RESUMEN
Fibroblast growth factors (FGFs) serve numerous regulatory functions in complex organisms, and their corresponding therapeutic potential is of growing interest to academics and industrial researchers alike. However, applications of these proteins are limited due to their low stability. Here we tackle this problem using a generalizable computer-assisted protein engineering strategy to create a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function. The data from the characterization of stabilized FGF2 showed a remarkable prediction potential of in silico methods and provided insight into the unfolding mechanism of the protein. The molecule holds a considerable promise for stem cell research and medical or pharmaceutical applications.
Asunto(s)
Diseño Asistido por Computadora , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ingeniería de Proteínas , Estabilidad Proteica , Secuencia de Aminoácidos , Animales , Simulación por Computador , Evolución Molecular Dirigida , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Humanos , Mutación Puntual , Pliegue de ProteínaRESUMEN
Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.
Asunto(s)
Linaje de la Célula , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/enzimología , Proteínas Tirosina Quinasas/metabolismo , Biomarcadores/metabolismo , Línea Celular , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/ß-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/ß-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/ß-catenin in suppression of chondrocyte differentiation.