Grb10 Expression Research Articles

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.

Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001).Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells).In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo.To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury.Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. DisclosuresHimburg:Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

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.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell Renewal

The mechanisms which regulate HSC regeneration following stress or injury remain poorly understood. Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. Following 550cGy TBI, BM KSL cells were depleted by 7 days post-TBI, whereas KSL cell recovery was evident at day+14. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells. We identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed in regenerating BM KSL cells compared to non-irradiated KSL cells (3.3 fold, p<0.0001).Grb10 is a member of the family of imprinted genes which are predominately expressed in numerous stem cell populations, including embryonic stem cells, skin and muscle stem cells. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) in detected in 7 day culture (p=0.03 and p=0.002, respectively). Furthermore, mice which were competitively transplanted with Grb10-deficient KSL cells had 10-fold decreased donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed > 10-fold deficit in long-term repopulating capacity in Grb10 deficient KSL cells compared to Grb10 expressing KSL cells (p=0.006 for %CD45.1+ donor cells in secondary mice).In order to examine the effect of Grb10-deficiency on HSC fate and hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8-week old Grb10m/+ (1 mutant allele, 1 wild type allele) were found to have 10-fold decreased Grb10 expression in BM lin- cells and had normal range complete blood counts. However, BM CFCs were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006) and competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (1/14, 7% vs. 5/14, 38% of mice with > 0.1% donor CD45.2+ cells). These results suggest that Grb10 regulates HSC self-renewal in vitro and in vivo.Mechanistically, Grb10m/+ mice displayed no alterations in the cell cycle status or frequency of apoptotic cells within BM HSCs compared to Grb10+/+ mice. However, single cytokine functional screening suggested that Grb10 regulates SCF-mediated proliferation of HSCs. Grb10m/+ BM KSL cells generated significantly less CFCs in culture in response to SCF treatment compared to Grb10+/+ KSL cells (p=0.008). Commensurate with this, SCF-mediated activation of mTOR was significantly increased in Grb10m/+ KSL cells compared to that observed in Grb10+/+ KSL cells (p=0.006). These data suggest that cytokine-mediated induction of mTOR signaling, which has been shown to deplete functional HSCs, is antagonized by Grb10, and that Grb10 is necessary to block cytokine-mediated HSC differentiation in vitro and in vivo. Grb10 represents a novel regulator of HSC fate determination and a new mechanistic target to facilitate HSC self-renewal. Studies are underway to determine whether Grb10 is also necessary for HSC regeneration after TBI. DisclosuresNo relevant conflicts of interest to declare.

307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (&lt;3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P &lt; 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P &lt; 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P &lt; 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P &lt; 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P &lt; 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

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.

Growth receptor binding protein 10 inhibits glucose‐stimulated insulin release from pancreatic β‐cells associated with suppression of the insulin/insulin‐like growth factor‐1 signalling pathway

Growth receptor binding protein 10 (Grb10) is an adaptor protein that interacts with the insulin receptor and insulin-like growth factor (IGF)-1 receptor. Overexpression of Grb10 in muscle cells and adipocytes inhibits insulin signalling, and transgenic mice overexpressing Grb10 exhibit impaired glucose tolerance. However, the roles of Grb10 in β-cells remain unknown. The aim of the present study was to explore the effect of Grb10 on β-cell function. The effects of Grb10 on glucose-stimulated insulin secretion (GSIS) and the insulin/IGF-1 signalling pathway were investigated in rat islets and/or dispersed islet cells with Grb10 overexpresion by adenovirus transfection. Protein expression was detected by western blot analysis. We found that Grb10 was expressed in both human and rat pancreas. Expression of Grb10 was increased in islets isolated from rats fed a high-fat plus high-sugar diet compared with islets isolated from rats fed normal chow diet, as well as in INS 832/13 cells exposed to high levels of glucose (20mmol/L), palmitate (1mmol/L) and interleukin-1β (50U/mL). Overexpression of Grb10 in INS 832/13 cells or rat islets impaired GSIS compared with the respective control (all P<0.05). Moreover, inhibition of GSIS by Grb10 overexpression was associated with a decrease in insulin- and IGF-1-induced Akt and extracellular signal-regulated kinase 1/2 phosphorylation. The results of the present study demonstrate that Grb10 is an important negative regulator of insulin/IGF-1 signalling in pancreatic β-cells and a potential target to improve β-cell function.

Growth Factor Receptor‐Bound Protein 10‐Mediated Negative Regulation of the Insulin‐Like Growth Factor‐1 Receptor‐Activated Signalling Pathway Results in Cognitive Disorder in Diabetic Rats

Growth factor receptor-bound protein 10 (Grb10) is a Src homology 2 domain-containing protein and one of the binding partners for several transmembrane tyrosine kinase receptors, including insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1-R). The hippocampus, which is critical for cognitive functions, is one of the main distribution areas of Grb10 in the central nervous system. In recent years, diabetic encephalopathy has been defined as a third type of diabetes and the IGF1-IR pathway was shown to be critical for the neuropathogenic process of cognitive disorder in diabetes. However, the role of endogenous Grb10 in regulating the IGF1-IR pathway and neurobehavioural changes is not explicit. The present study aimed to determine the in vivo function of endogenous Grb10 in diabetic encephalopathy and the underlying mechanisms. Using stereotaxic surgical techniques and lentiviral vectors expressing specific short hairpin RNA, we could steadily knockdown Grb10 expression in the hippocampus. More importantly, we demonstrated that hippocampus-specific modulation of Grb10 protein levels led to a prominent remission of cognitive disorder, including improvements in both ultrastructural pathology and abnormal neurobehavioural changes. Our findings indicate that endogenous overexpression of Grb10 functions as a suppressor of the IGF1-IR pathway, which may represent an important mechanism for regulating cognitive disorder in diabetes.

Abstract 4121: Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia.

Abstract The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. The type III receptor tyrosine kinase, FLT3 is widely expressed in AML and approximately 30% of AML patients carry an oncogenic FLT3-internal tandem duplication (ITD) mutation. We observed that Grb10 physically associates with normal and oncogenic FLT3 through FLT3 phospho-tyrosine 572 and 793 residues. Although Grb10 expression did not affect FLT3 activation or stability, its expression enhanced FLT3-mediated Akt and STAT5 phosphorylation without affecting Erk or p38 phosphorylation. Selective Grb10 depletion reduced Akt and STAT5 phosphorylation in Ba/F3 and OCI-AML-5 cells. Grb10 regulates cell cycle by accelerating G2-M transition followed by an increased cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation, survival and colony formation of Ba/F3-FLT3-ITD cells. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression correlated to relapse as well as to poor prognosis. In addition, Grb10 expression led to resistance to chemotherapy. These results suggest that Grb10 binds to FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells that furthermore initiates drug-resistance and poor prognosis in AML. Citation Format: Kazi J. Uddin, Lars Rönnstrand. Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2013-4121

Abstract 4312: Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate cancer.

Abstract Prostate cancer (PCa) is manageable in its initial stages and when localized, but as it progresses it metastasizes and also becomes resistant to conventional chemotherapies. Identifying mechanisms that are related to the rapid progression of the disease could result in development of appropriate intervention strategies. We analyzed protein and mRNA expression of Grb10 (also known as insulin receptor-binding protein) in various PCa cell lines and found a significant decrease when compared to normal prostate epithelial cells. We also analyzed the expression pattern of Grb10 in a human PCa tissue array and found significant loss of Grb10 mRNA in more than 90% of the samples. Based on Gleason Score, the loss of Grb10 mRNA correlated with the stage and grade of the disease. These initial observations led us to hypothesize that Grb10 may act as a tumor suppressor whose expression is lost during the progression of human PCa. Using immuno-histochemical analysis of human PCa tissue samples we found loss of Grb10 protein with increasing tumor grade. We next analyzed various components of insulin signaling, based on the evidence that Grb10 expression is negatively correlated with insulin signaling. We observed significant increase at both mRNA and protein levels of IR, IGF-1R, IRS-1 and IRS-2 in PCa cells and tissues that directly correlated with loss of Grb10. Recently, Grb10 expression was shown to be negatively correlated with PTEN levels in different cancer types including PCa. We determined the expression pattern of Grb10 mRNA in prostate tissue samples from 15-weeks old PTEN wild type (WT) and PTEN knockout (KO) mice and observed that Grb10 levels were significantly down-regulated in PTEN-KO mice suggesting that loss of PTEN results in loss of Grb10. Further analysis suggested increased insulin signaling in PTEN-KO mice compared to PTEN-WT mice confirming negative regulation of Grb10 with insulin signaling. To further understand the role of Grb10 in PCa, we overexpressed Grb10 in human PCa DU145 (PTEN-mutated) cells. Overexpression of Grb10 resulted in decrease in colony formation, migration and invasion that was associated with down-regulation of insulin signaling. In summary, our findings suggest that Grb10 is a negative regulator of insulin signaling whose expression is lost in PCa in humans and correlates well with disease progression and aggressiveness. We conclude that Grb10 is a tumor suppressor in human PCa whose loss promotes rapid progression of the disease. Identification of this novel role of Grb10 could open up new strategies for the management of human PCa. Citation Format: Mohammad Imran Khan, Vaqar M. Adhami, Rahul Kumar Lall, Deeba Nadeem Syed, Imtiaz Ahmad Siddiqui, Bilal Bin Hafeez, Ajit Kumar Verma, Hasan Mukhtar. Grb10, a negative regulator of insulin signaling, is a tumor suppressor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4312. doi:10.1158/1538-7445.AM2013-4312

Identification of gene sets and pathways associated with lactation performance in mice

Mammary transcriptome analyses across the lactation cycle and transgenic animal studies have identified candidate genes for mammogenesis, lactogenesis and involution; however, there is a lack of information on pathways that contribute to lactation performance. Previously we have shown significant differences in lactation performance, mammary gland histology, and gene expression profiles during lactation [lactation day 9 (L9)] between CBA/CaH (CBA) and the superior performing QSi5 strains of mice. In the present study, we compared these strains at midpregnancy [pregnancy day 12 (P12)] and utilized these data along with data from a 14th generation of intercross (AIL) to develop an integrative analysis of lactation performance. Additional analysis by quantitative reverse transcription PCR examined the correlation between expression profiles of lactation candidate genes and lactation performance across six inbred strains of mice. The analysis demonstrated that the mammary epithelial content per unit area was similar between CBA and QSi5 mice at P12, while differential expression was detected in 354 mammary genes (false discovery rate < 0.1). Gene ontology and functional annotation analyses showed that functional annotation terms associated with cell division and proliferation were the most enriched in the differentially expressed genes between these two strains at P12. Further analysis revealed that genes associated with neuroactive ligand-receptor interaction and calcium signaling pathways were significantly upregulated and positively correlated with lactation performance, while genes associated with cell cycle and DNA replication pathways were downregulated and positively correlated with lactation performance. There was also a significant negative correlation between Grb10 expression and lactation performance. In summary, using an integrative genomic approach we have identified key genes and pathways associated with lactation performance.

FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia

The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. We observed that Grb10 physically associates with FLT3 in response to FLT3-ligand (FL) stimulation through FLT3 phospho-tyrosine 572 and 793 residues and constitutively associates with oncogenic FLT3-ITD. Furthermore endogenous Grb10–FLT3 association was observed in OCI-AML-5 cells. Grb10 expression did not alter FLT3 receptor activation or stability in Ba/F3-FLT3 cells. However, expression of Grb10 enhanced FL-induced Akt phosphorylation without affecting Erk or p38 phosphorylation in Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD. Selective Grb10 depletion reduced Akt phosphorylation in Ba/F3-FLT3-WT and OCI-AML-5 cells. Grb10 transduces signal from FLT3 by direct interaction with p85 and Ba/F3-FLT3-ITD cells expressing Grb10 exhibits higher STAT5 activation. Grb10 regulates the cell cycle by increasing cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation and survival of Ba/F3-FLT3-ITD cells as well as increased colony formation in semisolid culture. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression partially correlated to relapse as well as to poor prognosis. These results suggest that Grb10 binds to both normal and oncogenic FLT3 and induces PI3K–Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells.

Disruption of Growth Factor Receptor–Binding Protein 10 in the Pancreas Enhances β-Cell Proliferation and Protects Mice From Streptozotocin-Induced β-Cell Apoptosis

Defects in insulin secretion and reduction in β-cell mass are associated with type 2 diabetes in humans, and understanding the basis for these dysfunctions may reveal strategies for diabetes therapy. In this study, we show that pancreas-specific knockout of growth factor receptor–binding protein 10 (Grb10), which is highly expressed in pancreas and islets, leads to elevated insulin/IGF-1 signaling in islets, enhanced β-cell mass and insulin content, and increased insulin secretion in mice. Pancreas-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high-fat diet and protected mice from streptozotocin-induced β-cell apoptosis and body weight loss. Our study has identified Grb10 as an important regulator of β-cell proliferation and demonstrated that reducing the expression level of Grb10 could provide a novel means to increase β-cell mass and reduce β-cell apoptosis. This is critical for effective therapeutic treatment of both type 1 and 2 diabetes.

Lmx1a is an activator of Rgs4 and Grb10 and is responsible for the correct specification of rostral and medial mdDA neurons

The LIM homeodomain transcription factor Lmx1a is a very potent inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyse the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A-overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other previously generated expression arrays and on their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (dr/dr) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a-mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A-overexpression cell system resulted in the identification of novel direct or indirect downstream targets of Lmx1a in mdDA neurons: Grb10, Rgs4 and Vmat2.

Abstract 1954: In vitro effects on gene expression of Casp9, GRB10, and CDKN1B following rG3 treatment of human breast cancer cells

Abstract Introduction: Integrins can stimulate cellular growth and proliferation, as well as apoptosis, and are important factors in AKT cell signaling. To assess their binding, a recombinant protein of the G3 domain of laminin-5 (rG3) that specifically binds the alpha subunit expressed on metastatic MDA-MB-231 cells in vitro was produced. Studying changes in expression of genes involved in cell signaling and the apoptosis cascade, specifically CDKN1B, GRB10, and Caspase 9, can help improve understanding of signaling pathways of metastatic cells and the AKT pathway. Methods: rG3 was produced using ArcticExpress Competent Cells (Stratagene). Plasmid DNA was transformed into competent cells and transferred to ampicillin plates for colony production of successful transformation. A single colony was used to inoculate LB broth containing gentamycin and ampicillin for an overnight incubation at 37°C. LB without antibiotics was inoculated with the overnight culture and incubated at 30°C for 3 hours. The temperature was lowered to 11.5°C before the expression was induced using IPTG at a final concentration of 1mM. SDS-PAGE/immunoblotting confirmed successful soluble rG3 protein expression. MDA-MB-231 human breast cancer cells from ATCC were grown in tissue culture flasks and maintained until confluent. Cells were plated at 3 × 106 cells/well in a 6-well tissue culture plate in a 2 ml volume of DMEM without pen/strep in duplicate. rG3 was added to wells at a final concentration of 175 µg/ml for treated cells and corresponding control buffer for untreated cells. After incubation overnight, cells were collected at time points within 0-60 hours and RNA was isolated using Qiagen RNeasy. An eppendorf Realplex 96-well plate reader was used for PCR analysis using a β-actin standard curve. Differences in gene expression were shown in fold change using Pfaffl's Method. Results: Our preliminary data show there are changes in gene expression after treatment with rG3.We are further investigating these changes in expression. Preliminary results suggest that changes in gene expression after treatment may be informative in understanding the AKT signaling pathway. Conclusion: The study of these changes in gene expression after altering the AKT pathway from treatment with rG3 could prove to be a helpful tool in understanding the signaling mechanisms of metastatic cells and the details of the signaling pathways involved in apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1954. doi:10.1158/1538-7445.AM2011-1954

Ovarian stimulation makes a disruption of imprinting genes of mouse oocyte and placenta

OBJECTIVE: To investigate the effect of ovarian stimulation (OS) on the expression of imprinting genes of oocytes and placenta. DESIGN: A randomized controlled study based on mouse model. MATERIALS AND METHODS: Eight-week-old C57 female mice were injected with pregnant mares' serum gonadotropin (PMSG) in different dose (0, 10, 20, 30 IU), and 10 IU human chorionic gonadotropin (HCG). Serum estradiol and FSH level were detected. MII oocytes were harvested at 14 hours post HCG injection. RNA extraction and RT-real time PCR were conducted in oocytes and ovarian tissues. C57 female mice were divided into OS group, which were injected with 10 IU PMSG and 10 IU HCG, and control group, whose estrous cycle were evaluated by vaginal smear, and was injected with 10 IU HCG at estrus day. The presence of a vaginal plug was designated at 0.5 day post coitum (dpc). Placentas and embryos were collected at 15dpc. Placentas were examined by gene microarrays, and RT-real time PCR was conducted to verify the expression of Kdm1b, Impact, Igf2, H19, Zac1, Grb10, Mest, and Snrpn. RESULTS: The serum estradiol and FSH level increased PMSG-dose-dependently. The expression of Zac1, H19 and Snrpn were increased, while Mest and Grb10 were decreased in 20 IU-PMSG-treated ovaries significantly. 10 IU PMSG treatment exhibited the best ovarian response (25 ± 2 oocytes/mouse), but 20 IUi PMSG dose resulted in decreased ovarian response (7 ± 1). Microarrays of placentas revealed up-regulation of more than 400 genes and down-regulation of approximately 200 genes in OS group than in control group. Following RT-real time PCR showed significantly increased expression of Igf2, H19, Zac1, and Grb10. The arrest of embryo development was increased, and the live birth rate was decreased significantlyin OS group than in control group. CONCLUSION: Ovarian stimulation makes a disruption of imprinting genes of mousue oocytes and placenta, and may adversely affect the developing potential of oocytes and embryos.

Temporal and spatial expression of a growth-regulated network of imprinted genes in growth plate

In mammals, the somatic growth rate is rapid during fetal and early postnatal life and then gradually declines and eventually stops. In search of the fundamental biological mechanism causing coordinated growth deceleration in multiple tissues, a network of imprinted genes was recently identified based on a coordinated decline in expression in several organs during postnatal growth. To explore a possible role in longitudinal bone growth, we characterized expression of the network during postnatal growth in microdissected metaphyseal bone and growth plate zones of 1-, 3-, and 9-week-old rats using real-time PCR. The expression pattern of the network is modified in growth plate. Similar to the coordinated decline previously observed in kidney, lung, liver, and heart, expression of all genes, except Gtl2, decreased with age in metaphyseal bone. On the contrary, Mest, Dlk1, H19, and Gtl2 decreased, and Cdkn1c, Grb10, and Slc38a4 increased with age in growth plate. During differentiation from resting to hypertrophic zone, Mest, Dlk1, Grb10, and Gtl2 expression decreased, whereas Slc38a4 expression increased. In particular, developmental changes in the expression of growth-promoting genes, Mest, Dlk1, Gtl2, and growth-inhibitory genes, Cdkn1c and Grb10, may contribute to the decline in longitudinal bone growth that occurs with age.

The expression analysis of &lt;I&gt;Grb10&lt;/I&gt; during mouse embryonic

Grb10 gene expression was analyzed in fetus and four organs during gestation process using the real-time RT-PCR and in situ hybridization. The real-time RT-PCR analysis result showed that the expression of Grb10 in whole embryos is throughout E8.5 to E19.5. The Grb10 gene expression level was gradually increased from E8.5 to E13.5, and then gradually reduced. The tissues specific expression levels were decreased in brain, heart, and lung from E12.5 to E19.5, but a peak expression was observed in liver at E18.5. Grb10 gene expression was also investigated at E13.5, E15.5, E16.5 and E18.5 sections by using in situ hybridization analysis. Strong signals of Grb10 were detected in brain, spine, kidney, and muscle tissues. These results suggest that Grb10 may play an important role during the development process in mouse.