Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene³. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele¹. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton^2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports^2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.     

Appl1 Is Dispensable for Mouse Development, and Loss of Appl1 Has Growth Factor-selective Effects on Akt Signaling in Murine Embryonic Fibroblasts

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1^−/− murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1^−/− MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1^−/− MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.     

缺失plcg1基因引起成纤维细胞PI—3K信号上调

磷脂酶C－γ1（phospholipase C－γ1,PLC－γ1）与磷脂酰肌醇3－激酶（phosphatidylinositol-3 kinase,PI-3K）是生长因子调控细胞生长与增殖的两个重要信号中介。为探讨PLC－γ1在表皮生长因子（EGF）介导的细胞分裂信号中的代偿机制,用磷脂酶C（phospholipase C,PLC）特异性抑制剂U73122及PI－3K牧场划必抑制剂wortmannin处理剔除PLC－δ1基因plcg1(PLC-γ1^-/-）及野生型（PLC－γ1^ / )小鼠胚胎成纤维细胞,发现未经处理情况下两种细胞的克隆形成能力、细胞活力及EGF引起的DNA合成能力相似,且均可被U73122与wortmannin抑制,但与PLC－γ1^ / 细胞相比,PLC－γ1^－／－更依赖于PI－3K,而对PLC的依赖性却减小。Western印迹也表明EGF刺激后PI－3K的p85α亚单位酷氨酸磷酸化程度比野生型显著增高,PI－3K信号通路的激活出现上调,且PLC－γ1^-/-中无基近亲PLC－γ2的代偿表达。因此PLC－γ1^-/-中PLC－γ1的功能可能被PI－3K通路代偿,而PLC－γ2或其他PLC同工酶并不代偿其功能。结果表明EGF介导的信号能路的冗余性及PLC－γ1信号通路的可代偿性。     

Epidermal Growth Factor in Synaptosomal Fractions of Mouse Cerebral Cortex

Using a specific and sensitive epidermal growth factor radioimmunoassay (EGF-RIA) we measured EGF concentrations in whole brain, cerebral cortex, and cerebral cortical synaptosomal (pinched-off presynaptic nerve terminals) fractions of 26-day-old mouse brain. The relative EGF concentration in synaptosomal fractions was significantly greater than the growth factor concentrations in whole brain or cerebral cortex. Intracerebral injection, in an amount of EGF, several-fold greater than whole brain EGF content, did not appreciably increase synaptosomal EGF concentration, suggesting that no artifact was involved. The high synaptosomal EGF content suggests a neurotransmitter or a neuromodulator role for EGF in the CNS.     

Placental and Embryonic Growth Restriction in Mice With Reduced Function Epidermal Growth Factor Receptor Alleles

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr^wa2 allele or heterozygous for the antimorphic Egfr^Wa5 allele. Egfr^wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr^Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr^wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr^Wa5 heterozygotes than in Egfr^wa2 homozygotes and suggest that Egfr^wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to strain-dependent differences in intrauterine growth restriction caused by reduced EGFR activity.EPIDERMAL growth factor receptor (EGFR) is the prototypical member of the ERBB family of receptor tyrosine kinases and is known to regulate many aspects of cellular biology including cell proliferation, survival, differentiation, and migration (reviewed in Yarden and Sliwkowski 2001). Eleven known ligands bind the extracellular region of ERBB-family receptors, and activation of the tyrosine kinase domain occurs following receptor homo- or heterodimerization. The resulting biological responses are dependent upon specific signaling cascades initiated by ERBBs and can be influenced by the particular ligand–ERBB combination (Yarden and Sliwkowski 2001). Studies using cultured cells have underscored the importance of EGFR in modulating various cellular processes, while animal models have been able to demonstrate that EGFR is required for numerous developmental and physiological processes (Casalini et al. 2004). In vivo studies have shown that EGFR is particularly important for normal placental development in mice; placentas from Egfr nullizygous (Egfr^{tm1Mag/tm1Mag}) embryos exhibit strain-specific defects that result in differential embryonic lethality (Sibilia and Wagner 1995; Threadgill et al. 1995). Two additional Egfr alleles result in reduced EGFR signaling in mice: the recessive hypomorphic Egfr^wa2 and dominant antimorphic Egfr^Wa5 alleles (Luetteke et al. 1994; Fowler et al. 1995; Du et al. 2004; Lee et al. 2004). These alleles can provide insight into the level of EGFR signaling required for normal placental development.Egfr^wa2 is a classical spontaneous mutation that arose in 1935 that causes a distinct wavy coat phenotype in the homozygote (Figure 1; Keeler 1935). This recessive mutation was subsequently found to be a single nucleotide transversion resulting in a valine → glycine substitution in the highly conserved kinase domain of EGFR (Luetteke et al. 1994; Fowler et al. 1995). Since mice homozygous for the Egfr^tm1Mag null allele die before or shortly after birth depending on genetic background, the hypomorphic Egfr^wa2 allele has been the primary model used to study the effect of attenuated EGFR signaling in a variety of adult physiological and disease states. In addition to eye and hair phenotypes, the adult Egfr^wa2 homozygous mouse exhibits delayed onset of puberty, abnormal ovulation, enlarged aortic valves and cardiac hypertrophy, decreased body size, defects in mammary gland development and lactation, increased susceptibility to colitis, and impaired intestinal adaptation following small bowel resection (Fowler et al. 1995; Helmrath et al. 1997; Chen et al. 2000; Egger et al. 2000; O''Brien et al. 2002; Prevot et al. 2005; Hsieh et al. 2007). Despite the widespread use of the Egfr^wa2 allele, there are limitations in using Egfr^wa2 homozygous mice to clearly define the physiological roles of EGFR. Egfr^wa2 has traditionally been maintained in cis, tightly linked with a hypomorphic Wnt3a allele, Wnt3a^vt (vestigal tail), making phenotypic analysis of reduced EGFR signaling by itself difficult. Furthermore, Egfr^wa2 has also typically been maintained on a mixed genetic background and since the Egfr nullizygous phenotype is similarly influenced by genetic modifiers, a mixed background could mask phenotypes that become evident when Egfr^wa2 mice are inbred.Open in a separate windowFigure 1.—Congenic 129 Egfr allelic series. Wild-type (left), Egfr^wa2 homozygote (middle), and Egfr^wa5 heterozygote (right) mice. As weanlings and adults, the Egfr^wa2 homozygotes and Egfr^wa5 heterozygotes are grossly indistinguishable.The Egfr^Wa5 allele arose in a large, genomewide N-ethyl-N-nitrosourea mutagenesis screen for dominant visible mutations in the mouse. Egfr^Wa5 heterozygous mice were first identified by their open eyelids at birth and by development of a wavy coat, similar to the phenotype of Egfr^wa2 homozygous mice (Figure 1). Egfr^Wa5 failed to complement the Egfr^tm1Mag null allele and was shown to function as an antimorph since Egfr^Wa5, but not Egfr^tm1Mag, heterozygotes exhibit eyelid and coat phenotypes (Lee et al. 2004). A single nucleotide missense mutation was found in the Egfr^Wa5 allele that results in an Asp → Gly substitution in the highly conserved DFG domain of the EGFR kinase catalytic loop (Du et al. 2004; Lee et al. 2004). Although Egfr^Wa5 heterozygotes are viable, Egfr^Wa5 homozygotes die prenatally and exhibit placental defects identical to those from Egfr^tm1Mag homozygous null embryos. Placentas from Egfr^Wa5 heterozygotes on a mixed background show variable reduction in the spongiotrophoblast layer and minor abnormalities in the labyrinth region, but no effects on embryo survival have been reported.In vitro studies with Egfr^Wa5 suggest that it encodes a kinase-dead EGFR since no phosphorylation of EGFR^Wa5 is detected following stimulation with ligands. In agreement with the genetic data showing that Egfr^Wa5 is an antimorph, in vitro studies have demonstrated that the EGFR^Wa5 receptor can inhibit phosphorylation of EGFR and MAPK in a dose-dependent manner (Lee et al. 2004). In Chinese hamster ovary cells expressing an equimolar ratio of EGFR and EGFR^Wa5 receptors, <10% of wild-type phosphorylation levels were observed by Western blot analysis.The Egfr allelic series available in the mouse has high utility for studying gene function since EGFR is involved in a multitude of developmental processes and human diseases. Although both Egfr^wa2 and Egfr^Wa5 alleles result in reduced EGFR signaling, the activity and phenotypic consequences of Egfr^wa2 homozygosity has not been compared to that of Egfr^Wa5 heterozygosity when both are on the same genetic backgrounds. Adult Egfr^Wa5 heterozygous mice appear highly similar to Egfr^wa2 homozygotes, but crosses with the Apc^Min intestinal tumor model have shown that a more substantial reduction in tumor number occurs when the Apc^Min mutation is bred onto the Egfr^wa2 homozygous vs. Egfr^Wa5 heterozygous background (Roberts et al. 2002; Lee et al. 2004). These results suggest that Egfr^Wa5 heterozygous mice retain higher levels of EGFR activity than Egfr^wa2 homozygous mice; however, the data are confounded by the fact that the crosses were performed using different mixed genetic backgrounds.This study reports a comprehensive genetic analysis of reduced EGFR signaling in Egfr^wa2 homozygotes and Egfr^Wa5 heterozygotes in placental development and embryonic growth for three congenic backgrounds, C57BL/6J (B6), 129S1/SvImJ (129), and BTBR/J-T+, tf/tf (BTBR). Wild-type placenta weight, embryo weight, and mRNA levels of genes selected for their trophoblast-specific expression were found to be highly strain dependent. Egfr^wa2 homozygous placentas are reduced in size in all three strains, and a proportion of 129-Egfr^wa2 homozygotes die before 15.5 days post-coitus (dpc). Egfr^wa2 homozygous embryos also display background-dependent intrauterine growth restriction (IUGR) in late gestation, which is most severe on 129 and BTBR backgrounds and models EGFR-associated IUGR in humans. Egfr^Wa5 heterozygous placentas exhibit a minor reduction in size on all three backgrounds with no impact on embryonic growth. These results suggest that reduced levels of EGFR signaling can interfere with normal placental development and that embryo development is affected only after placental size is sufficiently reduced. In addition, our data show that the level of EGFR signaling in Egfr^Wa5 heterozygous mice is higher than in Egfr^wa2 homozygotes and suggests that different Egfr allele combinations can be generated to “genetically titer” total EGFR activity in vivo.     

Complementation of Defective Colony-Stimulating Factor 1 Receptor Signaling and Mitogenesis by Raf and v-Src

Ras-activated signal transduction pathways are implicated in the control of cell proliferation, differentiation, apoptosis, and tumorigenesis, but the molecular mechanisms mediating these diverse functions have yet to be fully elucidated. Conditionally active forms of Raf, v-Src, and MEK1 were used to identify changes in gene expression that participate in oncogenic transformation, as well as in normal growth control. Activation of Raf, v-Src, and MEK1 led to induced expression of c-Myc and cyclin D1. Induction of c-Myc mRNA by Raf was an immediate-early response, whereas the induction of cyclin D1 mRNA was delayed and inhibited by cycloheximide. Raf activation also resulted in the induction of an established c-Myc target gene, ornithine decarboxylase (ODC). ODC induction by Raf was mediated, in part, by tandem E-boxes contained in the first intron of the gene. Activation of the human colony-stimulating factor 1 (CSF-1) receptor in NIH 3T3 cells leads to activation of the mitogen-activated protein (MAP) kinase pathway and induced expression of c-Fos, c-Myc, and cyclin D1, leading to a potent mitogenic response. By contrast, a mutated form of this receptor fails to activate the MAP kinases or induce c-Myc and cyclin D1 expression and fails to elicit a mitogenic response. The biological significance of c-Myc and cyclin D1 induction by Raf and v-Src was confirmed by the demonstration that both of these protein kinases complemented the signaling and mitogenic defects of cells expressing this mutated form of the human CSF-1 receptor. Furthermore, the induction of c-Myc and cyclin D1 by oncogenes and growth factors was inhibited by PD098059, a specific MAP kinase kinase (MEK) inhibitor. These data suggest that the Raf/MEK/MAP kinase pathway plays an important role in the regulation of c-Myc and cyclin D1 expression in NIH 3T3 cells. The ability of oncogenes such as Raf and v-Src to regulate the expression of these proteins reveals new lines of communication between cytosolic signal transducers and the cell cycle machinery.     

Loss of Dlg-1 in the Mouse Lens Impairs Fibroblast Growth Factor Receptor Signaling

Coordination of cell proliferation, differentiation and survival is essential for normal development and maintenance of tissues in the adult organism. Growth factor receptor tyrosine kinase signaling pathways and planar cell polarity pathways are two regulators of many developmental processes. We have previously shown through analysis of mice conditionally null in the lens for the planar cell polarity gene (PCP), Dlg-1, that Dlg-1 is required for fiber differentiation. Herein, we asked if Dlg-1 is a regulator of the Fibroblast growth factor receptor (Fgfr) signaling pathway, which is known to be required for fiber cell differentiation. Western blot analysis of whole fiber cell extracts from control and Dlg-1 deficient lenses showed that levels of the Fgfr signaling intermediates pErk, pAkt, and pFrs2α, the Fgfr target, Erm, and the fiber cell specific protein, Mip26, were reduced in the Dlg-1 deficient fiber cells. The levels of Fgfr2 were decreased in Dlg-1 deficient lenses compared to controls. Conversely, levels of Fgfr1 in Dlg-1 deficient lenses were increased compared to controls. The changes in Fgfr levels were found to be specifically in the triton insoluble, cytoskeletal associated fraction of Dlg-1 deficient lenses. Immunofluorescent staining of lenses from E13.5 embryos showed that expression levels of pErk were reduced in the transition zone, a region of the lens that exhibits PCP, in the Dlg-1 deficient lenses as compared to controls. In control lenses, immunofluorescent staining for Fgfr2 was observed in the epithelium, transition zone and fibers. By E13.5, the intensity of staining for Fgfr2 was reduced in these regions of the Dlg-1 deficient lenses. Thus, loss of Dlg-1 in the lens impairs Fgfr signaling and leads to altered levels of Fgfrs, suggesting that Dlg-1 is a modulator of Fgfr signaling pathway at the level of the receptors and that Dlg-1 regulates fiber cell differentiation through its role in PCP.     

Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.     

Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

表皮生长因子及受体在甲状腺肿瘤中的表达

目的:研究表皮生长因子(Epidermal Growth Factor,EGF)及受体(Epidermal Growth Factor Receptor,EGFR)及在甲状腺肿瘤中的表达。方法:应用免疫组织化学法检测91例甲状腺病变组织中EGFR和EGF的表达情况。结果:结节性甲状腺肿、甲状腺腺瘤、分化型甲状腺癌标本中EGFR表达的阳性率分别为15%、25%、68.62%,EGF表达的阳性率分别为10%、15%、68.62%,其中EGFR、EGF在分化型甲状腺癌与其余两组间差异均有统计学意义(P<0.05)。EGFR和EGF在甲状腺乳头状癌中的表达与性别、年龄、肿瘤大小、淋巴结转移、临床分期等临床因素无明显相关。结论:EGF和EGFR的表达可作为鉴别甲状腺肿瘤良恶性的一个指标。     

表皮生长因子和前列腺素E1对小鼠精原细胞增殖的促进作用

利用生殖细胞-体细胞无血清共培养模型研究了表皮生长因(EGF)和前列腺素E1(PGE1)对小鼠A型精原细胞增殖的影响.A型精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24 h后进行c-kit、EGF、表皮生长因子受体(EGFR)、环氧化酶-1(COX-1)及环氧化酶-2(COX-2)的免疫细胞化学检测,72 h后测定其形成集落教的情况.结果显示,A型精原细胞呈c-kit阳性,EGF、EGFR、COX-1及COX-2主要表达于精原细胞.EGF(10-7～10-6mol/L)或PGE,(10-8.10一mol/I_.)均可显著促进精原细胞集落的形成.此外,前列腺素(PG)受体拮抗剂SCl9220(10-6～10-5mol/L)可抑制PGE1对精原细胞的促增殖作用,COX-1抑制剂SC560(10-7～10-5mol/L)和COX-2抑制剂NS398(10-7～10-5mol/L)能抑制EGF促进精原细胞增殖的作用.因此,EGF可通过促进局部PG的产生而刺激精原细胞的增殖.     

Prolidase Directly Binds and Activates Epidermal Growth Factor Receptor and Stimulates Downstream Signaling

Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.     

Growth Factor erv1-like Modulates Drp1 to Preserve Mitochondrial Dynamics and Function in Mouse Embryonic Stem Cells

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission–fusion dynamics in pluripotent ESCs.     

表皮生长因子受体(EGFR)的细胞内信号传导

表皮生长因子受体（EGFR）是一种存在于细胞表面的多功能跨膜蛋白分子,具有酪氨酸蛋白激酶活性,EGFR与配体结合后启动细胞内信号传导通路,不同的通路之间存在交叉对话（Cross-talks）共同完成细胞生理功能.对EGFR的深入研究,不仅可阐明细胞生长和发育等重要的生命过程,而且在医药和工业上也将有广泛的应用.     

Targeting Human Epidermal Growth Factor Receptor Signaling with the Neuregulin's Heparin-binding Domain

A major limitation in biopharmaceutical development is selectively targeting drugs to diseased tissues. Growth factors and viruses have solved this problem by targeting tissue-specific cell-surface heparan sulfates. Neuregulin (NRG), a growth factor important in both nervous system development and cancer, has a unique heparin-binding domain (HBD) that targets to cell surfaces expressing its HER2/3/4 receptors (Esper, R. M., Pankonin, M. S., and Loeb, J. A. (2006) Brain Res. Rev. 51, 161–175). We have harnessed this natural targeting ability of NRG by fusing the HBD of NRG to soluble HER4. This fusion protein retains high affinity heparin binding to heparin and to cells that express heparan sulfates resulting in a more potent NRG antagonist. In vivo, it is targeted to peripheral nerve segments where it blocks the activity of NRG as a Schwann cell survival factor. The fusion protein also efficiently blocks autocrine and paracrine signaling and reduces the proliferation of MCF10CA1 breast cancer cells. These findings demonstrate the utility of the HBD of NRG in biopharmaceutical targeting and provide a new way to block HER signaling in cancer cells.     

Comprehensive Mapping of the Human Kinome to Epidermal Growth Factor Receptor Signaling

Disregulation of epidermal growth factor receptor (EGFR) signaling directly promotes bypass of proliferation and survival restraints in a high frequency of epithelia-derived cancer. As such, much effort is currently focused on decoding the molecular architecture supporting EGFR activation and function. Here, we have leveraged high throughput reverse phase protein lysate arrays, with a sensitive fluorescent nanocrystal-based phosphoprotein detection assay, together with large scale siRNA-mediated loss of function to execute a quantitative interrogation of all elements of the human kinome supporting EGF-dependent signaling. This screening platform has captured multiple novel contributions of diverse protein kinases to modulation of EGFR signal generation, signal amplitude, and signal duration. As examples, the prometastatic SNF1/AMPK-related kinase hormonally upregulated Neu kinase was found to support EGFR activation in response to ligand binding, whereas the enigmatic kinase MGC16169 selectively supports coupling of active EGFR to ERK1/2 regulation. Of note, the receptor tyrosine kinase MERTK and the pyrimidine kinase UCK1 were both found to be required for surface accumulation of EGFR and subsequent pathway activation in multiple cancer cell backgrounds and may represent new targets for therapeutic intervention.     

Transient Acceleration of Epidermal Growth Factor Receptor Dynamics Produces Higher-Order Signaling Clusters

Cell signaling depends on spatiotemporally regulated molecular interactions. Although the movements of signaling proteins have been analyzed with various technologies, how spatial dynamics influence the molecular interactions that transduce signals is unclear. Here, we developed a single-molecule method to analyze the spatiotemporal coupling between motility, clustering, and signaling. The analysis was performed with the epidermal growth factor receptor (EGFR), which triggers signaling through its dimerization and phosphorylation after association with EGF. Our results show that the few EGFRs isolated in membrane subdomains were released by an EGF-dependent increase in their diffusion area, facilitating molecular associations and producing immobile clusters. Using a two-color single-molecule analysis, we found that the EGF-induced state transition alters the properties of the immobile clusters, allowing them to interact for extended periods with the cytoplasmic protein, GRB2. Our study reveals a novel correlation between this molecular interaction and its mesoscale dynamics, providing the initial signaling node.     

鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。