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The aberrant expression of placental imprinted genes due to epigenetic alterations during pregnancy can impact fetal development. We investigated the impact of dietary modification of low vitamin B12 with varying doses of folic acid on the epigenetic control of imprinted genes and fetal development using a transgenerational model of C57BL/6J mice. The animals were kept on four distinct dietary combinations based on low vitamin B12 levels and modulated folic acid, mated in the F0 generation within each group. In the F1 generation, each group of mice is split into two subgroups; the sustained group was kept on the same diet, while the transient group was fed a regular control diet. After mating, maternal placenta (F1) and fetal tissues (F2) were isolated on day 20 of gestation. We observed a generation-wise opposite promoter CpG methylation and gene expression trend of the two developmental genes Dlk1 and Grb10, with enhanced gene expression in both the sustained and transient experimental groups in F1 placentae. When fetal development characteristics and gene expression were correlated, there was a substantial negative association between placental weight and Dlk1 expression (r = - 0.49, p < 0.05) and between crown-rump length and Grb10 expression (r = - 0.501, p < 0.05) in fetuses of the F2 generation. Consistent with these results, we also found that H3K4me3 at the promoter level of these genes is negatively associated with all fetal growth parameters. Overall, our findings suggest that balancing vitamin B12 and folic acid levels is important for maintaining the transcriptional status of imprinted genes and fetal development., Competing Interests: Declarations Ethics approval and consent to participate All the animal experiments were conducted after approval from the Institutional Animal Ethics Committee, Post Graduate Institute of Medical Education and Research, Chandigarh (PGIMER/IAEC/566). Consent for publication All authors provided consent for publication. Competing interests The authors declare that there are no competing interests., (© 2024. The Author(s).)

Background: The growth factor receptor bound protein 7 (Grb7) family of signalling adaptor proteins comprises Grb7, Grb10 and Grb14. Each can interact with the insulin receptor and other receptor tyrosine kinases, where Grb10 and Grb14 inhibit insulin receptor activity. In cell culture studies they mediate functions including cell survival, proliferation, and migration. Mouse knockout (KO) studies have revealed physiological roles for Grb10 and Grb14 in glucose-regulated energy homeostasis. Both Grb10 KO and Grb14 KO mice exhibit increased insulin signalling in peripheral tissues, with increased glucose and insulin sensitivity and a modestly increased ability to clear a glucose load. In addition, Grb10 strongly inhibits fetal growth such that at birth Grb10 KO mice are 30% larger by weight than wild type littermates., Results: Here, we generate a Grb7 KO mouse model. We show that during fetal development the expression patterns of Grb7 and Grb14 each overlap with that of Grb10. Despite this, Grb7 and Grb14 did not have a major role in influencing fetal growth, either alone or in combination with Grb10. At birth, in most respects both Grb7 KO and Grb14 KO single mutants were indistinguishable from wild type, while Grb7:Grb10 double knockout (DKO) were near identical to Grb10 KO single mutants and Grb10:Grb14 DKO mutants were slightly smaller than Grb10 KO single mutants. In the developing kidney Grb7 had a subtle positive influence on growth. An initial characterisation of Grb7 KO adult mice revealed sexually dimorphic effects on energy homeostasis, with females having a significantly smaller renal white adipose tissue depot and an enhanced ability to clear glucose from the circulation, compared to wild type littermates. Males had elevated fasted glucose levels with a trend towards smaller white adipose depots, without improved glucose clearance., Conclusions: Grb7 and Grb14 do not have significant roles as inhibitors of fetal growth, unlike Grb10, and instead Grb7 may promote growth of the developing kidney. In adulthood, Grb7 contributes subtly to glucose mediated energy homeostasis, raising the possibility of redundancy between all three adaptors in physiological regulation of insulin signalling and glucose handling., (© 2024. The Author(s).)

Background: Optimal size at birth dictates perinatal survival and long-term risk of developing common disorders such as obesity, type 2 diabetes and cardiovascular disease. The imprinted Grb10 gene encodes a signalling adaptor protein capable of inhibiting receptor tyrosine kinases, including the insulin receptor (Insr) and insulin-like growth factor type 1 receptor (Igf1r). Grb10 restricts fetal growth such that Grb10 knockout (KO) mice are at birth some 25-35% larger than wild type. Using a mouse genetic approach, we test the widely held assumption that Grb10 influences growth through interaction with Igf1r, which has a highly conserved growth promoting role., Results: Should Grb10 interact with Igf1r to regulate growth Grb10:Igf1r double mutant mice should be indistinguishable from Igf1r KO single mutants, which are around half normal size at birth. Instead, Grb10:Igf1r double mutants were intermediate in size between Grb10 KO and Igf1r KO single mutants, indicating additive effects of the two signalling proteins having opposite actions in separate pathways. Some organs examined followed a similar pattern, though Grb10 KO neonates exhibited sparing of the brain and kidneys, whereas the influence of Igf1r extended to all organs. An interaction between Grb10 and Insr was similarly investigated. While there was no general evidence for a major interaction for fetal growth regulation, the liver was an exception. The liver in Grb10 KO mutants was disproportionately overgrown with evidence of excess lipid storage in hepatocytes, whereas Grb10:Insr double mutants were indistinguishable from Insr single mutants or wild types., Conclusions: Grb10 acts largely independently of Igf1r or Insr to control fetal growth and has a more variable influence on individual organs. Only the disproportionate overgrowth and excess lipid storage seen in the Grb10 KO neonatal liver can be explained through an interaction between Grb10 and the Insr. Our findings are important for understanding how positive and negative influences on fetal growth dictate size and tissue proportions at birth., (© 2024. The Author(s).)

Background: Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism., Methods: Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed., Results: Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway., Conclusion: Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment., (© 2023. The Feinstein Institute for Medical Research.)

Leptin acts on hypothalamic neurons expressing agouti-related protein (AgRP) or pro-opiomelanocortin (POMC) to suppress appetite and increase energy expenditure, but the intracellular mechanisms that modulate central leptin signalling are not fully understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an adaptor protein that binds to the insulin receptor and negatively regulates its signalling pathway, can interact with the leptin receptor and enhance leptin signalling. Ablation of Grb10 in AgRP neurons promotes weight gain, while overexpression of Grb10 in AgRP neurons reduces body weight in male and female mice. In parallel, deletion or overexpression of Grb10 in POMC neurons exacerbates or attenuates diet-induced obesity, respectively. Consistent with its role in leptin signalling, Grb10 in AgRP and POMC neurons enhances the anorexic and weight-reducing actions of leptin. Grb10 also exaggerates the inhibitory effects of leptin on AgRP neurons via ATP-sensitive potassium channel-mediated currents while facilitating the excitatory drive of leptin on POMC neurons through transient receptor potential channels. Our study identifies Grb10 as a potent leptin sensitizer that contributes to the maintenance of energy homeostasis by enhancing the response of AgRP and POMC neurons to leptin., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Circular RNAs (circRNAs) are abundant in the brain and contribute to central nervous system diseases; however, the exact roles of circRNAs in human traumatic brain injury (TBI) have not been established. In this study, we used a competing endogenous RNA (ceRNA) chipset as well as in vitro and in vivo assays to characterize differentially expressed circRNAs in TBI. We detected 3035 differentially expressed circRNAs in the severe TBI group, 2362 in the moderate group, and 433 in the mild group. A ceRNA network was constructed. The circRNA has&#95;circ&#95;0020269 (circHtra1) was significantly upregulated after brain insults and was correlated with the severity of injury. circHtra1 inhibited cell proliferation and promoted apoptosis, and its knockdown reversed these effects. Further analyses revealed that circHtra1 functions as a miR-3960 sponge and increases the expression of GRB10 , which is involved in NK cell infiltration after TBI. circHtra1 was identified as a target of the IGF-1/ADAR1 axis. Reduced expression of ADAR1 (involved in A-to-I editing) after brain insults upregulated circHtra1. Our results show that circHtra1 promotes neuronal loss by sponging miR-3960 and regulating GRB10 and apoptosis during brain insults. In addition, A-to-I editing could regulate circRNA expression profiles after TBI, and circHtra1 is a potential therapeutic target., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Ping Zheng et al.)

Type 2 diabetes is a major factor contributing to cognitive decline and Alzheimer's disease (AD). Treadmill running is considered to be a critical approach for mice and rats to lower blood sugar and improve learning and memory capacity. The growth factor receptor-bound protein 10 (Grb10) has been proposed to inhibit insulin signaling and defective brain insulin signaling resulted in the cognitive deficits in patients with AD. However, the positive roles of treadmill training on diabetic- related impaired cognitive function and their molecular mechanisms remain unclear. Here, to investigate whether there was neuroprotective effects of treadmill training on impaired cognitive function caused by diabetes, the rats were injected intraperitoneally with streptozotocin at a dose of 30 mg/kg to establish diabetic model (DM). We found that higher Grb10, BACE1 and PHF10 protein levels in the hippocampus of DM rats, lower phosphorylation IGF-1Rβ and IRS-1( ser307 ). However, 8 weeks treadmill training effectively reduced abnormal Grb10, enhanced postsynaptic density protein PSD-93, PSD-95, SYN expressions of hippocampus, restored PI3K/Akt/ERK and mTOR/AMPK signaling, thus alleviated spatial learning and memory deficit, compared with DM group. Additionally, treadmill training also increased GLUT4 transportation. Overall, our findings suggest that treadmill intervention improved cognitive impairments caused by diabetes disease partly through modulating Grb10/ PI3K/Akt/ERK as well as mTOR/AMPK signaling., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling., Competing Interests: Conflict of interest The authors declare that no conflict of interest exists., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Insulin-like growth factor 1 receptor (IGF-1R) expression and signaling play important roles in promotion of skin cancer progression. Identification of signaling pathways that regulate IGF-1R is crucial for understanding the pathogenesis and therapeutic treatment of skin cancer. Methods: Molecular, cellular and genetic approaches were used to investigate the function of PINCH-1 in regulation of IGF-1R expression and skin cell behavior. Furthermore, conditional PINCH-1 knockout mouse and carcinogen (7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA))-induced skin cancer model were employed to determine the function of PINCH-1 in regulation of IGF-1R expression and skin carcinogenesis in vivo . Results: Knockdown of PINCH-1 from HaCaT keratinocytes or A431 squamous carcinoma cells diminished IGF-1R levels, suppressed cell proliferation and increased apoptosis. Re-expression of PINCH-1 in PINCH-1 knockdown cells restored IGF-1R expression, cell proliferation and survival. Furthermore, depletion of NEDD4 effectively reversed PINCH-1 deficiency-induced down-regulation of IGF-1R expression, cell proliferation and survival. Conditional knockout of PINCH-1 from keratin 5 (K5) positive keratinocytes in mice, like depletion of PINCH-1 from keratinocytes in culture, reduced the IGF-1R level. Using a mouse model of DMBA/TPA-induced skin cancer, we show that the levels of both PINCH-1 and IGF-1R were significantly increased in response to treatment with the carcinogens. Genetic ablation of PINCH-1 from the epidermis markedly reduced the IGF-1R expression and cell proliferation despite stimulation with DMBA/TPA, resulting in resistance to chemical carcinogen-induced skin cancer initiation and progression. Conclusions: Our results reveal a PINCH-1-NEDD4-IGF-1R signaling axis that is critical for promotion of skin tumorigenesis and suggest a new strategy for therapeutic control of skin cancer progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The exosomes are involved in intercellular communication via RNA trafficking in human diseases. Hsa&#95;circ&#95;0009910 (circ&#95;0009910) is a novel leukemia-related circular RNA. However, the mechanism of circ&#95;0009910 in acute myeloid leukemia (AML) cell-to-cell communication remained obscure. Expression of circ&#95;0009910, miRNA (miR)-5195-3p and growth factor receptor-bound protein 10 (GRB10) was detected by quantitative real-time polymerase chain reaction and Western blotting. A stable cell coculture model was established and functional experiment was performed using Cell Counting Kit-8 assay, flow cytometry, and Western blotting. The interaction among circ&#95;0009910, miR-5195-3p and GRB10 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. As a result, circ&#95;0009910 was upregulated in AML bone marrows and cells (HL-60 and MOLM-13), even higher in AML cells-derived exosomes. Functionally, blocking circ&#95;0009910 via small interfering RNA (siRNA) suppressed cell proliferation and cell cycle progression, but facilitated apoptosis rate of HL-60 and MOLM-13 cells, accompanied with lower B-cell lymphoma 2 (Bcl-2) level and higher Bcl-2-associated X protein (Bax) level. circ&#95;0009910 shuttled via exosomes negatively regulated miR-5195-3p expression by target binding. Furthermore, circ&#95;0009910 knockdown via exosomes and miR-5195-3p overexpression via mimic resulted in similar results of circ&#95;0009910 siRNA in proliferation, apoptosis and cell cycle progression of AML cells. Meanwhile, the role of circ&#95;0009910 knockdown in AML cells was partially reversed by miR-5195-3p deletion, and restoring GRB10 could abrogate miR-5195-3p effect as well. Notably, GRB10 was a downstream target of miR-5195-3p. circ&#95;0009910-containing exosomes mediated proliferation, apoptosis and cell cycle progression of AML cells partially through miR-5195-3p/GRB10 axis., (© 2021 John Wiley & Sons Ltd.)

Prostate cancer (PCa) progression is driven by androgen receptor (AR) signaling. Unfortunately, androgen-deprivation therapy and the use of even more potent AR pathway inhibitors (ARPIs) cannot bring about a cure. ARPI resistance (ie, castration-resistant PCa, CRPC) will inevitably develop. Previously, we demonstrated that GRB10 is an AR transcriptionally repressed gene that functionally contributes to CRPC development and ARPI resistance. GRB10 expression is elevated prior to CRPC development in our patient-derived xenograft models and is significantly upregulated in clinical CRPC samples. Here, we analyzed transcriptomic data from GRB10 knockdown in PCa cells and found that AR signaling is downregulated. While the mRNA expression of AR target genes decreased upon GRB10 knockdown, AR expression was not affected at the mRNA or protein level. We further found that phosphorylation of AR serine 81 (S81), which is critical for AR transcriptional activity, is decreased by GRB10 knockdown and increased by its overexpression. Luciferase assay using GRB10-knockdown cells also indicate reduced AR activity. Immunoprecipitation coupled with mass spectrometry revealed an interaction between GRB10 and the PP2A complex, which is a known phosphatase of AR. Further validations and analyses showed that GRB10 binds to the PP2Ac catalytic subunit with its PH domain. Mechanistically, GRB10 knockdown increased PP2Ac protein stability, which in turn decreased AR S81 phosphorylation and reduced AR activity. Our findings indicate a reciprocal feedback between GRB10 and AR signaling, implying the importance of GRB10 in PCa progression., (© 2020 Union for International Cancer Control.)

Aging induces cellular and molecular changes including modification of stem cell pools. In particular, alterations in aging neural stem cells (NSCs) are linked to age-related cognitive decline which can be modulated by lifestyle. Nutrient-sensing pathways provide a molecular basis for the link between lifestyle and cognitive decline. Adopting a back-translation strategy using stem cell biology to inform epidemiological analyses, here we show associations between cellular readouts of NSC maintenance and expression levels of nutrient-sensing genes following NSC exposure to aging human serum as well as morphological and gene expression alterations following repeated passaging. Epidemiological analyses on the identified genes showed associations between polymorphisms in SIRT1 and ABTB1 and cognitive performance as well as interactions between SIRT1 genotype and physical activity and between GRB10 genotype and adherence to a Mediterranean diet. Our study contributes to the understanding of neural stem cell molecular mechanisms underlying human cognitive aging and hints at lifestyle modifiable factors.

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis., Competing Interests: SZ, AM, JU, JV, VB, SL, VS, JC No competing interests declared, (© 2019, Zhang et al.)

Exposure to low concentration of the common food additive carrageenan (10 mg/L) for only six days led to glucose intolerance and insulin resistance in the C57BL/6J mouse. Longer exposure produced fasting hyperglycemia but with no increase in weight, in contrast to the HFD. Glucose intolerance was attributable to carrageenan-induced inflammation and to increased expression of GRB10. Both HFD and carrageenan increased p(Ser32)-I κ B α and p(Ser307)-IRS1, and the increases were greater following the combined exposure. The effects of carrageenan were inhibited by the combination of the free radical inhibitor Tempol and BCL10 siRNA, which had no impact on the HFD-mediated increase. In contrast, the PKC inhibitor sotrastaurin blocked the HFD-induced increases, without an effect on the carrageenan-mediated effects. HFD had no impact on the expression of GRB10. Both carrageenan and high fat increased hepatic infiltration by F4/80-positive macrophages. Serum galectin-3 and galectin-3 binding to the insulin receptor increased by carrageenan and by HFD. Tyrosine phosphorylation of the insulin receptor declined following either exposure and was further reduced by their combination. Carrageenan reduced the activity of the enzyme N-acetylgalactosamine-4-sulfatase (ARSB; arylsulfatase B), which was unchanged following HFD. Dietary exposure to both high fat and carrageenan can impair insulin signaling through both similar and distinct mechanisms.

Growth factor receptor-binding protein 10 (GRB10) is a well-known adaptor protein and a recently identified substrate of the mammalian target of rapamycin (mTOR). Depletion of GRB10 increases insulin sensitivity and overexpression suppresses PI3K/Akt signaling. Because the major reason for the limited efficacy of PI3K/Akt-targeted therapies in prostate cancer (PCa) is loss of mTOR-regulated feedback suppression, it is therefore important to assess the functional importance and regulation of GRB10 under these conditions. On the basis of these background observations, we explored the status and functional impact of GRB10 in PCa and found maximum expression in phosphatase and tensin homolog (PTEN)-deficient PCa. In human PCa samples, GRB10 inversely correlated with PTEN and positively correlated with pAKT levels. Knockdown of GRB10 in nontumorigenic PTEN null mouse embryonic fibroblasts and tumorigenic PCa cell lines reduced Akt phosphorylation and selectively activated a panel of receptor tyrosine kinases. Similarly, overexpression of GRB10 in PTEN wild-type PCa cell lines accelerated tumorigenesis and induced Akt phosphorylation. In PTEN wild-type PCa, GRB10 overexpression promoted mediated PTEN interaction and degradation. PI3K (but not mTOR) inhibitors reduced GRB10 expression, suggesting primarily PI3K-driven regulation of GRB10. In summary, our results suggest that GRB10 acts as a major downstream effector of PI3K and has tumor-promoting effects in prostate cancer.-Khan, M. I., Al Johani, A., Hamid, A., Ateeq, B., Manzar, N., Adhami, V. M., Lall, R. K., Rath, S., Sechi, M., Siddiqui, I. A., Choudhry, H., Zamzami, M. A., Havighurst, T. C., Huang, W., Ntambi, J. M., Mukhtar, H. Proproliferatve function of adaptor protein GRB10 in prostate carcinoma.

Purpose: Grb10 is a key imprinted gene that is suspected to have a role in the adverse outcomes of assisted reproductive technology (ART), but little is known about the effects of ART on it. Primary ART techniques, including superovulation, in vitro fertilization (IVF), and oocyte in vitro maturation (IVM), were analyzed in this study of the effects of ART on embryo quality and Grb10., Methods: Embryo development rates were determined. Blastocyst cell number and global methylation were analyzed at the single-embryo level, together with Grb10 methylation and mRNA expression of the imprinted genes., Results: Lower blastocyst cell number, higher genome and Grb10 CGI1 methylation, and variable mRNA expression were observed in the ART groups compared with the control group. Whether fertilization was in vivo or in vitro, the changes in the genome and Grb10 CGI1 methylation level and Grb10 and H19 expression were similar in the groups with superovulation and more significant than the IVM group., Conclusions: These results suggest that superovulation had a greater impact than IVF or IVM on the genome and Grb10 DNA methylation level, and Grb10 and H19 expression.

Serial cloning by somatic cell nuclear transfer (SCNT) is a critical tool for the expansion of precious transgenic lines or resetting the lifespan of primary transgenic cells for multiple genetic modifications. We successfully produced second-generation cloned goats using donor neonatal fibroblasts from first-generation clones. However, our attempts to produce any third-generation clones failed. SCNT efficiency decreased progressively with the clonal generations. The rate of pregnancy loss was significantly greater in recloning groups (P<0.05). While no pregnancy loss was observed during the first round of SCNT, 14 out of 21 pregnancies aborted in the second round of SCNT and all pregnancies aborted in the third round of SCNT. In this retrospective study, we also investigated the expression of 21 developmentally important genes in muscle tissue of cloned (G1) and recloned (G2) offspring. The expression of most of these genes in live clones was found to be largely comparable to naturally reproduced control goats, but fibroblast growth factor 10 (FGF10), methyl CpG binding protein 2 (MECP2) and growth factor receptor bound protein 10 (GRB10) were differentially expressed (P<0.05) in G2 goats compared with G1 and controls. To study the effects of serial cloning on DNA methylation, the methylation pattern of differentially methylated regions in imprinted genes H19 and insulin like growth factor 2 receptor (IGF2R) were also analysed. Aberrant H19 DNA methylation patterns were detected in G1 and G2 clones.

Background: Although androgen deprivation therapy is initially effective in controlling growth of hormone-naive prostate cancers (HNPCs) in patients, currently incurable castration-resistant prostate cancer (CRPC) inevitably develops., Objective: To identify CRPC driver genes that may provide new targets to enhance CRPC therapy., Design, Setting, and Participants: Patient-derived xenografts (PDXs) of HNPCs that develop CRPC following host castration were examined for changes in expression of genes at various time points after castration using transcriptome profiling analysis; particular attention was given to pre-CRPC changes in expression indicative of genes acting as potential CRPC drivers., Outcome Measurements and Statistical Analysis: The functionality of a potential CRPC driver was validated via its knockdown in cultured prostate cancer cells; its clinical relevance was established using data from prostate cancer patient databases., Results and Limitations: Eighty genes were found to be significantly upregulated at the CRPC stage, while seven of them also showed elevated expression prior to CRPC development. Among the latter, growth factor receptor bound protein 10 (GRB10) was the most significantly and consistently upregulated gene. Moreover, elevated GRB10 expression in clinical prostate cancer samples correlated with more aggressive tumor types and poorer patient treatment outcome. GRB10 knockdown markedly reduced prostate cancer cell proliferation and activity of AKT, a well-established CRPC mediator. A positive correlation between AKT activity and GRB10 expression was also found in clinical cohorts., Conclusions: GRB10 acts as a driver of CRPC and sensitizes androgen receptor pathway inhibitors, and hence GRB10 targeting provides a novel therapeutic strategy for the disease., Patient Summary: Development of castration-resistant prostate cancer (CRPC) is a major problem in the management of the disease. Using state-of-the-art patient-derived hormone-naive prostate cancer xenograft models, we found and validated the growth factor receptor bound protein 10 gene as a driver of CRPC, indicating that it may be used as a new molecular target to enhance current CRPC therapy., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

The growth factor receptor bound protein GRB10 is an imprinted gene product and a key negative regulator of the insulin, IGF1 and mTORC1 signaling pathways. GRB10 is highly expressed in mouse fetal liver but almost completely silenced in adult mice, suggesting a potential detrimental role of this protein in adult liver function. Here we show that the Grb10 gene could be reactivated in adult mouse liver by acute endoplasmic reticulum stress (ER stress) such as tunicamycin or a short-term high-fat diet (HFD) challenge, concurrently with increased unfolded protein response (UPR) and hepatosteatosis. Lipogenic gene expression and acute ER stress-induced hepatosteatosis were significantly suppressed in the liver of the liver-specific GRB10 knockout mice, uncovering a key role of Grb10 reactivation in acute ER stress-induced hepatic lipid dysregulation. Mechanically, acute ER stress induces Grb10 reactivation via an ATF4-mediated increase in Grb10 gene transcription. Our study demonstrates for the first time that the silenced Grb10 gene can be reactivated by acute ER stress and its reactivation plays an important role in the early development of hepatic steatosis., (© 2018 Society for Endocrinology.)

Imprinted genes are expressed from one parental allele only as a consequence of epigenetic events that take place in the mammalian germ line and are thought to have evolved through intragenomic conflict between parental alleles. We demonstrate, for the first time, oppositional effects of imprinted genes on brain and behavior. Specifically, we show that mice lacking paternal Grb10 make fewer impulsive choices, with no dissociable effects on a separate measure of impulsive action. Taken together with previous work showing that mice lacking maternal Nesp55 make more impulsive choices, this suggests that impulsive choice behavior is a substrate for the action of genomic imprinting. Moreover, the contrasting effect of these two genes suggests that impulsive choices are subject to intragenomic conflict and that maternal and paternal interests pull this behavior in opposite directions. Finally, these data may also indicate that an imbalance in expression of imprinted genes contributes to pathological conditions such as gambling and drug addiction, where impulsive behavior becomes maladaptive., (Copyright © 2018 Dent et al.)

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future., (Copyright © 2017. Published by Elsevier Inc.)

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls a wide spectrum of cellular processes, including cell growth, differentiation, and metabolism. mTOR forms two distinct multiprotein complexes known as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are characterized by the presence of raptor and rictor, respectively. mTOR controls insulin signaling by regulating several downstream components such as growth factor receptor-bound protein 10 (Grb10), insulin receptor substrate (IRS-1), F-box/WD repeat-containing protein 8 (Fbw8), and insulin like growth factor 1 receptor/insulin receptor (IGF-IR/IR). In addition, mTORC1 and mTORC2 regulate each other through a feedback loop to control cell growth. This review outlines the current understanding of mTOR regulation in insulin signaling in the context of whole body metabolism.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Infertility affects one in 6 couples in developed nations, resulting in an increasing use of assisted reproductive technologies (ART). Both ART and subfertility appear to be linked to lower birth weight outcomes, setting infants up for poor long-term health. Prenatal growth is, in part, regulated via epigenetically-controlled imprinted genes in the placenta. Although differences in DNA methylation between ART and control infants have been found, it remains unclear whether these differences are due to the ART procedures or to the underlying parental subfertility and how these methylation differences affect imprinted gene expression. In this study, we examined the expression of 108 imprinted genes in placental tissues from infants born to subfertile parents (n = 79), matched naturally-conceived controls (n = 158), and infants conceived using in vitro fertilization (IVF, n = 18). Forty-five genes were identified as having significantly different expression between the subfertile infants and controls, whereas no significant differences were identified between the IVF and control groups. The expression of 4 genes-IGF2, NAPIL5, PAX8-AS1, and TUBGCP5-was significantly downregulated in the IVF compared with the subfertile group. Three of the 45 genes significantly dysregulated between subfertile and control placentae-GRB10, NDN, and CD44 -were found to have a significant positive correlation between expression and birth weight. Methylation levels for these 3 genes and 4 others-MKRN3, WRB, DHCR24, and CYR61-were significantly correlated with expression. Our findings indicate that epigenetic differences in placentas resulting from IVF pregnancies may be related to the underlying subfertility in parents using IVF rather than the IVF procedure itself.

Somatic cell nuclear transfer is frequently associated with abnormal epigenetic modifications that may lead to the developmental failure of cloned embryos. BIX-01294 (a diazepine-quinazoline-amine derivative) is a specific inhibitor of the histone methyltransferase G9a. The aim of the present study was to investigate the effects of BIX-01294 on development, dimethylation of histone H3 at lysine 9 (H3K9), DNA methylation and the expression of imprinted genes in cloned mouse preimplantation embryos. There were no significant differences in blastocyst rates of cloned embryos treated with or without 0.1μM BIX-01294. Relative to clone embryos treated without 0.1μM BIX-01294, exposure of embryos to BIX-01294 decreased histone H3K9 dimethylation and DNA methylation in cloned embryos to levels that were similar to those of in vivo-fertilised embryos at the 2-cell and blastocyst stages. Cloned embryos had lower expression of octamer-binding transcription factor 4 (Oct4) and small nuclear ribonucleoprotein N (Snrpn), but higher expression of imprinted maternally expressed transcript (non-protein coding) (H19) and growth factor receptor-bound protein 10 (Grb10) compared with in vivo-fertilised counterparts. The addition of 0.1μM BIX-01294 to the activation and culture medium resulted in lower H19 expression and higher cyclin dependent kinase inhibitor 1C (Cdkn1c) and delta-like 1 homolog (Dlk1) expression, but had no effect on the expression of Oct4, Snrpn and Grb10. The loss of methylation at the Grb10 cytosine-phosphorous-guanine (CpG) islands in cloned embryos was partially corrected by BIX-01294. These results indicate that BIX-01294 treatment of cloned embryos has beneficial effects in terms of correcting abnormal epigenetic modifications, but not on preimplantation development.

We identified the Grb7 family members, Grb10 and Grb14, as Ca 2+ -dependent CaM-binding proteins using Ca 2+ -dependent CaM-affinity chromatography as we previously did with Grb7. The potential CaM-binding sites were identified and experimentally tested using fluorescent-labeled peptides corresponding to these sites. The apparent affinity constant of these peptides for CaM, and the minimum number of calcium ions bound to CaM that are required for effective binding to these peptides were also determined. We prepared deletion mutants of the three adaptor proteins lacking the identified sites and determined that they lost or strongly diminished their CaM-binding capacity following the sequence Grb7 > > Grb14 > Grb10. More than one CaM-binding site and/or accessory CaM-binding sites appear to exist in Grb10 and Grb14, as compared to a single one present in Grb7., (© 2017 Federation of European Biochemical Societies.)

Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10 m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10 +/+ mice. After total body irradiation (TBI), Grb10 m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10 +/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin effects on cell metabolism, growth, and survival are mediated by its binding to, and activation of, insulin receptor. With increasing prevalence of insulin resistance and diabetes there is considerable interest in identifying novel regulators of insulin signal transduction. The transmembrane protein endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a novel regulator of vascular remodeling and angiogenesis. Here, we investigate a potential role of ESDN in insulin signaling, demonstrating that Esdn gene deletion promotes insulin-induced vascular smooth muscle cell proliferation and migration. This is associated with enhanced protein kinase B and mitogen-activated protein kinase activation as well as insulin receptor phosphorylation. Likewise, insulin signaling in the liver, muscle, and adipose tissue is enhanced in Esdn(-/-) mice, and these animals exhibit improved insulin sensitivity and glucose homeostasis in vivo. The effect of ESDN on insulin signaling is traced back to its interaction with insulin receptor, which alters the receptor interaction with regulatory adaptor protein-E3 ubiquitin ligase pairs, adaptor protein with pleckstrin homology and Src homology 2 domain-c-Cbl and growth factor receptor bound protein 10-neuronal precursor cell-expressed developmentally downregulated 4. In conclusion, our findings establish ESDN as an inhibitor of insulin receptor signal transduction through a novel regulatory mechanism. Loss of ESDN potentiates insulin's metabolic and mitotic effects and provides insights into a novel therapeutic avenue.

Objective: Diabetes mellitus accelerates proatherogenic and proinflammatory phenotype of vascular smooth muscle cell (VSMC) associated with vascular complications. Evidence shows that microRNAs (miRNAs) play key roles in VSMC functions, but their role under diabetic conditions is unclear. We profiled miRNAs in VSMC from diabetic mice and examined their role in VSMC dysfunction., Approach and Results: High throughput small RNA-sequencing identified 135 differentially expressed miRNAs in VSMC from type 2 diabetic db/db mice (db/dbVSMC) versus nondiabetic db/+ mice. Several of these miRNAs were known to regulate VSMC functions. We further focused on miR-504, because it was highly upregulated in db/dbVSMC, and its function in VSMC is unknown. miR-504 and its host gene Fgf13 were significantly increased in db/dbVSMC and in aortas from db/db mice. Bioinformatics analysis predicted that miR-504 targets including signaling adaptor Grb10 and transcription factor Egr2 could regulate growth factor signaling. We experimentally validated Grb10 and Egr2 as novel targets of miR-504. Overexpression of miR-504 in VSMC inhibited contractile genes and enhanced extracellular signal-regulated kinase 1/2 activation, proliferation, and migration. These effects were blocked by miR-504 inhibitors. Grb10 knockdown mimicked miR-504 functions and increased inflammatory genes. Egr2 knockdown-inhibited anti-inflammatory Socs1 and increased proinflammatory genes. Furthermore, high glucose and palmitic acid upregulated miR-504 and inflammatory genes, but downregulated Grb10., Conclusions: Diabetes mellitus misregulates several miRNAs including miR-504 that can promote VSMC dysfunction. Because changes in many of these miRNAs are sustained in diabetic VSMC even after in vitro culture, they may be involved in metabolic memory of vascular complications. Targeting such mechanisms could offer novel therapeutic strategies for diabetic complications., (© 2016 American Heart Association, Inc.)

Introduction: Imprinted genes play an important role in mammalian fetoplacental growth and development. We have evaluated whether the placental expression of two imprinted genes, growth factor receptor-binding protein 10 (GRB10) and pleckstrin homology-like domain, family A, member 2 (PHLDA2) correlate with human fetoplacental growth parameters., Methods: Placentae (n = 77) were collected from small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies (n = 36 SGA and 41 AGA). Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of GRB10 and PHLDA2 normalized to a panel of reference genes., Results: Placental GRB10 transcript abundance associated positively with placental weight (r = 0.307, P = 0.007), birth weight (r = 0.267, P = 0.019) and neonatal head circumference (r = 0.280, P = 0.014). Placental GRB10 transcript levels were significantly lower in male SGA placentae compared to the male AGA placentae. Placental PHLDA2 transcript abundance did not show any associations with maternal, placental or neonatal parameters., Discussion: Placental GRB10 expression was found to be associated positively with placental weight, birth weight, and neonatal head circumference, especially in males. Hence, we speculate that placental GRB10 plays a role in regulating fetoplacental growth and thereby in the pathophysiology of fetal growth restriction in the context of fetal gender., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10's unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.

Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis.

Inflammation induced by exposure to the common food additive carrageenan leads to insulin resistance by increase in Ser(P)(307)-insulin receptor substrate 1 (IRS1) and subsequent decline in the insulin-stimulated increase in Ser(P)(473)-AKT. Inhibition of carrageenan-induced inflammation reversed the increase in Ser(P)(307)-IRS1 but did not completely reverse the carrageenan-induced decline in Ser(P)(473)-AKT. To identify the additional mechanism responsible for the decrease in Ser(P)(473)-AKT, studies were performed in human HepG2 cells and in C57BL/6J mice. Following carrageenan, expression of GRB10 (growth factor receptor-bound 10 protein), an adaptor protein that binds to the insulin receptor and inhibits insulin signaling, increased significantly. GRB10 silencing blocked the carrageenan-induced reduction of the insulin-stimulated increase in Tyr(P)-IRS1 and partially reversed the decline in Ser(P)(473)-AKT. The combination of GRB10 silencing with BCL10 silencing and the reactive oxygen species inhibitor Tempol completely reversed the decline in Ser(P)(473)-AKT. After carrageenan, GRB10 promoter activity was enhanced because of activation by GATA2. A direct correlation between Ser(P)(473)-AKT and Ser(P)(401)-GATA2 was evident, and inhibition of AKT phosphorylation by the PI3K inhibitor LY294002 blocked Ser(401)-GATA2 phosphorylation and the increase in GRB10 expression. Studies indicated that carrageenan inhibited insulin signaling by two mechanisms: through the inflammation-mediated increase in Ser(P)(307)-IRS1, a negative regulator of insulin signaling, and through a transcriptional mechanism leading to increase in GRB10 expression and GRB10-inhibition of Tyr(P)-IRS1, a positive regulator of insulin signaling. These mechanisms converge to inhibit the insulin-induced increase in Ser(P)(473)-AKT. They provide internal feedback, mediated by Ser(P)(473)-AKT, Ser(P)(401)-GATA2, and nuclear GATA2, which links the opposing effects of serine and tyrosine phosphorylations of IRS1 and can modulate insulin responsiveness., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The SH2-containing adaptor protein Grb10 was first identified in a yeast screen as a new binding partner for BCR-ABL and associates with BCR-ABL in a tyrosine-dependent manner. However, its function in BCR-ABL-mediated leukemogenesis in vivo is still unknown. Here we describe an important role of Grb10 in BCR-ABL-induced leukemia by using a versatile system for efficient oncogene expression and simultaneous Grb10 knockdown from a single vector. Primary bone marrow (BM) cells coexpressing Grb10-miR/BCR-ABL showed a significant decrease in colony formation and cell cycle progression. Transplantation of Grb10miR/BCR-ABL- or control-miR/BCR-ABL- transduced BM leads to a CML/B-ALL-like phenotype with significantly delayed disease onset and progression resulting in prolonged overall survival in Grb10-miR-transplanted mice. Methylcellulose experiments exhibit additive effects of imatinib treatment and Grb10 knockdown. Cell cycle analysis suggests an anti-proliferative effect of Grb10 knockdown in BCR-ABL(+) primary BM cells. However, Grb10 abrogation was not capable of completely abolishing the BCR-ABL-induced disease. Our findings were confirmed in the human BCR-ABL(+) cell line K562, where we demonstrate reduced viability, cell cycle progression and induction of apoptosis by stable Grb10 microRNA expression. Taken together, our results suggest that Grb10 knockdown in vivo leads to impaired proliferation, longer survival and reduced colony formation, suggesting an important role of Grb10 in BCR-ABL-mediated leukemogenesis.

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.

Background: Despite being a fundamental biological problem the control of body size and proportions during development remains poorly understood, although it is accepted that the insulin-like growth factor (IGF) pathway has a central role in growth regulation, probably in all animals. The involvement of imprinted genes has also attracted much attention, not least because two of the earliest discovered were shown to be oppositely imprinted and antagonistic in their regulation of growth. The Igf2 gene encodes a paternally expressed ligand that promotes growth, while maternally expressed Igf2r encodes a cell surface receptor that restricts growth by sequestering Igf2 and targeting it for lysosomal degradation. There are now over 150 imprinted genes known in mammals, but no other clear examples of antagonistic gene pairs have been identified. The delta-like 1 gene (Dlk1) encodes a putative ligand that promotes fetal growth and in adults restricts adipose deposition. Conversely, Grb10 encodes an intracellular signalling adaptor protein that, when expressed from the maternal allele, acts to restrict fetal growth and is permissive for adipose deposition in adulthood., Results: Here, using knockout mice, we present genetic and physiological evidence that these two factors exert their opposite effects on growth and physiology through a common signalling pathway. The major effects are on body size (particularly growth during early life), lean:adipose proportions, glucose regulated metabolism and lipid storage in the liver. A biochemical pathway linking the two cell signalling factors remains to be defined., Conclusions: We propose that Dlk1 and Grb10 define a mammalian growth axis that is separate from the IGF pathway, yet also features an antagonistic imprinted gene pair.

Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes., (© 2014 Wiley Periodicals, Inc.)

Heterozygous Gigyf2⁺/⁻ mice exhibits histopathological evidence of neurodegeneration such as motor dysfunction. Several lines of evidence have demonstrated the important role of insulin-like growth factor-1 receptor (IGF1R) signaling pathway in the neuropathogenic process of cognitive impairment, while decreased Grb10-Interacting GYF Protein 2 (GIGYF2) expression can alter IGF1R trafficking and its downstream signaling pathways. Growth factor receptor-bound protein 10 (Grb10), a suppressor of IGF1R pathway, has been shown to play a critical role in regulating diabetes-associated cognitive impairment. It remains unknown whether endogenous GIGYF2 expression contributes to the development of diabetes-associated cognitive impairment. Using streptozotocin (STZ)-induced diabetic mice model, we first demonstrated that a significantly increased level of GIGYF2 expression was correlated with a significant decrease in the expression of phosphorylated IGF1R as well as the phosphorylation of AKT and ERK1/2, two signaling pathways downstream of IGF1R, in the hippocampus of diabetic mice. On the contrary, in situ knockdown of GIGYF2 expression in hippocampus resulted in increased expression of phosphorylated IGF1R expression and correspondingly reversed the down-regulation of ERK1/2 phsophorylation but had no obvious effect on Grb10 expression. Functionally, knockdown of GIGYF2 expression markedly ameliorated diabetes-associated cognitive dysfunction as well as the ultrastructural pathology and abnormal neurobehavioral changes. These results suggest that increased expression of GIGYF2 might contribute to the development of diabetes-associated cognitive disorder via negatively regulating IGF1R signaling pathway. Therefore, down-regulation of GIGYF2 expression may provide a potential novel approach to treat diabetes-associated cognitive impairment caused by aberrant IGF1R signaling pathway.

The goal of the current work was to profile positive (mTORC1 activation, autocrine/paracrine growth factors) and negative [AMPK, unfolded protein response (UPR)] pathways that might regulate overload-induced mTORC1 (mTOR complex 1) activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supraphysiological model of hypertrophy. To achieve this, mTORC1-IRS-1/2 signaling, BiP/CHOP/IRE1α, and AMPK activation were determined in rat plantaris muscle following synergist ablation (SA). SA resulted in significant increases in muscle mass of ~4% per day throughout the 21 days of the experiment. The expression of the insulin-like growth factors (IGF) were high throughout the 21st day of overload. However, IGF signaling was limited, since IRS-1 and -2 were undetectable in the overloaded muscle from day 3 to day 9. The decreases in IRS-1/2 protein were paralleled by increases in GRB10 Ser(501/503) and S6K1 Thr(389) phosphorylation, two mTORC1 targets that can destabilize IRS proteins. PKB Ser(473) phosphorylation was higher from 3-6 days, and this was associated with increased TSC2 Thr(939) phosphorylation. The phosphorylation of TSC2 (Thr1345) (an AMPK site) was also elevated, whereas phosphorylation at the other PKB site, Thr(1462), was unchanged at 6 days. In agreement with the phosphorylation of Thr(1345), SA led to activation of AMPKα1 during the initial growth phase, lasting the first 9 days before returning to baseline by day 12. The UPR markers CHOP and BiP were elevated over the first 12 days following ablation, whereas IRE1α levels decreased. These data suggest that during supraphysiological muscle loading at least three potential molecular brakes engage to downregulate mTORC1., (Copyright © 2014 the American Physiological Society.)

MicroRNAs (miRNAs) are endogenous, noncoding, short, single-stranded RNAs that are evolutionarily conserved and believed to play a role in controlling a variety of biological processes. The roles of miRNAs in insulin resistance and liver steatosis, however, are largely unknown. The objective of this study was to evaluate the roles of miR-130a in the regulation of insulin sensitivity and liver steatosis. In our current study, we observed that overexpression of miR-130a-3p increases insulin signaling in both HepG2 cells and primary mouse hepatocytes, and silencing of miR-130a-3p has the opposite effects. However, miR-130a-5p has no effect in the regulation of insulin signaling. Consistently, whole-body and hepatic insulin sensitivity are improved in mice injected with adenoviruses that overexpress miR-130a-3p. Furthermore, we provided evidence showing that growth factor receptor-bound protein 10 is required for miR-130a-3p-regulated insulin sensitivity. On the other hand, we observed that expression of miR-130a-3p is decreased in the livers of db/db mice and that adenovirus-mediated overexpression of miR-130a-3p reverses insulin resistance and liver steatosis, the latter of which is achieved via suppressing fatty acid synthase expression in these mice. This study identifies a novel function for hepatic miR-130a-3p in the regulation of insulin sensitivity and liver steatosis., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Identification of key regulators of lipid metabolism and thermogenic functions has important therapeutic implications for the current obesity and diabetes epidemic. Here, we show that Grb10, a direct substrate of mechanistic/mammalian target of rapamycin (mTOR), is expressed highly in brown adipose tissue, and its expression in white adipose tissue is markedly induced by cold exposure. In adipocytes, mTOR-mediated phosphorylation at Ser501/503 switches the binding preference of Grb10 from the insulin receptor to raptor, leading to the dissociation of raptor from mTOR and downregulation of mTOR complex 1 (mTORC1) signaling. Fat-specific disruption of Grb10 increased mTORC1 signaling in adipose tissues, suppressed lipolysis, and reduced thermogenic function. The effects of Grb10 deficiency on lipolysis and thermogenesis were diminished by rapamycin administration in vivo. Our study has uncovered a unique feedback mechanism regulating mTORC1 signaling in adipose tissues and identified Grb10 as a key regulator of adiposity, thermogenesis, and energy expenditure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk., Competing Interests: The authors have declared that no competing interests exist.

Imprinted genes are defined by their parent-of-origin-specific monoallelic expression. Although the epigenetic mechanisms regulating imprinted gene expression have been widely studied, their functional importance is still unclear. Imprinted genes are associated with a number of physiologies, including placental function and foetal growth, energy homeostasis, and brain and behaviour. This review focuses on genomic imprinting in the brain and on two imprinted genes in particular, Nesp and paternal Grb10, which, when manipulated in animals, have been shown to influence adult behaviour. These two genes are of particular interest as they are expressed in discrete and overlapping neural regions, recognised as key "imprinting hot spots" in the brain. Furthermore, these two genes do not appear to influence placental function and/or maternal provisioning of offspring. Consequently, by understanding their behavioural function we may begin to shed light on the evolutionary significance of imprinted genes in the adult brain, independent of the recognised role in maternal care. In addition, we discuss the potential future directions of research investigating the function of these two genes and the behavioural role of imprinted genes more generally.

The adaptor protein Grb10 is a close homolog of Grb7 and Grb14. These proteins are characterized by an N-terminal proline-rich region, a Ras-GTPase binding domain, a PH domain, an SH2 domain and a BPS domain in between the PH and SH2 domains. Human Grb10 gene encodes three splice variants. These variants show differences in functionality. Grb10 associates with multiple proteins including tyrosine kinases in a tyrosine phosphorylation dependent or independent manner. Association with multiple proteins allows Grb10 to regulate different signaling pathways resulting in different biological consequences.

MiR199a was found to be differentially expressed in rat uteri between the prereceptive and receptive phase via microRNA (miRNA) microarray analysis in our previous study. However, the role of miR199a in rat embryo implantation remained unknown. In the study, northern blot results showed that the expression levels of miR199a were higher on gestation days 5 and 6 (g.d.5-6) in rat uteri than on g.d.3-4 and g.d.7-8. In situ localization of miR199a in rat uteri showed that miR199a was mainly localized in the stroma or decidua. The expression of miR199a was not significantly different in the uteri of pseudopregnant rats and evidently increased in the uteri of rats subjected to activation of delayed implantation and experimentally induced decidualization. Treatment with 17β-estradiol or both 17β-estradiol and progesterone significantly diminished miR199a levels. Gain of function of miR199a in endometrial stromal cells isolated from rat uteri inhibited cell proliferation and promoted cell apoptosis. Loss of function of miR199a displayed opposite roles on cell proliferation and apoptosis. Further investigation uncovered a significant inverse association between the expression of miR199a and growth factor receptor-bound protein 10 (Grb10), an imprinted gene, and miR199a could bind to the 3'UTR of Grb10 to inhibit Grb10 translation. In addition, in vivo analysis found that the immunostaining of GRB10 was attenuated in the stroma or decidua from g.d.4 to 6, contrary to the enhancement of miR199a. Collectively, upregulation of miR199a in rat uterus during the receptive phase is regulated by blastocyst activation and uterine decidualization. Enforced miR199a expression suppresses cell proliferation partially through targeting Grb10.

Objective: Individual sensitivity to recombinant human GH (r-hGH) is variable. Identification of genetic factors contributing to this variability has potential use for individualization of treatment. The objective of this study was to identify genetic markers and gene expression profiles associated with growth response on r-hGH therapy in treatment-naïve, prepubertal children with GH deficiency (GHD) or Turner syndrome (TS)., Design: A prospective, multicenter, international, open-label pharmacogenomic study., Methods: The associations of genotypes in 103 growth- and metabolism-related genes and baseline gene expression profiles with growth response to r-hGH (cm/year) over the first year were evaluated. Genotype associations were assessed with growth response as a continuous variable and as a categorical variable divided into quartiles., Results: Eleven genes in GHD and ten in TS, with two overlapping between conditions, were significantly associated with growth response either as a continuous variable (seven in GHD, two in TS) or as a categorical variable (four more in GHD, eight more in TS). For example, in GHD, GRB10 was associated with high response (≥ Q3; P=0.0012), while SOS2 was associated with low response (≤ Q1; P=0.006), while in TS, LHX4 was associated with high response (P=0.0003) and PTPN1 with low response (P=0.0037). Differences in expression were identified for one of the growth response-associated genes in GHD (AKT1) and for two in TS (KRAS and MYOD1)., Conclusions: Carriage of specific growth-related genetic markers is associated with growth response in GHD and TS. These findings indicate that pharmacogenomics could have a role in individualized management of childhood growth disorders.