabetic group compared with the non-diabetic groups. Serum IL-6 levels were found to be elevated in the diabetic compared with the non-diabetic groups. However, the diabetic RasGRF1 -/- mice exhibited lower serum IL-6 levels and better diastolic function than the diabetic WT mice. The diabetic RasGRF1 -/- mice were associated with reduced cardiac inflammation, which was shown by lower invading inflammation cells, lower expression of matrix metalloproteinase 9, and less chemokines compared to the diabetic WT mice. Furthermore, less oxidative stress as well as extracellular matrix deposition leading to a reduction in cardiac fibrosis was also found in the diabetic RasGRF1 -/- mice compared with the diabetic WT mice., Conclusion: The deletion of RasGRF1 attenuated myocardial fibrosis and improved cardiac function in diabetic mice through inhibiting inflammation and oxidative stress.

The mitotic spindle is a very dynamic structure that is built de novo and destroyed at each round of cell division. In order to perform its fundamental function during chromosome segregation, mitotic spindle dynamics must be tightly coordinated with other cell cycle events. These changes are driven by several protein kinases, phosphatases and microtubule-associated proteins. In budding yeast, the kinase Swe1 and the phosphatase Mih1 act in concert in controlling the phosphorylation state of Cdc28, the catalytic subunit of Cdk1, the major regulator of the cell cycle. In this study we show that Swe1 and Mih1 are also involved in the control of mitotic spindle dynamics. Our data indicate that Swe1 and the Polo-like kinase Cdc5 control the balance between phosphorylated and unphosphorylated forms of Mih1, which is, in turn, important for mitotic spindle elongation. Moreover, we show that the microtubule-associated protein Bik1 is a phosphoprotein, and that Swe1 and Mih1 are both involved in controlling phosphorylation of Bik1. These results uncover new players and provide insights into the complex regulation of mitotic spindle dynamics., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: This study aimed to assess heritability of myopia in Lithuania and evaluate both genes GJD2 (Gap Junction Protein, Delta 2) and RASGRF1 (RAS protein-specific guanine nucleotide-releasing factor 1) relation with myopia., Methods: In this study Lithuanian twin population aged between 18 and 40 (n = 460) were examined. Single-nucleotide polymorphisms of the RASGRF1 (rs8027411) and GJD2 (rs634990) genes were assessed by real-time polymerase chain reaction method., Results: Intrapair correlations for spherical equivalent in all twin pairs were significantly higher in MZ twin pairs r = 0.539 (p < 0.001, 95% CI 0.353-0.684) than in DZ twin pairs r = 0.203 (p < 0.01, 95% CI 0.0633-0.442) in myopia group. Correlations for spherical equivalent in emmetropia group were not significant in MZ twin pairs r = 0.091 (p > 0.05, 95% CI -0.215-0.381) and in DZ twin pairs r = - 0.220 (p > 0.05, 95% CI -0.587-0.222). The odds ratio (95% CI) were 2.7 (1.018-7.460) for combinations of genotypes of rs634990 CC and rs8027411 GT (p = 0.046)., Conclusions: Our studies have shown that the heritability of myopia makes 67.2% in Lithuania. Persons with combinations of genotypes rs634990 CC and rs8027411 GT have 2.7 times higher odds to have myopia.

Objective: RASGRF1 is a guanine nucleotide exchange factor, which promotes the release of GDP from inactive Ras and stabilizes the apoprotein. In this study, the methylation status of RASGRF1 promoter and its correlation with clinicopathological parameters were evaluated in colorectal cancer (CRC)., Methods: Methylation-specific PCR and bisulfate-sequencing PCR were carried out to investigate the promoter methylation status of the RASGRF1 gene in tumor tissues and adjacent non-tumor tissues of 68 CRC patients. The effect of RASGRF1 promoter hypermethylation on its mRNA and protein expression was evaluated by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry., Results: RASGRF1 was methylated in 58.6% tumor tissues and negatively correlated with its mRNA or protein expression. There was a statistical relationship between the degree of differentiation, lymph node metastasis and clinical stage and RASGRF1 hypermethylation. Furthermore, RASGRF1 mRNA and protein levels in tumor tissues were both significantly decreased compared with that in adjacent non-tumor tissues. In addition, the correlations between RASGRF1 hypermethylation, mRNA or protein expression, and overall survival were statistically significant., Conclusions: Collectively, our data suggest that RASGRF1 hypermethylation is involved in an epigenetic field defect in CRC and that it might be a potential risk factor for CRC., (© 2018 by the Association of Clinical Scientists, Inc.)

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.

Purpose: The aim is to evaluate the association between high myopia and genetic variant in the BicC family RNA binding protein 1 (BICC1) as well as its association with Ras protein specific guanine nucleotide releasing factor 1 (RASGRF1) genes in a Chinese Han population with a case-control study., Methods: Five TagSNPs in BICC1 and RASGRF1 genes were selected and genotyped in 821 unrelated subjects, which composed of 419 controls (spherical equivalent within ±1.00 D in both eyes and axial length ≦24.0 mm) and 402 cases (spherical equivalent ≤-6.0D in at least one eye and axial length ≥26.0 mm). Statistical analysis was performed with SNPstats., Results: After an analysis adjusted by age and sex, rs4245599 in BICC1 was found to be significantly associated with high myopia under the codominant, dominant, recessive and log-additive model (P = 0.001, 0.0015, 0.0045 and 2e-04, odds ratio [OR] = 2.15, 1.59, 1.73 and 1.46, respectively), and rs10763559 in BICC1 was associated with high myopia and under the dominant and log-additive model (P = 0.032 and 0.036, OR = 0.72 and 0.78, respectively). Rs4778879 in RASGRF1 was found to be significantly associated with high myopia under codominant, dominant, recessive, and log-additive model (P = 0.0088, 0.0065, 0.026, and 0.0021, OR = 1.87, 1.48, 1.56, and 1.37, respectively). However, no significant association was found between rs745030 in RASGRF1 and high myopia, neither was there any association of rs745029 in RASGRF1., Conclusion: The present study indicated that genetic variants in BICC1 and RASGRF1 are closely associated with high myopia, which appears to be a potential candidate for high myopia in Chinese Han population. Considering the small sample size of this study, further work is needed to validate our results. The function of BICC1 and RASGRF1 in the process of developing high myopia needs to be explored in the future.

DNA methylation, one of the mechanisms of epigenetic regulation, has been suggested to be related with epilepsy. RASgrf1 is a paternally imprinted gene and has a differentially methylated region (DMR) at the promoter that can silence gene expression. We have previously observed the down-regulation of RASgrf1 in the temporal neocortex of epilepsy patients and in the hippocampus of epileptic animals. Here, we further explored the dynamic change (1-day acute period, 10-day latent period and 45-day chronic phase) of DNA methylation and RASgrf1 expression after acute epileptic seizures in kainic acid (KA)-treated mice, and we observed the impact of N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase (DNMT) inhibitor, on an acute epileptic model by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR (BSP). The results directly showed that the methylation of the RASgrf1 promoter gradually increased and reached a maximal level at the latent period, with subsequent suppression of RASgrf1 mRNA and protein expression levels, which reached a minimum level in the chronic phase. RG108 inhibited the increased methylation of the RASgrf1 gene, with significant inhibition occurring at the latent period, and restored RASgrf1 expression levels in the chronic phase. In addition, we demonstrated that RG108 could suppress acute epileptic seizures in KA-treated mice and epileptic discharges in 4-aminopyridine (4-AP)-treated hippocampal slices. These findings demonstrate that RASgrf1 is closely associated with epilepsy via the aberrant methylation of RASgrf1, and regulating the methylation status of relevant genes might be an intriguing topic in future research on epilepsy.

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all- trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML., Competing Interests: The authors declare no conflict of interest.

We describe a regulatory mechanism that controls the activity of retromer, an evolutionarily conserved sorting device that orchestrates cargo export from the endosome. A spontaneously arising mutation that activates the yeast ( Saccharomyces cerevisiae ) CDC25 family phosphatase, Mih1, results in accelerated turnover of a subset of endocytosed plasma membrane proteins due to deficient sorting into a retromer-mediated recycling pathway. Mih1 directly modulates the phosphorylation state of the Vps26 retromer subunit; mutations engineered to mimic these states modulate the binding affinities of Vps26 for a retromer cargo, resulting in corresponding changes in cargo sorting at the endosome. The results suggest that a phosphorylation-based gating mechanism controls cargo selection by yeast retromer, and they establish a functional precedent for CDC25 protein phosphatases that lies outside of their canonical role in regulating cell cycle progression.

Background: Genome-wide association studies of patients have linked the Rasgrf1 gene with myopia. The aim of this study was to investigate the messenger RNA (mRNA) expression of Rasgrf1 in the eyes of guinea pigs with induced myopia., Methods: The myopia was induced by form deprivation in 24 guinea pigs, while additional 12 animals served as a control. Biometric measurements were used to monitor myopic progression. The animals were sacrificed at two, three and four weeks after beginning of the monocular form deprivation, followed by dissection of the retina, and the sclera, as well as mRNA isolation from both layers. A quantitative reverse transcriptase-polymerase chain reaction was performed to detect the expression of Rasgrf1., Results: The spherical equivalent in eyes subjected to form deprivation differed from the fellow eyes, with measurements of -3.80 ± 0.08 D, -3.96 ± 0.94 D and -4.00 ± 0.94 D at the two-, three- and four-week times, respectively, significantly more myopia than the inter-ocular difference in the control group (p < 0.05). The form-deprived eyes also had a longer axial length compared with the fellow eye: 1.37 ± 0.76 mm, 1.32 ± 0.65 mm and 0.92 ± 0.80 mm at two, three and four weeks, respectively, significantly different from the control group (p < 0.05). In contrast, there was no difference in the corneal curvature, anterior chamber depth or lens thickness between the two eyes at any time (p > 0.05). The increase of Rasgrf1 expression was observed in the sclera, with a fold change of 6.596, 4.379 after three weeks and 6.788, 5.711 after four weeks of treatment, compared with the fellow eyes and the control group, respectively (p < 0.05)., Conclusion: Rasgrf1 up-regulation was found in the sclera of myopic eyes; however, further investigation is needed to determine whether Rasgrf1 plays a causative role or is a consequence of myopia-induced scleral remodelling., (© 2016 Optometry Australia.)

Objectives: In response to endoplasmic reticulum (ER) stress, endothelial cells initiate corrective pathways such as the unfolded protein response. Recent studies suggest that reactive oxygen species produced on the ER may participate in homeostatic signaling through Ras in response to ER stress. We sought to identify mechanisms responsible for this focal signaling pathway., Approach and Results: In endothelial cells, we found that ER stress induced by tunicamycin activates the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 focally on the ER surface but not on the plasma membrane. Ras activation is also restricted to the ER, occurs downstream of Nox4, and is required for activation of the unfolded protein response. In contrast, treatment with the growth factor VEGF (vascular endothelial growth factor) results in Ras activation and reactive oxygen species production confined instead to the plasma membrane and not to the ER, demonstrating local coupling of reactive oxygen species and Ras signals. We further identify the calcium-responsive, ER-resident guanyl exchange factors RasGRF1 and RasGRF2 as novel upstream mediators linking Nox4 with Ras activation in response to ER stress. Oxidation of the sarcoendoplasmic reticulum calcium ATPase and increases in cytosolic calcium caused by ER stress are blocked by Nox4 knockdown, and reduction in cytosolic free calcium prevents both Ras activation and the unfolded protein response., Conclusions: ER stress triggers a localized signaling module on the ER surface involving Nox4-dependent calcium mobilization, which directs local Ras activation through ER-associated, calcium-responsive RasGRF., (© 2016 American Heart Association, Inc.)

Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction.

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase., (Copyright © 2016 by the Genetics Society of America.)

Astrocytoma is one of the most common primary central nervous system tumors and has both high mortality and a poor 5-year survival rate. MicroRNAs (miRNAs) play important roles in carcinogenesis by acting on multiple signaling pathways. Although we have demonstrated that miR-137 is downregulated in astrocytoma tissues, the role of miR-137 in astrocytoma still remains unknown. In the present study, we aimed to investigate the function of miR-137 and its possible target genes in astrocytoma. miR-137 was significantly downregulated in astrocytoma tissues, and its expression level was inversely correlated with the clinical stage. Restoring miR-137 was able to dramatically inhibit cell proliferation, migration, and invasion and enhance apoptosis in vitro, whereas silencing its expression inhibited these processes. By overexpressing or inhibiting miR-137 in cancer cells, we experimentally confirmed that miR-137 directly recognized the 3'-UTR (3'-untranslated region) of the RASGRF1 (Ras protein-specific guanine nucleotide-releasing factor 1) transcript and regulated RASGRF1 expression. Furthermore, an inverse correlation was observed between miR-137 levels and RASGRF1 protein levels, but not mRNA levels, in astrocytoma samples. The silencing of RASGRF1 resulted in similar effects to miR-137 restoration in cancer cells. Finally, overexpression of RASGRF1 rescued the inhibitory effects of miR-137. Taken together, our results indicate that miR-137 acts as a tumor suppressor in astrocytoma by targeting RASGRF1. These findings suggest that miR-137 may serve as a novel therapeutic target in astrocytoma treatment.

Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling"., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The accumulation of amyloid-β (Aβ) leads to the loss of dendritic spines and synapses, which is hypothesized to cause cognitive impairments in Alzheimer's disease (AD) patients. In our previous study, we demonstrated that a novel mercaptoacetamide-based class II histone deacetylase inhibitor (HDACI), known as W2, decreased Aβ levels and improved learning and memory in mice. However, the underlying mechanism of this effect is unknown., Objective: Because dendritic spine formation is associated with cognitive performance, here we investigated whether HDACI W2 regulates dendritic spine density and its molecular mechanism of action., Methods: To examine the effect of HDACI W2 on dendritic spine density, we conducted morphological analysis of dendritic spines using GFP transfection and Golgi staining. In addition, to determine the molecular mechanism of W2 effects on spines, we measured the levels of mRNAs and proteins involved in the Ras signaling pathway using quantitative real-time PCR, immunocytochemistry, and western analysis., Results: We found that HDACI W2 altered dendritic spine density and morphology in vitro and in vivo. Additionally, W2 increased the mRNA or protein levels of Ras GRF1 and phospho-ERK. Moreover, knockdown of RasGRF1 and inhibition of ERK activity prevented the W2-mediated spinogenesis in primary hippocampal neurons., Conclusion: Our Class II-selective HDACI W2 promotes the formation and growth of dendritic spines in a RasGRF1 and ERK dependent manner in primary hippocampal neurons.

Purpose: To evaluate the association between polymorphisms of RASGRF1 rs8027411 and high myopia in Chinese and Japanese populations., Methods: All eligible studies investigating the association between the RASGRF1 gene and high myopia listed in PubMed, EMBASE, the Cochrane Library, Web of Science, and the China Biologic Medical Database were retrieved. The effects were assessed with the pooled odds ratio (OR) and 95% confidence interval (CI). Heterogeneity between studies was evaluated with the Q-statistic test. Publication bias was tested with Begg's and Egger's linear regression tests. Subgroup analysis and sensitivity analysis were performed to identify the sources of heterogeneity., Results: In the present meta-analysis, 2,529 individuals with high myopia and 3,127 controls from four studies were included and divided into seven groups. The results indicated that RASGRF1 rs8027411 was significantly associated with high myopia in Chinese and Japanese populations. Carriers of the rs8027411 G allele had a lower risk of high myopia compared to carriers with the T allele (G versus T, OR=0.83, 95% CI=0.77-0.89; p<0.001). Low but not significant heterogeneity was found in a recessive model. No heterogeneity was found in other genetic models. The subgroup analysis indicated that the protective effect of rs8027411 variants was more prominent in Chinese populations (G versus T, OR=0.80 in Chinese and OR=0.86 in Japanese; GG versus TT, OR=0.65 in Chinese and OR=0.77 in Japanese; GT versus TT, OR=0.76 in Chinese and OR=0.81 in Japanese; (GG+GT) versus TT, OR=0.73 in Chinese and OR=0.80 in Japanese; and GG versus (GT+TT), OR=0.77 in Chinese and OR=0.87 in Japanese). Sensitivity analysis indicated that the study results were stable in allelic, homozygote, heterozygote, and dominant models but were not stable in the recessive model. No evidence of publication bias was found., Conclusions: Carriers of the rs8027411 G allele in the RASGRF1 gene may be at a lower risk of high myopia in Chinese and Japanese populations. The RASGRF1 gene may play a role in the development of high myopia, especially in Asians. Additional studies are required to validate these results.

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the induction and prolongation of a variety of psychiatric disorders. As such, much effort has been made to understand the molecular mechanisms involved in its control. However, the vast majority of the studies on the HPA axis have used adult animals, and among these the majority has used males. Here we show that in knockout mice lacking the guanine nucleotide exchange factor, RasGRF1, habituation to 30 min/day of restraint stress is markedly accelerated, such that these mice do not display elevated corticosterone levels or enhanced locomotion after 7 days of stress exposure, like WT mice do. Strikingly, this phenotype is present in early-adolescent female RasGRF1 knockout mice, but not in their early-adolescent male, mid-adolescent female, adult female or adult male counterparts. Moreover, not only is there a clear response to restraint stress in early-adolescent female RasGRF1 knockout mice, their response after one, three and five exposures is magnified approximately threefold compared to WT mice. These findings imply that distinct mechanisms exist to regulate the HPA axis in early-adolescent females that involves RasGRF1. A full understanding of how RasGRF1 controls the HPA axis response to stress may be required to design effective strategies to combat stress-associated psychiatric disorders initiated in young females., (© 2015 Society for Endocrinology.)

Background: This study investigated the association of the 27 SNPs located in RASGRF1. GJD2, and ACTC1 genes with pathological myopia in a Chinese Han population., Methods: Myopia patients were stratified according to whether they did (n = 274) or did not (n = 131) have myopic macular degeneration (MMD). The SNPbrowser software was used to identify specific SNPs for analysis and minimal allele frequency of >20%, and a pairwise r(2) < 0.85 were genotyped using MALDI-TOF mass spectrometry., Results: Before controlling for false discovery rate, the frequency of the rs1867315 C/C genotype compared with healthy controls was lower in the myopia group (p = 0.006) and in myopia patients without macular degeneration (p = 0.019). The frequency of the rs670957A/A genotype was also lower in patients without MMD compared with controls (p = 0.045). For rs2070664, the frequency of the A allele was higher in the patients with MMD compared to those without MMD (p = 0.032). After controlling for a false discovery rate of 5%, there was no significant difference in genotype and allele frequencies between these groups., Conclusion: In this study, there was no association of the analyzed SNPs located in RASGRF1. GJD2, and ACTC1 with pathological myopia, suggesting that SNPs included in our study have no or a limited role in causing pathologic myopia in this Chinese Han population.

Background: Bidirectional long-term plasticity at the corticostriatal synapse has been proposed as a central cellular mechanism governing dopamine-mediated behavioral adaptations in the basal ganglia system. Balanced activity of medium spiny neurons (MSNs) in the direct and the indirect pathways is essential for normal striatal function. This balance is disrupted in Parkinson's disease and in l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID), a common motor complication of current pharmacotherapy of Parkinson's disease., Methods: Electrophysiological recordings were performed in mouse cortico-striatal slice preparation. Synaptic plasticity, such as long-term potentiation (LTP) and depotentiation, was investigated. Specific pharmacological inhibitors or genetic manipulations were used to modulate the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, a signal transduction cascade implicated in behavioral plasticity, and synaptic activity in different subpopulations of striatal neurons was measured., Results: We found that the Ras-ERK pathway, is not only essential for long-term potentiation induced with a high frequency stimulation protocol (HFS-LTP) in the dorsal striatum, but also for its reversal, synaptic depotentiation. Ablation of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal activator of Ras proteins, causes a specific loss of HFS-LTP in the medium spiny neurons in the direct pathway without affecting LTP in the indirect pathway. Analysis of LTP in animals with unilateral 6-hydroxydopamine lesions (6-OHDA) rendered dyskinetic with chronic L-DOPA treatment reveals a complex, Ras-GRF1 and pathway-independent, apparently stochastic involvement of ERK., Conclusions: These data not only demonstrate a central role for Ras-ERK signaling in striatal LTP, depotentiation, and LTP restored after L-DOPA treatment but also disclose multifaceted synaptic adaptations occurring in response to dopaminergic denervation and pulsatile administration of L-DOPA., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to determine the imprinting status of the Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1) gene in domestic pigs. In this study, a 228-bp partial sequence located in exon 14 and a 193-bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A novel single nucleotide polymorphism, a G/A transition, was identified in Rasgrf1 exon 14, and then the reciprocal Berkshire x Wannan black F1 hybrid model and the reverse transcription-polymerase chain reaction-restriction fragment length polymorphism method were used to detect the imprinting status of the porcine Rasgrf1 gene at the 1-day-old developmental stage. Imprinting analysis showed that, compared to the imprinted expression of the Rasgrf1 gene in mouse and rat, a variable imprinting status was observed in domestic pigs. In principle, the porcine Rasgrf1 gene was maternally expressed in the liver and small intestine, paternally expressed in the lung, and biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, fat, testis, ovary, longissimus dorsi, and pituitary tissues. In conclusion, our results indicated that the Rasgrf1 gene shows both species- and tissue-specific variation in imprinted expression.

Rasgrf-1 is a guanine exchange factor (GEF) that catalyzes the exchange of GDP for GTP. In a RNA microarray analysis of chronic lymphocytic leukemia (CLL) specimens (n = 5), this gene was found to be overexpressed in CLL as compared to normal peripheral blood mononuclear cell (PBMC) CD19 + B cells (n = 3). CLL specimens (n = 29) expressed Rasgrf-1 RNA at levels 5-300-fold higher as compared to normal B cells. CLL specimens expressed a 75 kDa isoform that was smaller than the expected full-length protein (140 kDa) and the truncated variant had higher GEF activity. Knockdown of Rasgrf-1 in CLL specimens inhibited active GTP-bound Ras and the Ras/Erk/mitogen-activated protein kinase (MAPK) pathway. Rasgrf-1 was phosphorylated and activated by B cell receptor (BCR) signaling that increased its GEF function, and this phosphorylation was blocked by Src and Bruton's tyrosine kinase (BTK) inhibitors. Rasgrf-1 is a novel GEF protein that has a role in BCR signaling and its overexpression further activates the Ras/Erk/MAPK pathway in CLL specimens.

Background: Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those "pancreatic" phenotypes., Results: Here, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins.Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice., Conclusions: The specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.

The small GTPase Cdc42 is a key regulator of polarity, but little is known in mammals about its spatial regulation and the relevance of spatial Cdc42 pools for polarity. Here we report the identification of a GM130-RasGRF complex as a regulator of Cdc42 at the Golgi. Silencing GM130 results in RasGRF-dependent inhibition of the Golgi pool of Cdc42, but does not affect Cdc42 at the cell surface. Furthermore, active Cdc42 at the Golgi is important to sustain asymmetric front-rear Cdc42-GTP distribution in directionally migrating cells. Concurrent to Cdc42 inhibition, silencing GM130 also results in RasGRF-dependent Ras-ERK pathway activation. Moreover, depletion of GM130 is sufficient to induce E-cadherin downregulation, indicative of a loss in cell polarity and epithelial identity. Accordingly, GM130 expression is frequently lost in colorectal and breast cancer patients. These findings establish a previously unrecognized role for a GM130-RasGRF-Cdc42 connection in regulating polarity and tumorigenesis.

Background: Refractive errors and high myopia are the most common ocular disorders, and both of them are leading causes of blindness in the world. Recently, genetic association studies in European and Japanese population identified that common genetic variations located in 15q14 and 15q25 were associated with high myopia. To validate whether the same variations conferred risk to high myopia in the Han Chinese population, we genotyped 1,461 individuals (940 controls and 521 cases samples) recruited of Han Chinese origin., Result: We found rs8027411 in 15q25 (P = 0.012 after correction, OR = 0.78) was significantly associated with high myopia but rs634990 in 15q14 (P = 0.54 after correction), OR = 0.88) was not., Conclusions: Our findings supported that 15q25 is a susceptibility locus for high myopia, and gene RASGRF1 was possible to play a role in the pathology of high myopia.

The dentate gyrus of the hippocampus plays a pivotal role in pattern separation, a process required for the behavioral task of contextual discrimination. One unique feature of the dentate gyrus that contributes to pattern separation is adult neurogenesis, where newly born neurons play a distinct role in neuronal circuitry. Moreover,the function of neurogenesis in this brain region differs in adolescent and adult mice. The signaling mechanisms that differentially regulate the distinct steps of adult neurogenesis in adolescence and adulthood remain poorly understood. We used mice lacking RASGRF1(GRF1), a calcium-dependent exchange factor that regulates synaptic plasticity and participates in contextual discrimination performed by mice, to test whether GRF1 plays a role in adult neurogenesis.We show Grf1 knockout mice begin to display a defect in neurogenesis at the onset of adulthood (~2 months of age), when wild-type mice first acquire the ability to distinguish between closely related contexts. At this age, young hippocampal neurons in Grf1 knockout mice display severely reduced dendritic arborization. By 3 months of age, new neuron survival is also impaired. BrdU labeling of new neurons in 2-month-old Grf1 knockout mice shows they begin to display reduced survival between 2 and 3 weeks after birth, just as new neurons begin to develop complex dendritic morphology and transition into using glutamatergic excitatory input. Interestingly, GRF1 expression appears in new neurons at the developmental stage when GRF1 loss begins to effect neuronal function. In addition, we induced a similar loss of new hippocampal neurons by knocking down expression of GRF1 solely in new neurons by injecting retrovirus that express shRNA against GRF1 into the dentate gyrus. Together, these findings show that GRF1 expressed in new neurons promotes late stages of adult neurogenesis. Overall our findings show GRF1 to be an age-dependent regulator of adult hippocampal neurogenesis, which contributes to ability of mice to distinguish closely related contexts.

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F1 hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F1 hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Costello syndrome is linked to activating mutations of a residue in the p-loop or the NKCD/SAK motifs of Harvey Ras (HRas). More than 10 HRas mutants that induce Costello syndrome have been identified; G12S HRas is the most prevalent of these. However, certain HRas p-loop mutations also are linked to cancer formation that are exemplified with G12V HRas. Despite these relations, specific links between types of HRas mutations and diseases evade definition because some Costello syndrome HRas p-loop mutations, such as G12S HRas, also often cause cancer. This study established novel kinetic parameter-based equations that estimate the value of the cellular fractions of the GTP-bound active form of HRas mutant proteins. Such calculations differentiate between two basic kinetic mechanisms that populate the GTP-bound form of Ras in cells. (i) The increase in the level of GTP-bound Ras is caused by the HRas mutation-mediated perturbation of the intrinsic kinetic characteristics of Ras. This generates a broad spectrum of the population of the GTP-bound form of HRas that typically causes Costello syndrome. The upper end of this spectrum of HRas mutants, as exemplified by G12S HRas, can also cause cancer. (ii) The increase in the level of GTP-bound Ras occurs because the HRas mutations perturb the action of p120GAP on Ras. This causes production of a significantly high population of the only GTP-bound form of HRas linked merely to cancer formation. HRas mutant G12V belongs to this category.

Purpose: We investigated the association of genetic variations, which were identified recently in a large-scale genome-wide association study (GWAS) to confer risk of refractive error and common myopia in Caucasians, with high myopia in Japanese subjects., Methods: The 5 single-nucleotide polymorphisms (SNPs) from the 5 genes TOX, RDH5, ZIC2, RASGRF1, and SHISA6, were genotyped in 1339 unrelated highly myopic Japanese patients and 3248 healthy Japanese participants in the Nagahama Study. In addition, genotypes were compared between high myopia patients without choroidal neovascularization (CNV) and patients with myopic CNV., Results: Significant associations between rs8000973 near ZIC2 (P = 7.16 × 10(-7)), rs4778879 in RASGRF1 (P = 3.40 × 10(-7)), and rs2969180 in SHISA6 (P = 0.033) and high myopia were observed. Odds ratios (95% confidence intervals) were 1.33 (1.19-1.49), 0.78 (0.71-0.86), and 1.11 (1.01-1.22) for the rs8000973 C allele, rs4778879 A allele, and rs2969180 G allele, respectively. The effect of the rs2969180 allele G contrasted with that observed in the original report, whereas the effect of the other 2 SNPs agreed. Further analysis using controls with -1.0 diopter (D) ≤ spherical equivalent ≤ +1.0 D showed a significant association between ZIC2 and RASGRF1, but not SHISA6. Among the patients with high myopia, 516 had myopic CNV in either eye, while 823 patients did not have myopic CNV in eyes. No evaluated genes showed a significant association with the development of myopic CNV., Conclusions: ZIC2 and RASGRF1 are susceptibility genes, not only for common myopia, but also for high myopia.

Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). FAK is recruited to adhesions and activated by integrin receptors binding to matrix proteins, such as fibronectin (FN). Canonical models place Rgnef downstream of integrin-FAK signaling in regulating Rho GTPase activity and cell movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK localization to early peripheral adhesions and promoting FAK activation upon FN binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires Rgnef-FAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the Rgnef-FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion localization, but not paxillin-Y118 phosphorylation. This suggests that, downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a mechanism distinct from adhesion formation and FAK activation. These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner.

The Ras family of small G-proteins plays an essential role in the regulation of a variety of signal transduction processes, ranging from cell cycle control to the regulation of exocytosis. Signalling by the Ras G-proteins is initiated by the CDC25 homology domain (CDC25-HD) containing guanine nucleotide exchange factors (GEFs); each GEF, with its specific selectivity profile towards G-proteins, commonly acts on only a small subset of the Ras family members. Thus, GEFs play a pivotal part in establishing the activation of the downstream signalling routes. The structural basis for the establishment of selectivity in the GEF-G-protein interaction is only partially understood, and several controversies on the selectivity of GEFs are discussed in the literature. In the present study, we undertook a systematic approach to determine the selectivity of CDC25-HD for members of the Ras family. We generated a data set of 126 pairs using a standardised in vitro approach encompassing purified recombinant proteins, and a comprehensive mutational study analysed the basis of the selectivity. Together, these data highlight the distinct selectivity of various GEFs and allow for predictions of untested combinations of GEFs and G-proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)