RESUMO
We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development.
Assuntos
Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Proteína ADAM10 , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Regulação para Baixo/fisiologia , Feminino , Imunofluorescência , Proteínas Ligadas por GPI , Immunoblotting , Imunoprecipitação , Ligantes , Luciferases/biossíntese , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Plasmídeos/genética , Gravidez , Interferência de RNA , Proteínas Recombinantes/genética , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The tumor suppressor protein RECK has been implicated in the regulation of matrix metalloproteinases (MMPs), NOTCH-signaling and WNT7-signaling. It remains unclear, however, how broad the spectrum of RECK targets extends. To find novel RECK binding partners, we took the unbiased approach of yeast two-hybrid screening. This approach detected ADAMTS10 as a RECK-interactor. ADAMTS10 has been characterized as a metalloproteinase involved in fibrillin-rich microfibril biogenesis, and its mutations have been implicated in the connective tissue disorder Weill-Marchesani syndrome. Experiments in vitro using recombinant proteins expressed in mammalian cells indicated that RECK indeed binds ADAMTS10 directly, that RECK protects ADAMTS10 from fragmentation following chemical activation and that ADAMTS10 interferes with the activity of RECK to inhibit MT1-MMP. In cultured cells, RECK increases the amount of ADAMTS10 associated with the cells. Hence, the present study has uncovered novel interactions between two molecules of known clinical importance, RECK and ADAMTS10.This article has an associated First Person interview with the first author of the paper.
RESUMO
The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.