肝素结合性表皮生长因子在心肌重塑中的作用

肝素结合性表皮生长因子（HB-EGF）是表皮生长因子家族成员之一。HB-EGF是多种细胞的有丝分裂原,参与一系列生理和病理过程,包括心肌细胞肥大,成纤维细胞增生,胶原纤维表达增多,是心肌重塑发生发展过程中的一个重要生长因子。本文综述了HB-EGF在心肌重塑过程中的研究进展。     

Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.     

Hepatoma-Derived Growth Factor-Related Protein-3 Is a Novel Angiogenic Factor

Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.     

The Actin Binding Domain of the Epidermal Growth Factor Receptor Is Required for EGF-Stimulated Tissue Invasion

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.     

Interactions among the Epidermal Growth Factor-like Modules of Thrombospondin-1

Epidermal growth factor (EGF)-like modules are defined in part by six cysteines joined by disulfides in a 1–3, 2–4, and 5–6 pattern. Thrombospondin-1 (TSP-1) is a multimodular glycoprotein with three EGF-like modules, E1, E2, and E3, arranged in tandem. These modules likely propagate conformational changes between surrounding C-terminal and N-terminal elements of TSP-1 and interact with other extracellular molecules. E1, E2, and their homologs in other TSPs are unique among EGF-like modules in having two residues rather than one between Cys-4 and Cys-5. In addition, E2 has a calcium-binding site and an unusually long loop between Cys-5 and Cys-6. The structure of E1, E2, or E3 expressed alone changed little upon heating as monitored by far-UV CD, whereas more marked changes occurred in E12, E23, and E123 tandem constructs. The individual modules denatured in differential scanning calorimetry experiments only at >85 °C. E12, E23, or E123 tandem constructs, however, had a transition in the range of 44–70 °C. The temperature of the transition was higher when calcium was present and higher with E123 than with E12 or E23. Isothermal titration calorimetry demonstrated K_D values of binding of calcium to E2, E12, E23, or E123 at 25 °C of 11.5, 2.9, 2.2, or 0.3 μm, respectively. Monoclonal antibodies HB8432 and C6.7, which recognize epitopes in E2, bound to E12, E23, or E123 with greater affinity than to E2 alone. These results indicate that interactions among the modules of E123 influence the tertiary structure and calcium binding of E2.Thrombospondins (TSPs)² are multimodule, calcium-binding extracellular glycoproteins with various functions (1). TSP-1, which was the first TSP to be discovered and remains the best characterized, and TSP-2 are trimers. Each subunit is composed of an N-terminal module, oligomerization domain, von Willebrand factor type C module, three properdin or TSP type 1 modules, and the C-terminal signature domain that includes three EGF-like modules (E123), 13 aspartate-rich calcium-binding repeats of the wire module, and a lectin-like module (2–4). The five mammalian TSPs fall into two groups, trimeric (TSP-1 and TSP-2) and pentameric (TSP-3, TSP-4, and TSP-5) (1). All have a signature domain, with the major difference being the presence of four rather than three EGF-like modules in the signature domain of pentameric TSPs.EGF-like modules exist in more than 300 human extracellular proteins and play important roles in biological processes such as blood clotting and cell-cell signaling (5–7). The modules are 30–50 residues long and characterized by six cysteine residues that form three disulfide bonds in the order 1–3, 2–4, and 5–6 (Fig. 1) (6, 7). The backbone structure of the EGF-like modules consists of two submodules, referred to as the major (N-terminal) and minor (C-terminal) submodules (6, 8, 9).Open in a separate windowFIGURE 1.Model of the structure of E123. The model is built based on the crystal structure of EGF modules in the TSP-2 signature domain (Protein Data Bank code 1YO8) using SYBYL 7.0. E1 is shown in red, E2 in pink, and E3 in purple. The cysteines are colored yellow; the backbones of the residues between the fourth and fifth Cys are in blue; Glu-609 recognized by HB8432 and C6.7 is shown in green; and the long loop in E2 between the fifth and sixth Cys is hot pink. Ca²⁺ bound to the binding site on E2 near the interface between E1 and E2 is depicted as a red ball.The crystal structure of the three EGF-like modules of TSP-2 has been solved as part of the TSP-2 signature domain in 2 mm calcium (Ca²⁺) (Fig. 1) (4). All have the 1–3, 2–4, and 5–6 disulfide pattern. There is one Ca²⁺-binding site in the second EGF-like module (E2), located near the interface between the first and second EGF-like modules (E1 and E2) (Fig. 1). There is only one residue between the fourth and fifth cysteines in most EGF-like modules (6). However, E1 and E2 of TSP-1 and TSP-2 and three of the four EGF-like modules (E1, E2, and E2′) of pentameric TSPs have two residues between the fourth and fifth Cys. This difference is potentially important because the N-terminal major submodule of the repeat containing the 1–3 and 2–4 disulfides and the C-terminal submodule with the 5–6 disulfide have the potential to undergo hinge-like motions around the residues between the fourth and fifth Cys (6, 8, 9). Having two rather than one residue between these two Cys increases the potential flexibility. In addition, E2 modules in all five TSPs contain an unusually long loop of 23 residues between the fifth and sixth Cys (Fig. 1). In the TSP-2 signature domain structure, residues from the long loop interact with repeat 12N of the wire module (4). E3, which has one residue between the fourth and fifth Cys, interacts with the wire and the lectin-like module (3, 4). A common polymorphism (N700S) in wire repeat 1C of human TSP-1 influences the stability of the EGF-like modules (10). This finding suggests that the interactions between the EGF-like modules and more C-terminal elements of the signature domain allow conformational changes in the more C-terminal elements to be propagated N-terminally.The EGF-like modules (E123) of TSP-1 denature in differential scanning calorimetry (DSC) with a melting temperature of ∼68 °C in 2 mm Ca²⁺ (10), although most EGF-like modules are stable to heating (7). We have investigated this transition in detail to learn its origins and the influence of Ca²⁺. The results indicate interactions among the modules of E123 that enhance Ca²⁺ binding and influence the tertiary structure of E2.     

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。     

Interferon-β Is a Potent Promoter of Nerve Growth Factor Production by Astrocytes

Abstract: Recent clinical evidence has suggested that interferon-β is efficacious in the treatment of the demyelinating disease, multiple sclerosis. The mechanism of its efficacy remains unclear, and suggested modes of action have focused on immune modulation. Nonimmune effects of interferon-β may also contribute to its efficacy. Given that astrocytes produce a range of neurotrophic factors, we examined the possibility that interferon-β could increase the astrocytic production of nerve growth factor (NGF), which has been reported to cause oligodendrocytes to proliferate and to extend their processes; these phenotypes can impact favorably on remyelination. When the recombinant form of mouse interferon-β was added to mouse astrocyte cultures, a dose-dependent increase in NGF mRNA was obtained. The 40-fold increase in NGF mRNA elicited by 1,000 U/ml interferon-β was far more potent than that produced by other NGF-elevating agents in this study. In concordance, the protein for NGF was elevated by interferon-β. The production of NGF by interferon-β may be relevant to its clinical efficacy in multiple sclerosis. Furthermore, we suggest the potential utility of interferon-β in Alzheimer's disease.     

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.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes

In the skin epidermis, keratinocytes undergo anchorage-dependent cornification, which gives rise to stratified multilayers, each with a distinct differentiation feature. The active formation of the cornified cell envelope (CCE), an important element in the skin barrier, occurs in keratinocytes of the upper epidermal layers and impacts their terminal differentiation. In the present study, we identified the extracellularly extruded syntaxin-4 as a potent differentiation regulator of epidermal keratinocytes. We found that differentiation stimuli led to the acceleration of syntaxin-4 exposure at the keratinocyte cell surface and that the artificial control of extracellular syntaxin-4, either by the forced expression of several syntaxin-4 mutants with structural alterations at the putative functional core site (AIEPQK), or by using antagonistic circular peptides containing this core sequence, dramatically influenced the CCE formation, with spatial misexpression of TGase1 and involucrin. We also found that the topical application of a peptide that exerted the most prominent antagonistic activity for syntaxin-4, named ST4n1, evidently prevented the formation of the hyperplastic and hyperkeratotic epidermis generated by physical irritation in HR-1 mice skin. Collectively, these results demonstrate that extracellularly extruded syntaxin-4 is a potent regulator of CCE differentiation, and that ST4n1 has potential as a clinically applicable reagent for keratotic skin lesions.     

HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。     

Schwannoma-Derived Growth Factor Interacts with the Epidermal Growth Factor Receptor

Abstract: Schwannoma-derived growth factor (SDGF) is a potent mitogen and neuronal differentiation factor. Because of its relationship to epidermal growth factor (EGF) and the heregulins, it was asked if SDGF interacts with the EGF receptor or HER2/neu. SDGF binds to and causes the phosphorylation on tyrosine of the EGF receptor but not HER2/neu.     

Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.     

A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.     

Epidermal Growth Factor Induces Internalization But Not Degradation of the Epidermal Growth Factor Receptor in a Human Breast Cancer Cell Line

Abstract

Metabolism of the epidermal growth factor (EGF) receptor was studied in the MDA-MB-231 human breast cancer cell line. As in normal fibroblasts the EGF receptor from MDA-MB-231 cells was synthesized from a M_r =160,000 precursor and tunicamycin treatment of cells resulted in accumulation of a M_r =130,000 polypeptide. Unlike normal fibroblasts in which a M_r =170,000 mature form of the EGF receptor was found, MDA-MB-231 cells contained a M_r =172,000 mature form. Addition of EGF to MDA-MB-231 cells led to rapid internalization of EGF receptors, however, internalization did not affect receptor half-life and receptors did not recycle to the cell surface. EGF receptors could be visualized by immunofluorescence and remained sequestered in intracellular membranous structures following internalization. EGF was degraded slowly by MDA-MB-231 cells relative to degradation of EGF by normal cells. A high endogenous level of in vivo phosphorylation of threonine 654 of the EGF receptor was found in MDA-MB-231 cells and treatment of cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further stimulated phosphorylation of this residue. EGF induced receptor internalization resulted in dephosphorylation of threonine 654. The significance of these unusual properties of EGF receptor metabolism in MDA-MB-231 cells is discussed.     

The DNA Replication Licensing Factor Miniature Chromosome Maintenance 7 Is Essential for RNA Splicing of Epidermal Growth Factor Receptor,c-Met,and Platelet-derived Growth Factor Receptor

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221–248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.     

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.     

人表皮生长因子基因转染原代角质形成细胞

目的:将带信号肽的人表皮生长因子基因转染原代成人角质形成细胞, 证实细胞活性及hEGF的有效表达, 为后期的皮肤修复研究打下基础.方法:先酶切验证pcDNA3.1-hEGF, 后消化成人皮肤组织, 以Defined Keratinocyte-SFM(DKSFM)传代培养角质形成细胞并鉴定, 脂质体转染质粒pcDNA3.1-hEGF入细胞, 转基因细胞培养48 h后作RT-PCR和Western-blot分析, 上清分别进行放射免疫测定和MTT测定.结果:质粒pcDNA3.1-hEGF上的hEGF序列经测序证实,双酶切后获得约230 bp和5.4 kb条带;成人角质形成细胞体外培养可快速稳定增殖, 转hEGF基因细胞经RT-PCR扩增出一条约230 bp的特异性条带, Western-blot检测到hEGF表达明显升高;放射免疫法和MTT实验证实转基因细胞有稳定的hEGF蛋白分泌.结论:质粒pcDNA3.1-hEGF在脂质体介导下成功转染成人皮肤角质形成细胞, 转基因细胞能分泌有生物活性的EGF;体外培养的成人皮肤角质形成细胞可见少量EGF分泌.