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1.
Mol Cell Proteomics ; 17(7): 1410-1425, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29669734

RESUMEN

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.


Asunto(s)
Proteínas ADAMTS/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Morfogénesis , Proteolisis , Proteómica/métodos , Esferoides Celulares/metabolismo , Células 3T3 , Proteínas ADAMTS/química , Secuencia de Aminoácidos , Animales , Colágeno/metabolismo , Perros , Fibroblastos/metabolismo , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Dominios Proteicos , Especificidad por Sustrato , Regulación hacia Arriba
2.
Nat Aging ; 4(3): 350-363, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38472454

RESUMEN

Melanoma, the most lethal form of skin cancer, often has worse outcomes in older patients. We previously demonstrated that an age-related decrease in the secreted extracellular matrix (ECM) protein HAPLN1 has a role in slowing melanoma progression. Here we show that HAPLN1 in the dermal ECM is sufficient to maintain the integrity of melanoma-associated blood vessels, as indicated by increased collagen and VE-cadherin expression. Specifically, we show that HAPLN1 in the ECM increases hyaluronic acid and decreases endothelial cell expression of ICAM1. ICAM1 phosphorylates and internalizes VE-cadherin, a critical determinant of vascular integrity, resulting in permeable blood vessels. We found that blocking ICAM1 reduces tumor size and metastasis in older mice. These results suggest that HAPLN1 alters endothelial ICAM1expression in an indirect, matrix-dependent manner. Targeting ICAM1 could be a potential treatment strategy for older patients with melanoma, emphasizing the role of aging in tumorigenesis.


Asunto(s)
Melanoma , Neoplasias Cutáneas , Anciano , Animales , Humanos , Ratones , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/genética , Molécula 1 de Adhesión Intercelular/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Regulación hacia Arriba
3.
Adv Clin Chem ; 106: 1-32, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35152971

RESUMEN

A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) are major mediators in extracellular matrix (ECM) turnover and have gained increasing interest over the last years as major players in ECM remodeling during tissue homeostasis and the development of diseases. Although, ADAMTSs are recognized in playing important roles during tissue remodeling, and loss of function in various member of the ADAMTS family could be associated with the development of numerous diseases, limited knowledge is available about their specific substrates and mechanism of action. In this chapter, we will review current knowledge about ADAMTSs and their use as disease biomarkers.


Asunto(s)
Proteínas ADAMTS , Matriz Extracelular , Proteínas ADAMTS/genética , Biomarcadores , Humanos
4.
Adv Sci (Weinh) ; 9(22): e2201483, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35657074

RESUMEN

Aging is a major risk factor in microvascular dysfunction and disease development, but the underlying mechanism remains largely unknown. As a result, age-mediated changes in the mechanical properties of tissue collagen have gained interest as drivers of endothelial cell (EC) dysfunction. 3D culture models that mimic age-mediated changes in the microvasculature can facilitate mechanistic understanding. A fibrillar hydrogel capable of changing its stiffness after forming microvascular networks is established. This hydrogel model is used to form vascular networks from induced pluripotent stem cells under soft conditions that mimic young tissue mechanics. Then matrix stiffness is gradually increased, thus exposing the vascular networks to the aging-mimicry process in vitro. It is found that upon dynamic matrix stiffening, EC contractility is increased, resulting in the activation of focal adhesion kinase and subsequent dissociation of ß-catenin from VE-Cadherin mediated adherens junctions, leading to the abruption of the vascular networks. Inhibiting cell contractility impedes the dissociation of ß-catenin, thereby preventing the deconstruction of adherens junctions, thus partially rescuing the age-mediated vascular phenotype. The findings provide the first direct evidence of matrix's dynamic mechano-changes in compromising microvasculature with aging and highlight the importance of hydrogel systems to study tissue-level changes with aging in basic and translational studies.


Asunto(s)
Uniones Adherentes , beta Catenina , Uniones Adherentes/metabolismo , Células Endoteliales/metabolismo , Hidrogeles , Fenotipo , beta Catenina/metabolismo
5.
Methods Mol Biol ; 2043: 275-284, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31463920

RESUMEN

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.


Asunto(s)
Proteínas ADAMTS/metabolismo , Medios de Cultivo Condicionados/química , Matriz Extracelular/metabolismo , Animales , Células 3T3 BALB , Sistema Libre de Células , Cromatografía Liquida , Células HEK293 , Humanos , Ratones , Espectrometría de Masas en Tándem
6.
Cell Stem Cell ; 27(5): 798-812.e6, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-32931729

RESUMEN

Matrix dynamics influence how individual cells develop into complex multicellular tissues. Here, we develop hydrogels with identical polymer components but different crosslinking capacities to enable the investigation of mechanisms underlying vascular morphogenesis. We show that dynamic (D) hydrogels increase the contractility of human endothelial colony-forming cells (hECFCs), promote the clustering of integrin ß1, and promote the recruitment of vinculin, leading to the activation of focal adhesion kinase (FAK) and metalloproteinase expression. This leads to the robust assembly of vasculature and the deposition of new basement membrane. We also show that non-dynamic (N) hydrogels do not promote FAK signaling and that stiff D- and N-hydrogels are constrained for vascular morphogenesis. Furthermore, D-hydrogels promote hECFC microvessel formation and angiogenesis in vivo. Our results indicate that cell contractility mediates integrin signaling via inside-out signaling and emphasizes the importance of matrix dynamics in vascular tissue formation, thus informing future studies of vascularization and tissue engineering applications.


Asunto(s)
Hidrogeles , Ingeniería de Tejidos , Células Endoteliales , Humanos , Morfogénesis , Transducción de Señal
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