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1.
J Cell Sci ; 136(13)2023 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-37282854

RESUMEN

Tylosis with oesophageal cancer (TOC) is a rare familial disorder caused by cytoplasmic mutations in inactive rhomboid 2 (iRhom2 or iR2, encoded by Rhbdf2). iR2 and the related iRhom1 (or iR1, encoded by Rhbdf1) are key regulators of the membrane-anchored metalloprotease ADAM17, which is required for activating EGFR ligands and for releasing pro-inflammatory cytokines such as TNFα (or TNF). A cytoplasmic deletion in iR2, including the TOC site, leads to curly coat or bare skin (cub) in mice, whereas a knock-in TOC mutation (toc) causes less severe alopecia and wavy fur. The abnormal skin and hair phenotypes of iR2cub/cub and iR2toc/toc mice depend on amphiregulin (Areg) and Adam17, as loss of one allele of either gene rescues the fur phenotypes. Remarkably, we found that iR1-/- iR2cub/cub mice survived, despite a lack of mature ADAM17, whereas iR2cub/cub Adam17-/- mice died perinatally, suggesting that the iR2cub gain-of-function mutation requires the presence of ADAM17, but not its catalytic activity. The iR2toc mutation did not substantially reduce the levels of mature ADAM17, but instead affected its function in a substrate-selective manner. Our findings provide new insights into the role of the cytoplasmic domain of iR2 in vivo, with implications for the treatment of TOC patients.


Asunto(s)
Queratodermia Palmar y Plantar Difusa , Queratodermia Palmoplantar , Neoplasias , Animales , Ratones , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Proteínas Portadoras/genética , Queratodermia Palmoplantar/genética , Proteínas de la Membrana/genética
2.
J Biol Chem ; 296: 100733, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33957124

RESUMEN

A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17-/- mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.


Asunto(s)
Proteína ADAM17/química , Proteína ADAM17/metabolismo , Citoplasma/metabolismo , Proteína ADAM17/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistemas CRISPR-Cas , Femenino , Fibroblastos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Dominios Proteicos , Estabilidad Proteica , Eliminación de Secuencia
3.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-33227998

RESUMEN

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell-cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2-/-) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2-/-iR1∆Ch mice had retarded bone growth compared to iR2-/- mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2-/-iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2-/-iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα-/- mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.


Asunto(s)
Proteína ADAM17/genética , Proteínas Portadoras/genética , Condrocitos/metabolismo , Condrogénesis/genética , Proteínas de la Membrana/genética , Osteogénesis/genética , Proteína ADAM17/metabolismo , Animales , Calcificación Fisiológica/genética , Proteínas Portadoras/metabolismo , Comunicación Celular , Diferenciación Celular , Proliferación Celular , Condrocitos/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Placa de Crecimiento/crecimiento & desarrollo , Placa de Crecimiento/metabolismo , Integrasas/genética , Integrasas/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismo
4.
J Biol Chem ; 295(13): 4350-4358, 2020 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-32060096

RESUMEN

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17.


Asunto(s)
Proteína ADAM17/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Membrana Celular , Receptores ErbB/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , Receptores de Interleucina-6/genética , Transducción de Señal/genética
5.
Biochem J ; 474(9): 1467-1479, 2017 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-28264989

RESUMEN

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.


Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Membrana Celular/enzimología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Animales , Células COS , Catálisis , Membrana Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ratones
6.
J Cell Sci ; 130(5): 868-878, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28104813

RESUMEN

A disintegrin and metalloproteinase 17 (ADAM17) controls the release of the pro-inflammatory cytokine tumor necrosis factor α (TNFα, also known as TNF) and is crucial for protecting the skin and intestinal barrier by proteolytic activation of epidermal growth factor receptor (EGFR) ligands. The seven-membrane-spanning protein called inactive rhomboid 2 (Rhbdf2; also known as iRhom2) is required for ADAM17-dependent TNFα shedding and crosstalk with the EGFR, and a point mutation (known as sinecure, sin) in the first transmembrane domain (TMD) of Rhbdf2 (Rhbdf2sin) blocks TNFα shedding, yet little is known about the underlying mechanism. Here, we used a structure-function analysis informed by structural modeling to evaluate the interaction between the TMD of ADAM17 and the first TMD of Rhbdf2, and the role of this interaction in Rhbdf2-ADAM17-dependent shedding. Moreover, we show that double mutant mice that are homozygous for Rhbdf2sin/sin and lack Rhbdf1 closely resemble Rhbdf1/2-/- double knockout mice, highlighting the severe functional impact of the Rhbdf2sin/sin mutation on ADAM17 during mouse development. Taken together, these findings provide new mechanistic and conceptual insights into the critical role of the TMDs of ADAM17 and Rhbdf2 in the regulation of the ADAM17 and EGFR, and ADAM17 and TNFα signaling pathways.


Asunto(s)
Proteína ADAM17/química , Proteína ADAM17/metabolismo , Proteínas Portadoras/metabolismo , Modelos Moleculares , Proteolisis , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Células de la Médula Ósea/citología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Placa de Crecimiento/metabolismo , Válvulas Cardíacas/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Relación Estructura-Actividad
7.
Circ Res ; 119(4): 519-31, 2016 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-27354212

RESUMEN

RATIONALE: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. OBJECTIVE: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. METHODS AND RESULTS: Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. CONCLUSIONS: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.


Asunto(s)
Proteína ADAM10/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Circulación Sanguínea/fisiología , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptor Notch1/fisiología , Receptores Notch/fisiología , Flujo Sanguíneo Regional/fisiología , Transducción de Señal/fisiología , Proteína ADAM10/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Animales , Células Endoteliales/fisiología , Femenino , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Ratones Transgénicos , Embarazo , Proteínas Proto-Oncogénicas/deficiencia , Receptor Notch1/deficiencia , Receptor Notch4 , Receptores Notch/deficiencia
8.
Proc Natl Acad Sci U S A ; 112(19): 6080-5, 2015 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-25918388

RESUMEN

The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17(-/-) mice die perinatally with open eyes, like Egfr(-/-) mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2(-/-) mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2(-/-) double knockout mice resemble Adam17(-/-) and Egfr(-/-) mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2(-/-) tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.


Asunto(s)
Proteínas ADAM/metabolismo , Proteínas Portadoras/metabolismo , Receptores ErbB/metabolismo , Proteína ADAM17 , Animales , Separación Celular , Células Madre Embrionarias/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Fibroblastos/metabolismo , Citometría de Flujo , Heterocigoto , Selectina L/metabolismo , Leucocitos/metabolismo , Ligandos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microglía/metabolismo , Neoplasias/metabolismo , Fenotipo , Fosforilación , Regiones Promotoras Genéticas , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo
9.
J Orthop Res ; 32(2): 224-30, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24108673

RESUMEN

Mice lacking ADAM10 in endothelial cells (Adam10ΔEC mice) have shorter femurs, tibiae, and humeri than controls, raising questions about how endothelial cells could control long bone growth. We performed a histopathological evaluation of the femur and tibia growth plates at different postnatal stages, and assessed the distribution of TRAP-positive osteoclasts and endothelial cells at the growth plate. The growth plates in Adam10ΔEC mice appeared normal at P7 and P14, but a thickened zone of hypertrophic chondrocytes and increased trabecular bone density were apparent by P21 and later. The number of TRAP+ cells at the COJ was normal at P7 and P14, but was strongly reduced at P21 and later. Moreover, the density of endomucin-stained endothelial cells at the COJ was increased starting at P7. The defects in long bone growth in Adam10ΔEC mice could be caused by a lack of osteoclastogenesis at the COJ. Moreover, ADAM10 appears to regulate endothelial cell organization in the developing bone vasculature, perhaps in a similar manner as in the developing retinal vascular tree, where ADAM10 is thought to control Notch-dependent endothelial cell fate decisions. This study provides evidence for the regulation of osteoclast function by endothelial cells in vivo.


Asunto(s)
Proteínas ADAM/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Desarrollo Óseo/fisiología , Células Endoteliales/fisiología , Placa de Crecimiento/fisiología , Proteínas de la Membrana/deficiencia , Osteoclastos/fisiología , Proteína ADAM10 , Fosfatasa Ácida/metabolismo , Envejecimiento , Animales , Huesos/fisiología , Fémur/irrigación sanguínea , Fémur/crecimiento & desarrollo , Isoenzimas/metabolismo , Ratones , Fosfatasa Ácida Tartratorresistente , Tibia/crecimiento & desarrollo
10.
Invest Ophthalmol Vis Sci ; 54(1): 864-70, 2013 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-23299479

RESUMEN

PURPOSE: Pathological neovascularization is a crucial component of proliferative retinopathies. Previous studies showed that inactivation of A disintegrin and metalloproteinase 17 (ADAM17), a membrane-anchored metalloproteinase that regulates epidermal growth factor receptor (EGFR) signaling, reduces pathological retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Here, we tested how genetic inactivation of a physiological ADAM17 inhibitor, the tissue inhibitor of matrix metalloproteinases-3 (TIMP3), or intravitreal injection of TIMP3 or of the EGFR inhibitor erlotinib influenced the outcome of OIR. METHODS: Wild-type mice were subjected to OIR in a chamber with 75% oxygen for 5 days beginning at postnatal day 7 (P7). Upon removal from the oxygen chamber at P12, they received a single intravitreal injection of TIMP3, erlotinib, or control. The central avascular area and neovascular tufts were measured after 5 days in room air (21% oxygen) at P17. Moreover, OIR experiments were performed with Timp3-/- mice and littermate controls. RESULTS: Timp3-/- mice showed greater revascularization of the central avascular area and developed equal or fewer neovascular tufts compared to littermate controls, depending on the genetic background. Wild-type mice injected with TIMP3 or erlotinib developed fewer neovascular tufts when compared to untreated littermates. Moreover, vessel regrowth into the avascular area was reduced in TIMP3-injected mice, but not in erlotinib-injected mice. CONCLUSIONS: These studies demonstrate that TIMP3 and erlotinib inhibit pathological neovascularization in the mouse retina, most likely due to inactivation of ADAM17 and the EGFR, respectively. Thus, TIMP3 and erlotinib emerge as attractive candidate antiangiogenic compounds for prevention and treatment of proliferative retinopathies.


Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Quinazolinas/farmacología , Enfermedades de la Retina/tratamiento farmacológico , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Femenino , Inyecciones Intravítreas , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/etiología , Neovascularización Patológica/metabolismo , Oxígeno/toxicidad , Inhibidores de Proteínas Quinasas/farmacología , Enfermedades de la Retina/etiología , Enfermedades de la Retina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Inhibidor Tisular de Metaloproteinasa-3/genética
11.
J Neurosci ; 30(14): 4833-44, 2010 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-20371803

RESUMEN

The metalloproteinase and major amyloid precursor protein (APP) alpha-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10(-/-) mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that alpha-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP alpha-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.


Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Proteínas de la Membrana/fisiología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Corteza Cerebral/crecimiento & desarrollo , Femenino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neurogénesis/genética , Neurogénesis/fisiología , Embarazo , Receptores Notch/biosíntesis , Receptores Notch/metabolismo
12.
J Mol Med (Berl) ; 88(5): 497-505, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20119708

RESUMEN

ADAM8 is a member of the "a disintegrin and metalloproteinase" (ADAM) family of membrane-anchored metalloproteinases. ADAM8-deficient mice have no evident spontaneous developmental or pathological defects, and little is currently known about the role of ADAM8 in disease. Here, we investigated the contribution of ADAM8 to pathological neovascularization in mice using an oxygen-induced retinopathy (OIR) model and heterotopical injection of tumor cells. We found an increase in retinal re-vascularization but fewer neovascular tufts in the OIR model and increased growth of heterotopically injected tumor cells in Adam8-/- mice compared with wild-type controls. These results suggest that ADAM8 functions to limit both of these processes in wild-type mice. In cell-based assays, overexpression of ADAM8 increased the ectodomain shedding of several co-expressed membrane proteins with roles in angiogenesis (CD31, Tie-2, Flk-1, Flt-1, EphrinB2, EphB4, VE-cadherin, KL-1, E-selectin, and neuregulin-1beta2). Thus, dysregulated expression of ADAM8 in endothelial cells in vivo could potentially increase the processing of these and other substrate proteins. Taken together, our findings suggest that inhibiting ADAM8 could be useful for promoting re-vascularization and thereby preventing formation of neovascular tufts in proliferative retinopathies. On the other hand, blocking ADAM8 could be detrimental in the context of rapidly growing tumors.


Asunto(s)
Proteínas ADAM/metabolismo , Antígenos CD/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Retina/patología , Neovascularización Retiniana/metabolismo , Proteínas ADAM/genética , Animales , Antígenos CD/genética , Línea Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Eliminación de Gen , Melanoma/patología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Retina/citología , Regulación hacia Arriba
13.
Circ Res ; 106(5): 932-40, 2010 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-20110534

RESUMEN

RATIONALE: Pathological neovascularization is a critical component of diseases such as proliferative retinopathies, cancer and rheumatoid arthritis, yet much remains to be learned about the underlying causes. Previous studies showed that vascular endothelial growth factor (VEGF)-A activates the membrane-anchored metalloproteinase ADAM17 (a disintegrin and metalloproteinase 17) in endothelial cells, thereby stimulating crosstalk between VEGF receptor 2 and extracellular signal-regulated kinase. These findings raised interesting questions about the role of ADAM17 in angiogenesis and neovascularization in vivo. OBJECTIVE: The objective of this study was to inactivate ADAM17 in endothelial cells or in pericytes to determine how this affects developmental angiogenesis, pathological retinal neovascularization and heterotopic tumor growth. METHODS AND RESULTS: We generated animals in which floxed ADAM17 was removed by Tie2-Cre in endothelial cells, or by smooth muscle (sm) Cre in smooth muscle cells and pericytes. There were no evident developmental defects in either conditional knockout strain, but pathological retinal neovascularization and growth of heterotopically injected tumor cells was reduced in Adam17flox/flox/Tie2-Cre mice, although not in Adam17flox/flox/sm-Cre mice. Moreover, lack of ADAM17 in endothelial cells decreased ex vivo chord formation, and this could be largely restored by addition of the ADAM17 substrate HB-EGF (heparin-binding epidermal growth factor-like growth factor). Finally we found that ADAM17 is important for the VEGF receptor 2 stimulated processing of several receptors with known functions in endothelial cell biology. CONCLUSIONS: These results provide the first evidence for a role for ADAM17 in pathological neovascularization in vivo. Because ADAM17 does not appear to be required for normal developmental angiogenesis or vascular homeostasis, it could emerge as a good target for treatment of pathological neovascularization.


Asunto(s)
Proteínas ADAM/deficiencia , Células Endoteliales/enzimología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/prevención & control , Neovascularización Patológica/prevención & control , Pericitos/enzimología , Neovascularización Retiniana/prevención & control , Proteínas ADAM/genética , Proteína ADAM17 , Actinas/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Crecimiento Similar a EGF de Unión a Heparina , Integrasas/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/enzimología , Neovascularización Patológica/genética , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas Receptoras/genética , Receptor TIE-2 , Neovascularización Retiniana/enzimología , Neovascularización Retiniana/genética , Porcinos , Factores de Tiempo , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
14.
Circ Res ; 103(9): 916-8, 2008 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-18818406

RESUMEN

Vascular endothelial growth factor (VEGF)-A and the VEGF receptors are critical for regulating angiogenesis during development and homeostasis and in pathological conditions, such as cancer and proliferative retinopathies. Most effects of VEGF-A are mediated by the VEGFR2 and its coreceptor, neuropilin (NRP)-1. Here, we show that VEGFR2 is shed from cells by the metalloprotease disintegrin ADAM17, whereas NRP-1 is released by ADAM10. VEGF-A enhances VEGFR2 shedding by ADAM17 but not shedding of NRP-1 by ADAM10. VEGF-A activates ADAM17 via the extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase pathways, thereby also triggering shedding of other ADAM17 substrates, including tumor necrosis factor alpha, transforming growth factor alpha, heparin-binding epidermal growth factor-like growth factor, and Tie-2. Interestingly, an ADAM17-selective inhibitor shortens the duration of VEGF-A-stimulated ERK phosphorylation in human umbilical vein endothelial cells, providing evidence for an ADAM17-dependent crosstalk between the VEGFR2 and ERK signaling. Targeting the sheddases of VEGFR2 or NRP-1 might offer new opportunities to modulate VEGF-A signaling, an already-established target for treatment of pathological neovascularization.


Asunto(s)
Proteínas ADAM/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células Endoteliales/enzimología , Fibroblastos/enzimología , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Neuropilina-1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Porcinos , Factores de Tiempo , Transfección , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
15.
Nat Immunol ; 7(12): 1293-8, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17072319

RESUMEN

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.


Asunto(s)
Proteínas ADAM/inmunología , Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptores de IgE/inmunología , Receptores de IgE/metabolismo , Proteína ADAM10 , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/inmunología , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Ratones , Ratones Noqueados , Transfección
16.
Methods Mol Biol ; 327: 99-113, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16780215

RESUMEN

All ligands of the epidermal growth factor receptor (EGFR) are made as membrane anchored precursors that can be proteolytically processed and released from the plasma membrane. This process, which is referred to as protein ectodomain shedding, is emerging as a key regulator of the function of EGFR ligands. In light of the important roles of EGFR signaling in development and disease, it will be important to understand more about the regulation of proteolytic processing of EGFR ligands. This chapter describes a sensitive and semiquantitative method to measure ectodomain shedding of EGFR ligands that was designed to facilitate studies of this process in cells.


Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Receptores ErbB/análisis , Fibroblastos/metabolismo , Ligandos , Ratones , Sensibilidad y Especificidad
17.
J Cell Biol ; 164(5): 769-79, 2004 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-14993236

RESUMEN

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.


Asunto(s)
Endopeptidasas/metabolismo , Receptores ErbB/metabolismo , Metaloendopeptidasas/metabolismo , Fenilalanina/análogos & derivados , Proteínas ADAM , Proteína ADAM12 , Proteína ADAM17 , Anfirregulina , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Betacelulina , Células Cultivadas , Desintegrinas/genética , Desintegrinas/metabolismo , Familia de Proteínas EGF , Embrión de Mamíferos/anatomía & histología , Endopeptidasas/genética , Factor de Crecimiento Epidérmico/metabolismo , Epirregulina , Fibroblastos/citología , Fibroblastos/metabolismo , Genotipo , Glicoproteínas/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fenilalanina/metabolismo , Inhibidores de Proteasas/metabolismo , Estructura Terciaria de Proteína , Acetato de Tetradecanoilforbol/metabolismo , Tiofenos/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo
18.
Mol Cell Biol ; 24(1): 96-104, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14673146

RESUMEN

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.


Asunto(s)
Sistema Cardiovascular/embriología , Desintegrinas/metabolismo , Proteínas de la Membrana/metabolismo , Metaloproteasas/metabolismo , Proteínas ADAM , Animales , Western Blotting , Desarrollo Óseo/fisiología , Encéfalo/metabolismo , Pulmón/crecimiento & desarrollo , Ratones
19.
J Biol Chem ; 279(6): 4241-9, 2004 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-14638693

RESUMEN

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.


Asunto(s)
Metaloendopeptidasas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células Cultivadas , Cricetinae , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas , Metaloendopeptidasas/química , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Células PC12 , Estructura Terciaria de Proteína , Ratas , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido
20.
Mol Cell Biol ; 23(16): 5614-24, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12897135

RESUMEN

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/genética , Metaloendopeptidasas/fisiología , Neovascularización Patológica , Células Tumorales Cultivadas , Proteínas ADAM , Alelos , Animales , Western Blotting , Encéfalo/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , ADN Complementario/metabolismo , Endotelio Vascular/citología , Citometría de Flujo , Eliminación de Gen , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Fenotipo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Enfermedades de la Retina/patología , Factores de Tiempo , Distribución Tisular , Venas Umbilicales/citología
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