RESUMEN
In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-ß and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.
Asunto(s)
Matriz Extracelular/metabolismo , Fibrinolisina/metabolismo , Queratinocitos/metabolismo , Proteolisis , Transducción de Señal , Activinas/metabolismo , Uniones Adherentes/metabolismo , Animales , Membrana Basal/metabolismo , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Retroalimentación Fisiológica , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Queratinocitos/patología , Ratones , Plasminógeno/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Unión Proteica , Proteómica , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Cicatrización de HeridasRESUMEN
In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.e., expression of specific molecular markers, lipolytic response to agonists of beta-adrenoreceptors (beta2-AR agonist > beta1-AR agonist >> beta3-AR agonist) and to the atrial natriuretic peptide, insulin-stimulated glucose transport, and secretion of leptin and adiponectin. hMADS cells were able to respond to drugs as inhibition of adipocyte differentiation was observed in the presence of prostaglandin F2alpha, tumour necrosis factor-alpha, and nordihydroguaiaretic acid, a natural polyhydroxyphenolic antioxidant. Thus, for the first time, human adipose cells with normal karyotype and indefinite life span have been established. They represent a novel and valuable tool for studies of fat tissue development and metabolism.