RESUMEN
The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor-related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)-tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor165 (rhVEGF165). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF165 induced expression of active ß-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF165 induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.
Asunto(s)
Diferenciación Celular/fisiología , Pulpa Dental/citología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Transducción de Señal , Células Madre/fisiología , Animales , Células Cultivadas , Humanos , Ratones , Factor A de Crecimiento Endotelial Vascular , Vía de Señalización WntRESUMEN
Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/ß-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/ß-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/ß-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling.
Asunto(s)
Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Sindecano-4/metabolismo , Trombospondinas/metabolismo , beta Catenina/metabolismo , Animales , Técnicas de Cultivo de Célula , Fibronectinas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Ratones , Sindecano-4/genética , Trombospondinas/genética , Transfección , Vía de Señalización WntRESUMEN
We previously found that single nucleotide polymorphisms in the low-density lipoprotein receptor-related protein 6 (LRP6) gene are associated with Alzheimer's disease (AD). Here, we studied the posttranscriptional metabolism of the LRP6 message scanning sequentially the 23 LRP6 exons in human tissues and found a novel LRP6 isoform that completely skips exon 3 (LRP6Δ3) in all tissues examined and was also conserved in mice. Expression levels of the LRP6 isoforms were determined in 47 cortical brain messenger (m)RNA samples including 22 AD cases, 11 control subjects, and 14 individuals with other neurological disorders. LRP6Δ3 mRNA levels were significantly augmented in AD brains compared with controls (1.6-fold; p = 0.037) or other pathological samples (2-fold; p = 0.007). Functional analysis in Wnt/ß-catenin signaling assays revealed that skipping of exon 3 reduced significantly the signaling activity of the LRP6 coreceptor. We conclude that the LRP6Δ3 isoform is a novel splice variant, which shows diminished Wnt/ß-catenin signaling activity and might have a functional role in individuals with AD.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Estudios de Asociación Genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Isoformas de Proteínas/genética , Vía de Señalización Wnt/genética , Anciano , Anciano de 80 o más Años , Empalme Alternativo/genética , Animales , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca(2+) transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest cross-talk between canonical and Wnt/Ca(2+) signaling in central neurons.