RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway.

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.

PINCH-1 promotes IGF-1 receptor expression and skin cancer progression through inhibition of the GRB10-NEDD4 complex.

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.

Treadmill training attenuate STZ-induced cognitive dysfunction in type 2 diabetic rats via modulating Grb10/IGF-R signaling.

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-1Rß 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.

Circ_0009910 shuttled by exosomes regulates proliferation, cell cycle and apoptosis of acute myeloid leukemia cells by regulating miR-5195-3p/GRB10 axis.

The exosomes are involved in intercellular communication via RNA trafficking in human diseases. Hsa_circ_0009910 (circ_0009910) is a novel leukemia-related circular RNA. However, the mechanism of circ_0009910 in acute myeloid leukemia (AML) cell-to-cell communication remained obscure. Expression of circ_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_0009910, miR-5195-3p and GRB10 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. As a result, circ_0009910 was upregulated in AML bone marrows and cells (HL-60 and MOLM-13), even higher in AML cells-derived exosomes. Functionally, blocking circ_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_0009910 shuttled via exosomes negatively regulated miR-5195-3p expression by target binding. Furthermore, circ_0009910 knockdown via exosomes and miR-5195-3p overexpression via mimic resulted in similar results of circ_0009910 siRNA in proliferation, apoptosis and cell cycle progression of AML cells. Meanwhile, the role of circ_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_0009910-containing exosomes mediated proliferation, apoptosis and cell cycle progression of AML cells partially through miR-5195-3p/GRB10 axis.

GRB10 sustains AR activity by interacting with PP2A in prostate cancer cells.

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.

Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes.

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)-IκBα 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.

Proproliferative function of adaptor protein GRB10 in prostate carcinoma.

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.

Patient-derived Hormone-naive Prostate Cancer Xenograft Models Reveal Growth Factor Receptor Bound Protein 10 as an Androgen Receptor-repressed Gene Driving the Development of Castration-resistant Prostate Cancer.

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.

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways.

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.

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization.

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.

Placental imprinting variation associated with assisted reproductive technologies and subfertility.

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.

Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration.

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 (Grb10m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10+/+ mice. After total body irradiation (TBI), Grb10m/+ 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.

The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2.

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.

The neuropilin-like protein ESDN regulates insulin signaling and sensitivity.

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.

Monoallelic loss of the imprinted gene Grb10 promotes tumor formation in irradiated Nf1+/- mice.

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.

Carrageenan Inhibits Insulin Signaling through GRB10-mediated Decrease in Tyr(P)-IRS1 and through Inflammation-induced Increase in Ser(P)307-IRS1.

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.

Grb10 is involved in BCR-ABL-positive leukemia in mice.

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.

Negative regulation of Grb10 Interacting GYF Protein 2 on insulin-like growth factor-1 receptor signaling pathway caused diabetic mice cognitive impairment.

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.

Grb10 promotes lipolysis and thermogenesis by phosphorylation-dependent feedback inhibition of mTORC1.

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.

A novel function of microRNA 130a-3p in hepatic insulin sensitivity and liver steatosis.

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.