RESUMO
Clefts of the palate and/or lip are among the most common human craniofacial malformations and involve multiple genetic and environmental factors. Defects can only be corrected surgically and require complex life-long treatments. Our studies utilized the well-characterized Pax9-/- mouse model with a consistent cleft palate phenotype to test small-molecule Wnt agonist therapies. We show that the absence of Pax9 alters the expression of Wnt pathway genes including Dkk1 and Dkk2, proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 are shown by the genetic rescue of secondary palate clefts in Pax9-/-Dkk1f/+;Wnt1Cre embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant Pax9+/- mice restored Wnt signaling and led to the growth and fusion of palatal shelves, as marked by an increase in cell proliferation and osteogenesis in utero, while other organ defects were not corrected. This work underscores the importance of Pax9-dependent Wnt signaling in palatogenesis and suggests that this functional upstream molecular relationship can be exploited for the development of therapies for human cleft palates that arise from single-gene disorders.
Assuntos
Fissura Palatina/genética , Fatores de Transcrição Box Pareados/genética , Palato/embriologia , Proteína Wnt1/agonistas , Proteína Wnt1/genética , Animais , Padronização Corporal , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Transgênicos , Morfogênese , Osteogênese , Fator de Transcrição PAX9 , Fenótipo , Ligação Proteica , Via de Sinalização WntRESUMO
Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSC-OVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents. Stem Cells 2016;34:2625-2634.
Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Microftalmia/genética , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição/genética , Proteína Wnt1/genética , Substituição de Aminoácidos , Benzotiazóis/farmacologia , Biomarcadores/metabolismo , Diferenciação Celular , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Microftalmia/metabolismo , Microftalmia/patologia , Mutação , Fenótipo , Cultura Primária de Células , Piridinas/farmacologia , Pirimidinas/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt1/agonistas , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt1/metabolismoRESUMO
BACKGROUND/AIMS: We have reported in a separate study that alcohol exposure triggers activation of the TNF-α signaling pathway leading to an adverse shift of multipotential mesenchymal stem cells in bone marrow (BMSCs) away from osteogenesis towards adipogenesis. However, inhibition of TNF-α signaling only yielded moderate inhibition of adipogenesis. Here we showed that in addition to promoting the TNF-α signaling, alcohol also suppressed the Wnt1/ß-catenin signaling pathway. METHODS: We treated primary BMSCs from human subjects with alcohol for 24 days. We measured changes of genes related to endoplasmic reticulum (ER) stress, adipogenic markers and osteogenic markers using quantitative real-time RT-PCR and Western blot analysis. We performed Alizarin red staining for osteogenesis. We also conducted assays for osteogenic biomarkers alkaline phosphatase, collagen-I and osteocalcin. RESULTS: Wnt/ß-catenin signaling was markedly activated in BMSCs treated with osteogenic inducers relative to the control cells, as indicated by the increased levels of nuclear ß-catenin along with reduced levels of cytosolic ß-catenin, as well as increased protein levels of Wnt1. Activation of Wnt/ß-catenin signaling was significantly suppressed in BMSCs exposed to alcohol, which was reflected by downregulated expression of osteogenic marker genes Osf2/Cbfa1, osteopontin and osteocalcin, upregulated adipogenic marker PPARγ2 and aP2, and reduced number of calcifcation nodules. In contrast, activation of Wnt/ß-catenin signaling by BIO favored osteogenesis even in the presence of alcohol. Simultaneous activation of Wnt1 by BIO and inhibition of TNF-α by 3,6'-dithiothalidomide produced synergetic suppression of ethanol-induced adipogenic lineage compared to interference with either of them alone. CONCLUSION: This remarkable shift of BMSCs towards osteoblast lineage suggests the superiority of concordant and reciprocal interferences of the TNF-α and Wnt/ß-catenin pathways for promoting osteogenesis.
