RESUMEN
Wnt/ß-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/ß-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/ß-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/ß-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/ß-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7.
Asunto(s)
Receptores Frizzled/genética , Células Madre Mesenquimatosas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Adulto , Células Cultivadas , Receptores Frizzled/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Interferencia de ARN , ARN Mensajero/genética , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismoRESUMEN
WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the ß-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced ß-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the ß-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.
Asunto(s)
Receptores Frizzled/genética , Genes Reporteros , Proteínas Recombinantes de Fusión/genética , Proteínas Wnt/genética , Vía de Señalización Wnt , Secuencia de Bases , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMEN
Human mesenchymal stem cells (hMSC) are subjected to the control of several signal transduction pathways during regeneration processes, whereby Wnt/ß-catenin signaling is of pivotal importance. Since there exists only fragmentary knowledge concerning the molecular function of the Wnt-coreceptors LRP5 and LRP6 (low-density lipoprotein receptor-related protein) in hMSC, we studied their impact on Wnt/ß-catenin signal transduction by RNA interference. For monitoring changes in ß-catenin-dependent transcription in a highly sensitive and specific manner, hMSC were stably transfected with a TCF/LEF reporter gene plasmid. In the presence of the activator Wnt3a, knockdown of LRP6 led to a strong decreased Wnt/ß-catenin signaling, while RNAi against LRP5 exhibited no effect in this setting. In a reverse approach, ectopic expression of LRP6 resulted in a strong enhancement of Wnt/ß-catenin signaling, whereas overexpression of LRP5 exhibited no increased signaling capacity. Furthermore, only the ectopic expression of LRP6--but not that of LRP5--was able to restore Wnt3a-mediated ß-catenin signaling after knockdown of endogenously expressed LRP6. These results demonstrate LRP6 as the predominant Wnt3a LRP-receptor in hMSC, which cannot be substituted by LRP5. In addition, we observed enhanced differentiation toward the adipogenic lineage after RNAi against LRP6 which was associated with the induction of PPAR-γ and fat vacuole formation. Thus, LRP6 is not only indispensable for Wnt3a/ß-catenin signaling, but also for the suppression of differentiation of hMSC into the adipogenic lineage. Based on these observations, LRP6 may represent an attractive drug target for manipulating hMSC in cell and tissue regeneration approaches.