RESUMEN
Growth factors (GFs) are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Co-immobilizing GFs on materials while preserving their bioactivity still represents a major challenge in the field of tissue regeneration and bioactive implants. In this study, we explore the potential of an oriented immobilization technique based on two high affinity peptides, namely the Ecoil and Kcoil, to allow for the simultaneous capture of the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) on a chondroitin sulfate coating. This glycosaminoglycan layer was selected as it promotes cell adhesion but reduces non-specific adsorption of plasma proteins. We demonstrate here that both Ecoil-tagged GFs can be successfully immobilized on chondroitin sulfate surfaces that had been pre-decorated with the Kcoil peptide. As shown by direct ELISA, changing the incubation concentration of the various GFs enabled to control their grafted amount. Moreover, cell survival studies with endothelial and smooth muscle cells confirmed that our oriented tethering strategy preserved GF bioactivity. Of salient interest, co-immobilizing EGF and VEGF led to better cell survival compared to each GF captured alone, suggesting a synergistic effect of these GFs. Altogether, these results demonstrate the potential of coiled-coil oriented GF tethering for the co-immobilization of macromolecules; it thus open the way to the generation of biomaterials surfaces with fine-tuned biological properties. STATEMENT OF SIGNIFICANCE: Growth factors are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Controlled coimmobilization of growth factors on biomaterials while preserving their bioactivity represents a major challenge in the field of tissue regeneration and bioactive implants. This study demonstrates the potential of an oriented immobilization technique based on two high affinity peptides to allow for the simultaneous capture of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Our system allowed an efficient control on growth factor immobilization by adjusting the incubation concentrations of EGF and VEGF. Of salient interest, co-immobilizing of specific ratios of EGF and VEGF demonstrated a synergistic effect on cell survival compared to each GF captured alone.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas Inmovilizadas/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacología , Sinergismo Farmacológico , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/química , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Proteínas Inmovilizadas/agonistas , Proteínas Inmovilizadas/química , Factor A de Crecimiento Endotelial Vascular/agonistas , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.
Asunto(s)
Proliferación Celular , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/metabolismo , Fibroblastos/metabolismo , Proteínas Mutantes/farmacología , Ingeniería de Proteínas , Secuencia de Aminoácidos , Células Cultivadas , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fibroblastos/citología , Prepucio/citología , Prepucio/metabolismo , Biblioteca de Genes , Humanos , Masculino , Datos de Secuencia Molecular , Mutación/genética , Reacción en Cadena de la Polimerasa , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1ß (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Factor de Crecimiento Epidérmico/agonistas , Fibroblastos/metabolismo , Modelos Biológicos , Proteína Quinasa C/metabolismo , Proteínas ADAM/genética , Animales , Línea Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Fibroblastos/citología , Isoenzimas/genética , Isoenzimas/metabolismo , Ligandos , Ratones , Ratones Noqueados , Proteína Quinasa C/genética , Especificidad por SustratoRESUMEN
Agonist and antagonist drugs acting on epidermal growth factor receptor (EGFR) signaling are emerging as a new possibility for pharmaceutical study and clinical manipulation of some skin and corneal disorders. EGFR activation appears to be effective in reducing the time of reepithelialization after corneal wound healing, with potential uses in penetrating keratoplasty, refractive surgery, alkali burns, diabetic keratopathy, keratopathy following chemotherapy, cornea transplantation, and dry eye. Most of the studies show therapeutic advantages of human recombinant epidermal growth factor (hrEGF) eye drops without showing adverse effects. In contrast, EGFR inhibition delays epithelial cell proliferation and stratification during corneal regeneration.The aim of this review is to summarize the most seminal discoveries and recent advances so as to clarify the role of the EGFR system in corneal physiology and pharmacology. Epidermal growth factor eye drops could be a first-choice treatment for promoting regeneration in numerous epithelial defects in the medium to long term.
Asunto(s)
Córnea/metabolismo , Lesiones de la Cornea , Factor de Crecimiento Epidérmico , Receptores ErbB/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Córnea/efectos de los fármacos , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Epitelio Corneal/efectos de los fármacos , Receptores ErbB/farmacología , Humanos , Proteínas Recombinantes , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
The dynamic behaviors of human skeletal muscle myoblasts were investigated in the culture on a laminin-coated surface in the presence of 100 ng/ml epidermal growth factor (EGF) in medium. The coexistence of laminin and EGF caused the enhancement of myoblast migration, giving an average migration rate of 62.0 microm/h, which was 2.7 times that on a plain surface. This encouraged migration could be a driving force to separate the dividing cells from each other, accompanied by shortened disjunction time of daughter cells to complete cytokinesis. In addition, the synergic effect of laminin and EGF led to the promotion of myoblast growth with keeping a relatively high fraction of proliferative cells during the culture for 150 h, which is considered to arise from the reduced frequency of cell-cell contacts during cytokinesis and thereby suppressing the process towards myotube formation after cell division.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citocinesis/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Laminina/farmacología , Mioblastos Esqueléticos/fisiología , Animales , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Factor de Crecimiento Epidérmico/agonistas , Humanos , Laminina/agonistas , Ratones , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/fisiología , Mioblastos Esqueléticos/citologíaRESUMEN
Transforming growth factor (TGF)-beta1 has a biphasic effect on rat intestinal epithelial (RIE) cells. By itself, TGF-beta1 functions as a tumor suppressor by inhibiting the growth, migration and invasion of RIE cells. We show in this study that in conjunction with epidermal growth factor (EGF), TGF-beta1 helped to augment migration, invasion and anchorage-independent growth (AIG) compared to that by EGF alone. EGF plus TGF-beta1 induced a dramatic morphological change characteristic of epithelial-mesenchymal transition (EMT). The mechanism for this enhanced effect of TGF-beta1 and EGF on oncogenic properties was explored by analysis of EGF- and TGF-beta1-mediated signaling pathways and complementary DNA arrays. TGF-beta1 augmented EGF-mediated signaling of mitogen-activated protein kinase (MAPK) and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. Inhibition of MAPK, but not phosphoinositide-3 kinase (PI3K), abolished TGF-beta1 plus EGF-induced EMT and downregulation of E-cadherin at mRNA and protein levels. By contrast, cell migration and invasion were sensitive to inhibition of either MAPK or PI3 kinase. TGF-beta1 plus EGF-induced AIG was significantly more resistant to inhibition of PI3K and MAPK compared to that induced by EGF alone. EGF and TGF-beta1 synergistically induced the expression of a series of proteases including matrix metalloproteinase (MMP) 1 (collagenase), MMP3, MMP9, MMP10, MMP14 and cathepsin. Among them, the expression of MMP1, MMP3, MMP9 and MMP10 was MAPK dependent. Inhibition of the MMPs or cathepsin significantly blocked EGF plus TGF-beta1-induced invasion, but had no effect on colony formation. Phospholipase C (PLC) and Cox2 induced by EGF plus TGF-beta1 also played a significant role in invasion, whereas PLC was also important for colony formation. Our study reveals specific signaling functions and induction of genes differentially required for enhanced effect of EGF- and TGF-beta1-induced oncogenic properties, and helps to explain the tumor-promoting effect of TGF-beta1 in human cancer with elevated expression or activation of TGF-beta1 and receptor protein tyrosine kinases.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Supresoras de Tumor/agonistas , Animales , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Sinergismo Farmacológico , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Mucosa Intestinal/patología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Factor de Crecimiento Transformador beta1/agonistas , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
We have shown that one of the principle mechanisms of chemotherapy resistance involves the activation of nuclear factor kappa-B (NF-kappaB). In an effort to identify NF-kappaB-regulated chemotherapy response genes, we performed a microarray assay and observed that heparin-binding EGF-like growth factor (HB-EGF) was significantly upregulated by SN38 (a strong inducer of NF-kappaB activity) in colon cancer cells. Further studies revealed that HB-EGF was rapidly induced following a variety of chemotherapy treatments. Using RNA interference, we demonstrated that the chemotherapy-induced HB-EGF was largely dependent on activator protein-1 (AP-1) and NF-kappaB activation. Constitutive HB-EGF expression rescued AP-1/NF-kappaB small interfering RNA (siRNA) cells from chemotherapy-induced apoptosis. Meanwhile, we found that the enzymatic shedding of HB-EGF was also regulated by chemotherapy treatment, resulting in the elevated release of soluble HB-EGF from the cellular membrane. Induction of HB-EGF expression and ectodomain shedding synergistically led to robust epidermal growth factor receptor (EGFR) phosphorylation, whereas inhibition of HB-EGF expression by use of the HB-EGF inhibitor (CRM197) or siRNA resulted in the suppression of chemotherapy-induced EGFR phosphorylation. These results suggest that the chemotherapy-induced EGFR activation is regulated by HB-EGF. Finally, we demonstrated that overexpression of HB-EGF led to apoptotic resistance to chemotherapy, whereas suppression of HB-EGF expression by siRNA resulted in a dramatic increase in cell death. In summary, our study suggests that chemotherapy-induced HB-EGF activation represents a critical mechanism of inducible chemotherapy resistance. Therefore, therapeutic intervention aimed at inhibiting HB-EGF activity may be useful in cancer prevention and treatments.
Asunto(s)
Resistencia a Antineoplásicos/genética , Factor de Crecimiento Epidérmico/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias/tratamiento farmacológico , Apoptosis/genética , Proteínas Bacterianas/farmacología , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/genética , Expresión Génica , Genes fos , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , FN-kappa B/metabolismo , Neoplasias/genética , ARN Interferente Pequeño/farmacología , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA/genéticaRESUMEN
Transition Therapeutics (through its acquisition of Waratah Pharmaceuticals), in collaboration with Novo Nordisk, is developing E1-INT, an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog, for the treatment of insulin-dependent (type 1) and non-insulin-dependent (type 2) diabetes. The compound is currently undergoing phase II clinical trials.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Factor de Crecimiento Epidérmico/uso terapéutico , Gastrinas/uso terapéutico , Hipoglucemiantes/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Industria Farmacéutica , Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/química , Gastrinas/agonistas , Gastrinas/química , Humanos , Hipoglucemiantes/química , Relación Estructura-ActividadRESUMEN
The epidermal growth factor (EGF) receptor is activated not only by EGF-like ligands, but also by stimuli that do not directly act on the receptor, including agonists of G protein--coupled receptors and certain environmental stresses such as ionizing radiation. Carpenter discusses two reports that indicate EGF receptor activation by such heterologous stimuli may occur through the action of proteases that release cell surface EGF-like growth factor precursors. This mechanism of EGF receptor transactivation appears to involve the generation of soluble agonists.
Asunto(s)
Factor de Crecimiento Epidérmico/agonistas , Factor de Crecimiento Epidérmico/biosíntesis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Activación Transcripcional , Animales , Factor de Crecimiento Epidérmico/fisiología , Humanos , Transducción de Señal/genética , Transducción de Señal/fisiología , Activación Transcripcional/genética , Activación Transcripcional/fisiologíaRESUMEN
Numerous epidermal growth factor (EGF)-related peptide binding members of the ErbB family of receptor tyrosine kinases have been described. While several EGF agonists bind and activate ErbB-1/EGF receptor, neu differentiation factor (NDF) functions as a ligand for ErbB-3 and ErbB-4. However, it is currently unknown which specific subsets of ErbB receptors become activated in response to each of these ligands. The present study addresses this issue using the T47D breast tumor cell line, which expresses moderate levels of all the presently known ErbB receptors. We show that all the EGF agonists, but not NDF, stimulated tyrosine phosphorylation of ErbB-1. In contrast, all the EGF-related factors except amphiregulin were able to induce tyrosine phosphorylation of ErbB-2. The ability to induce tyrosine phosphorylation of ErbB-3 varied dramatically among the different EGF-related peptides. While EGF, transforming growth factor (TGF)-alpha, and amphiregulin only had a moderate effect, NDF dramatically increased the ErbB-3 phosphotyrosine content. Most notably, heparin binding EGF-related growth factor (HB-EGF) and betacellulin (BTC) were more effective than other EGF agonists. Consequently, only NDF, HB-EGF, and BTC significantly stimulated association of phosphatidylinositol kinase activity with ErbB-3. Among the EGF agonists, HB-EGF induced a low level of ErbB-4 tyrosine phosphorylation, while BTC was as efficient as NDF in activating ErbB-4. The BTC activation of ErbB-4 appears to be independent of ErbB-1, as shown by pretreatment of cells with an antibody that inhibits binding of EGF agonists to ErbB-1. As a result of the differential activation of ErbB receptors, most of the EGF-related growth factors had distinguishable biological activities on cultured mammary epithelial cell lines.