RNA silencing experiments showed that knocking down Gab1 adaptor protein in BL41 human Burkitt lymphoma cells significantly reduced B cell receptor (BCR)-induced Erk phosphorylation, indicating that Gab1 plays a pivotal role in regulating Erk activity in B cells.

Obesity and type 2 diabetes mellitus are independent risk factors for the onset and progression of hepatocellular carcinoma (HCC). This study aimed to analyze the association of DNA methylation signatures at HCC pathway genes with obesity and related metabolic disturbances. A population of 474 adults within the Methyl Epigenome Network Association (MENA) project was included. DNA methylation levels were measured in white blood cells by microarray. The identification and discrimination of HCC pathway genes were performed using KEGG and PathDIP databases. Anthropometry measurements, the blood metabolic profile, and clinical data were analyzed. The methylation patterns of 20 CpG sites at HCC pathway genes strongly correlated with BMI (FDR <0.0001). These genes encompassed GADD45A, MTOR, FRAT2, E2F3, WNT7B, FRAT1, LRP5, DPF3, GSTA2, APC, MYC, WNT10B, ARID1B, AKT1, GSTA1, WNT5A, CDK4, GAB1, TCF7, which statistically contributed to the regulation of the HCC pathway (P = 2.10e-07). The main biological process where these genes were implicated included uncontrolled cell proliferation, DNA damage, increased survival, and altered oncogenic expression. Interestingly, 9 out of 20 BMI-associated CpGs also correlated with waist circumference and HOMA-IR index. In conclusion, pathway analysis revealed potential associations of DNA methylation signatures at HCC pathway genes with adiposity and insulin resistance phenotypes.

Studies of primate lentiviruses continue to provide information about the evolution of simian immunodeficiency viruses (SIVs) and the origin and emergence of HIV since chimpanzees in west-central Africa (Pan troglodytes troglodytes) were recognized as the reservoir of SIVcpzPtt viruses, which have been related phylogenetically to HIV-1. Using in-house peptide ELISAs to study SIV prevalence, we tested 104 wild-born captive chimpanzees from Gabon and Congo. We identified two new cases of SIVcpz infection in Gabon and characterized a new SIVcpz strain, SIVcpzPtt-Gab4. The complete sequence (9093 bp) was obtained by a PCR-based 'genome walking' approach to generate 17 overlapping fragments. Phylogenetic analyses of separated genes (gag, pol-vif and env-nef) showed that SIVcpzPtt-Gab4 is closely related to SIVcpzPtt-Gab1 and SIVcpzPtt-Gab2. No significant variation in viral load was observed during 3 years of follow-up, but a significantly lower CD4+ T cells count was found in infected than in uninfected chimpanzees (p<0.05). No clinical symptoms of SIV infection were observed in the SIV-positive chimpanzees. Further field studies with non-invasive methods are needed to determine the prevalence, geographic distribution, species association, and natural history of SIVcpz strains in the chimpanzee habitat in Gabon.

Huntington's disease (HD) is caused due to expansion of CAG repeats in the gene huntingtin (Htt). Adaptor protein Grb2, involved in Ras-MAP kinase pathway, is a known interactor of Htt. Mutant Htt-Grb2 interaction reduces Ras-MAPK signaling in HD models. In normal cells Grb2 forms Grb2-Sos1-Gab1 complex through its N-SH3 and C-SH3 domains respectively, essential for sustained activation of Ras. We found that C-SH3 of Grb2 mediates the interaction with mutant Htt and this interaction being stronger could replace Gab1, with mutant Htt becoming the preferred partner. This would have immense effect on downstream signaling events.

Coxsackievirus type B3 (CV-B3), an enterovirus associated with the pathogenesis of several human diseases, subverts, or employs the host intracellular signaling pathways to support effective viral infection. We have previously demonstrated that Grb2-associated binding protein 1 (GAB1), a signaling adaptor protein that serves as a platform for intracellular signaling assembly and transduction, is cleaved upon CV-B3 infection, resulting in a gain-of-pro-viral-function via the modification of GAB1-mediated ERK1/2 pathway. GAB2 is a mammalian homolog of GAB1. In this study, we aim to address whether GAB2 plays a synergistic role with GAB1 in the regulation of CV-B3 replication. Here, we reported that GAB2 is also a target of CV-B3-encoded viral proteinase. We showed that GAB2 is cleaved at G238 during CV-B3 infection by viral proteinase 2A, generating two cleaved fragments of GAB2-N1-237 and GAB2-C238-676. Moreover, knockdown of GAB2 significantly inhibits the synthesis of viral protein and subsequent viral progeny production, accompanied by reduced levels of phosphorylated p38, suggesting a pro-viral function for GAB2 linked to p38 activation. Finally, we examined whether the cleavage of GAB2 can promote viral replication as observed for GAB1 cleavage. We showed that expression of neither GAB2-N1-237 nor GAB2-C238-676 results in enhanced viral infectivity, indicating a loss-of-function, rather than a gain-of-function of GAB2 cleavage in mediating virus replication. Taken together, our findings in this study suggest a novel host defense machinery through which CV-B3 infection is limited by the cleavage of a pro-viral protein.

OBJECTIVE

To investigate the influence of Qingkailing (QKL) on learning and memory abilities, global neurotransmitter and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway of senescence accelerated mouse-prone/8 (SAMP8) mice with Alzheimer's dementia (AD).

METHODS

SAMP mice were modeled and divided into the model group, the QKL group and the doneppezil hydrochloride group, all treated for 90 days. And a control group was set up with senescence accelerated mouse-resistance/1 (SAMR1) mice. Morris water maze was used to test the learning and memory abilities of mice; contents of acetylcholine (Ach) and monoamine neurotransmitters in brain were measured by HPLC; levels of Grb2-associated binder-1 (Gab1), AKT and phospho-serine/threonine protein kinase B (PAKT473) were evaluated by Western-blot.

RESULTS

Compared with the control group, in the model group, the average escape latency detected by hidden platform trial and reverse trial on the 3rd day was higher (P < 0.01); levels of Ach, 5-hydroxytryptamine (5-HT) and Gab1 were lower (P < 0.01, P < 0.05 and P < 0.01), respectively. As compared with the model group, the escape latency (within the 2nd to 5th day) decreased (P < 0.01), levels of Ach and 5-HT increased (P < 0.05), and Gab1 protein expression increased (P < 0.01) in the QKL treated group after treatment, in addition, the level of phosphorylated AKT protein significantly increased (P < 0.05).

CONCLUSIONS

QKL could improve the learning and memory ability of AD model mice, which is probably related to its function in increasing cerebral Ach, 5-HT and activating PI3K/AKT pathway.

The neuregulin-1 (NRG1) signaling pathway plays an important role in the development of the peripheral neuromuscular system, including in muscle spindle and postnatal myelination. We previously showed that NRG1 on the axonal membrane regulates peripheral nerve myelination through Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling. Here, we determined the role of Gab1 in the development of muscles and the muscle spindle using muscle-specific conditional Gab1 knockout mice. The mutant mice showed general retardation in muscular growth and hypotrophy of extrafusal muscle fibers. In addition, the muscle-specific Gab1 knockout mutant exhibited significant underdevelopment of muscle spindles, which are normally regulated by NRG1, and abnormal proprioceptive behavior. Furthermore, the selective knockdown of Gab1 in C2C12 muscle cells reduced NRG1-induced expression of Egr3, a critical transcription factor for muscle spindle development. However, Gab2 knockout mice did not show any defects in the development of muscles or muscle spindles. Our findings suggest that Gab1 is an essential signaling molecule in mediating axonal NRG1 signaling for the development of both extrafusal and intrafusal muscle fibers.

The pancreatic stellate cell (PSC) is the principal cell type of the desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC). PSCs interact with cancer cells and influence the progression of the disease through a complex network of signaling molecules including hepatocyte growth factor (HGF). Functional heterogeneity of PSCs within a tumor might conceivably influence tumor progression. We investigated PSC populations isolated from different human PDACs and examined the effects of PSC-conditioned medium on BxPC-3 and AsPC-1 pancreatic cancer cells. The different PSC populations exhibited a wide range of variation (120-3,000 pg/ml) in their ability to secrete HGF. Media from high-HGF-producing PSCs stimulated phosphorylation of Met, Gab1, and ERK in the cancer cells and induced increases in DNA synthesis and migration which were blocked by the Met inhibitor SU11274, indicating a role of HGF as a mediator. HGF levels produced by PSCs and the effects of PSC media on the cancer cells were increased by IL-1α and inhibited by TGFβ. The functional heterogeneity of PSCs in terms of HGF-mediated tumor-stroma interactions suggests that inhibition of the HGF pathway as a novel treatment approach in PDAC might have different effects in different subsets of patients.

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7-67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion-genes (AHRR-NCOA2 and GTF2I-NCOA2) were examined using RT-PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT-PCR for the most frequently observed fusions, an AHRR-NCOA2 fusion transcript was found in 9/14 cases. GTF2I-NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR-NCOA2 fusion and one case a novel GAB1-ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR-NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I-NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1-ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms.

The receptor tyrosine kinases EGFR and Met induce phosphorylation of the docking protein Gab1, and there is evidence that Gab1 may have a role in the signaling from these receptors. Studying hepatocytes, we previously found that although Gab1 mechanistically interacted in different ways with EGFR and Met, it was involved in mitogenic signaling induced by both EGF and HGF. It has been reported that in EGFR, Gab1 is required particularly at a low dose of EGF. Whether this also applies to HGF/Met signaling has not been investigated. We have studied the role of Gab1 in activation of the Akt and ERK pathways at low- and high-intensity stimulation with EGF and HGF in cultured hepatocytes. In cells where Gab1 was depleted by a specific Gab1-directed siRNA, the EGF-induced phosphorylation of ERK was lowered and HGF-induced phosphorylation of both ERK and Akt was substantially reduced. These effects were more marked at low-dose HGF stimulation. The inhibitory consequence of Gab1 depletion was particularly pronounced for HGF-induced Akt phosphorylation. The results suggest that Gab1 is an important signal amplifier for low-intensity stimulation by HGF.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 (Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been identified in two dominantly inherited skin diseases, Vohwinkel syndrome with ichthyosis and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. We generated stable HaCaT cell lines that express wild-type (WT) loricrin and a mutant form found in Vohwinkel syndrome with ichthyosis, using an ecdysone-inducible promoter system. The cells expressing the mutant loricrin grew more rapidly than those expressing WT loricrin after induction for 5 days. Confocal immunofluorescence microscopy revealed that phospho-Akt occurred in the nucleolus where the mutant loricrin was also located. The level of activity of Akt kinase was about nine times higher in cells with the mutant than in those with WT loricrin. ERK1/2, the epidermal growth factor receptor, vascular endothelial growth factor (VEGF) receptor 2 and Stat3 were all phosphorylated in cells with the mutant loricrin. The docking proteins, Gab1 and c-Cbl, were also tyrosine-phosphorylated in these cells. Furthermore, chromatin immunoprecipitation assays showed that Stat3 protein bound to the VEGF promoter in cells with the mutant. Thus, this study suggests that VEGF release and the subsequent activation of VEGF receptor 2 link loricrin gene mutations to rapid cell proliferation in a cellular model of loricrin keratoderma.

Cholangiocarcinoma is a malignant neoplasm arising from the epithelial cells lining the biliary ducts and its occurrence can be anatomically classified as within the liver (intrahepatic) or outside the liver (extrahepatic). Extrahepatic cholangiocarcinoma, which can be called as biliary tract cancer (BTC), is the most common form of this malignancy, and its etiology is still unclear. In this study, we tried to elucidate the complicated association between receptor tyrosine kinase (RTK) gene polymorphisms and susceptibility of BTC by analyzing frequency distribution of genotypes and alleles of GRB2-associated-binding protein 1 (Gab1), endothelial growth factor receptor (EGFR), and endothelial growth factor (EGF) and identified potential risk of BTC for people carrying specific genotype of Gab1 and EGFR. Two hundred twenty-five and 300 patients with BTC and cholelithiasis (gallstone (GS)), respectively, and 300 controls matched by age, sex, and ethnicity with patients were recruited from Shengjing Hospital of China Medical University from January 2008 to July 2011 with informed consents. Genomic DNA of BTC group was extracted and purified from formalin-fixed, paraffin-embedded tumor tissue sections using QiAamp DNA FFPE Tissue kit. For GS group and controls, DNA was extracted from peripheral blood leukocytes using genomic DNA extraction kit from Aid Lab. Target genes of RTK family were identified from National Center of Biotechnology Information (NCBI) SNP database and Japanese Single Nucleotide Polymorphisms (JSNP) database. Frequency distribution of genotypes and alleles was analyzed using HapMap Project database. All of the statistical analysis was conducted with SPSS 13.0 software. Eight loci were identified for Gab1 (4), EGFR (3), and EGF (1) as the target single-nucleotide polymorphisms (SNPs) for the association of gene polymorphisms and BTC. A/A genotype and A allele of rs3805246 in Gab1 and G/G genotype and G allele of rs2017000 in EGFR were significantly higher in BTC group than in GS group or controls. After controlling for BMI, age, gender, and smoking habit, patients with "A/A + G/A" had 2.154 times odds to have BTC; as for patients with "A/A" only, they still had 1.976 times odds to have BTC. In the rs2017000 of EGFR, patients with "G/G + G/A" had 1.772 times odds to have BTC, and patients with "G/G" only had 1.530 times odds to have BTC. Furthermore, patients with A/A in rs3805246 and G/G in rs2017000 simultaneously had 1.620 times chance to have BTC than people with other genotypes. This study explored the independent potential effect of EGFR signaling transduction pathway and its downstream element Gab1 and the gene-gene interaction on the disease mechanism of BTC in the perspective of genetics and molecular epidemiology.

The Src homology phosphotyrosyl phosphatase, SHP2, is a positive effector of EGFR signaling. However, the molecular mechanism and biological functions of SHP2 regulation are still not completely known. To better understand the cellular processes in which SHP2 participates, we carried out mass spectrometry to find SHP2 binding proteins. FLAG-SHP2 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by LC/MS/MS mass spectrometry. Among the identified proteins, we focus in this report on the heat shock protein 70 (HSP70). Physical interactions of SHP2 with HSP70 were confirmed in vivo. Further experiments demonstrate that EGF does not activate binding of SHP2 with HSP70 rather the binding appears to be constitutive. However, the formation of an HSP70/SHP2 complex affected the binding of SHP2 with EGFR and (or) GAB1. These data suggest that binding of HSP70 with SHP2 regulates to some extent the EGF signaling pathway. In addition, immunostaining experiments indicated that SHP2 and HSP70 co-localized in the cell membrane region after EGF treatment. Our findings propose a possible involvement of HSP70 in the regulation of EGF signaling pathway by SHP2.

Receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor (EGFR) and Met lead to activation of intracellular signals including Akt, a critical regulator of cell survival, metabolism and proliferation. Upon binding their respective ligands, each of these receptors is recruited into clathrin coated pits (CCPs) eventually leading to endocytosis. We have recently shown that phosphorylation of Gab1 and Akt following EGFR activation requires clathrin, but does not require receptor endocytosis. We examined whether clathrin regulates Akt signaling downstream of Met, as it does for EGFR signaling. Stimulation with the Met ligand Hepatocyte Growth Factor (HGF) leads to enrichment of phosphorylated Gab1 (pGab1) within CCPs in ARPE-19 cells. Perturbation of clathrin using the inhibitor pitstop2 decreases HGF-stimulated Akt phosphorylation. These results indicate that clathrin may regulate Met signaling leading to Akt phosphorylation similarly as it does for EGFR signaling.

The timely orchestration of multiple signalling pathways is crucial for the integrity of an organism and therefore tightly controlled. Gab family proteins coordinate signal transduction at the plasma membrane (PM) by acting as docking platforms for signalling components involved in MAP kinase (MAPK), PI3 kinase (PI3K), phospholipase C (PLC) and Rho family GTPase signalling. The interaction with these components as well as the targeting of the docking platform to the PM underlies complex spatial and temporal regulatory mechanisms. Deregulated Gab1 activation and membrane binding have been observed in some haematopoietic malignancies and solid tumours, thereby contributing, for example, to the development of Philadelphia chromosome-negative myeloproliferative neoplasms and certain lung cancers. Previously, we could demonstrate that the presence of PIP3 in the PM, which is increased in many cancer cells, is not sufficient for constitutive Gab1 membrane recruitment. In addition, MAPK-dependent phosphorylation of Gab1 at serine 552 (Ser552) is vital for Gab1 membrane binding. Here, we confirm our hypothesis that in the absence of MAPK activity an intrinsic part of Gab1 prevents binding to PIP3 at the PM. This epitope of Gab1, which encompasses Ser552, interacts directly with the Gab1 PH domain. Two arginines located in positions +4 and +8 of Ser552 are essential for the interaction with the PH domain, as well as for the inhibition of membrane recruitment of unphosphorylated Gab1. Ser552 phosphorylation is dispensable in respective arginine to alanine mutants of Gab1. Gab1 recruitment to the PM is highly dynamic and continuous PI3K and MAPK activities are both essential for sustained Gab1 membrane localisation. Our data document the existence of a sophisticated and robust control mechanism that prevents Gab1 translocation and signalling complex assembly after the activation of either MAPK or PI3K alone.

Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT.

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.

Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.

The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.

BACKGROUND

The mammary gland is key to all mammal species; in particular in multiparous species like pigs the number and the shape of functional mammary gland complexes are major determinants of fitness. Accordingly, we aimed to catalog the genes relevant to mammogenesis in pigs. Moreover, we aimed to address the hypothesis that the extent and timing of proliferation, differentiation, and maturation processes during prenatal development contribute to postnatal numerical, morphological and functional properties of the mammary gland. Thus we focused on differentially expressed genes and networks relevant to mammary complex development in two breeds that are subject to different selection pressure on number, shape and function of teats and show largely different prevalence of non-functional inverted teats. The expression patterns of fetal mammary complexes obtained at 63 and 91 days post conception (dpc) from German Landrace (GL) and Pietrain (PI) were analyzed by Affymetrix GeneChip Porcine Genome Arrays.

RESULTS

The expression of 11,731 probe sets was analysed between the two stages within and among breeds. The analysis showed the largest distinction of samples of the breed GL at 63 dpc from all other samples. According to Ingenuity Pathways Analysis transcripts with abundance at the four comparisons made (GL63-GL91, PI63-PI93, GL63-PI63 and GL91-PI91) were predominantly assigned to biofunctions relevant to 'cell maintenance, proliferation, differentiation and replacement', 'organismal, organ and tissue development' and 'genetic information and nucleic acid processing'. Moreover, these transcripts almost exclusively belong to canonical pathways related to signaling rather than metabolic pathways. The accumulation of transcripts that are up-regulated in GL compared to PI indicate a higher proliferating activity in GL, whereas processes related to differentiation, maturation and maintenance of cells are more prominent in PI. Differential expression was validated by quantitative RT-PCR of five genes (GAB1, MAPK9, PIK3C2B, PIK3C3 and PRKCH) that are involved in several relevant signaling pathways.

CONCLUSIONS

The results indicate that mammary complex development in PI precedes GL. The differential expression between the two breeds at fetal stages likely reflects the prenatal initiation of postnatal phenotypes concerning the number and shape as well as functionality of teats.

Fat-tailed sheep have a unique characteristic of depositing fat in their tails. In the present study, we conducted genome-wide association studies (GWAS) on traits related to tail fat deposition and body size in the Hulun Buir sheep. A total number of 300 individuals belonging to two fat-tailed lines of the Hulun Buir sheep breed genotyped with the Ovine Infinium HD SNP BeadChip were included in the current study. Two mixed models, one for continuous and one for binary phenotypic traits, were employed to analyse ten traits, that is, body length (BL), body height (BH), chest girth (CG), tail length (TL), tail width (TW), tail circumference (TC), carcass weight (CW), tail fat weight (TF), ratio of CW to TF (RCT) and tail type (TT). We identified 7, 6, 7, 2, 10 and 1 SNPs significantly associated with traits TF, CW, RCT, TW, TT and CG, respectively. Their associated genomic regions harboured 42 positional candidate genes. Out of them, 13 candidate genes including SMURF2, FBF1, DTNBP1, SETD7 and RBM11 have been associated with fat metabolism in sheep. The RBM11 gene has already been identified in a previous study on signatures of selection in this specific sheep population. Two more genes, that is, SMARCA5 and GAB1 were associated with body size in sheep. The present study has identified candidate genes that might be implicated in tail fat deposition and body size in sheep.

Sprouty (Spry) proteins have been implicated in cancer progression, but their role in triple-negative breast cancer (TNBC), a subtype of lethal and aggressive breast cancer, is unknown. Here, we reported that Spry1 is significantly expressed in TNBC specimen and MDA-MB-231 cells. To understand Spry1 regulation of signaling events controlling breast cancer phenotype, we used lentiviral delivery of human Spry1 shRNAs to suppress Spry1 expression in MDA-MB-231, an established TNBC cell line. Spry1 knockdown MDA-MB-231 cells displayed an epithelial phenotype with increased membrane E-cadherin expression. Knockdown of Spry1 impaired MDA-MB-231 cell migration, Matrigel invasion, and anchorage-dependent and -independent growth. Tumor xenografts originating from Spry1 knockdown MDA-MB-231 cells grew slower, had increased E-cadherin expression, and yielded fewer lung metastases compared to control. Furthermore, suppressing Spry1 in MDA-MB-231 cells impaired the induction of Snail and Slug expression by EGF, and this effect was associated with increased EGFR degradation and decreased EGFR/Grb2/Shp2/Gab1 signaling complex formation. The same phenotype was also observed in the TNBC cell line MDA-MB-157. Together, our results show that unlike in some tumors, where Spry may mediate tumor suppression, Spry1 plays a selective role in at least a subset of TNBC to promote the malignant phenotype via enhancing EGF-mediated mesenchymal phenotype.