RESUMO
Hirschsprung (HSCR) disease is a complex genetic disorder attributed to a failure of the enteric neural crest cells (ENCCs) to form ganglia in the hindgut. Hedgehog and Notch are implicated in mediating proliferation and differentiation of ENCCs. Nevertheless, how these signaling molecules may interact to mediate gut colonization by ENCCs and contribute to a primary etiology for HSCR are not known. Here, we report our pathway-based epistasis analysis of data generated by a genome-wide association study on HSCR disease, which indicates that specific genotype constellations of Patched (PTCH1) (which encodes a receptor for Hedgehog) and delta-like 3 (DLL3) (which encodes a receptor for Notch) SNPs confer higher risk to HSCR. Importantly, deletion of Ptch1 in mouse ENCCs induced robust Dll1 expression and activation of the Notch pathway, leading to premature gliogenesis and reduction of ENCC progenitors in mutant bowels. Dll1 integrated Hedgehog and Notch pathways to coordinate neuronal and glial cell differentiation during enteric nervous system development. In addition, Hedgehog-mediated gliogenesis was found to be highly conserved, such that Hedgehog was consistently able to promote gliogenesis of human neural crest-related precursors. Collectively, we defined PTCH1 and DLL3 as HSCR susceptibility genes and suggest that Hedgehog/Notch-induced premature gliogenesis may represent a new disease mechanism for HSCR.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Hedgehog/metabolismo , Doença de Hirschsprung/genética , Doença de Hirschsprung/fisiopatologia , Neuroglia/fisiologia , Receptores Notch/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/fisiologia , Epistasia Genética , Trato Gastrointestinal/citologia , Trato Gastrointestinal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteínas Hedgehog/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Crista Neural/citologia , Crista Neural/fisiologia , Neurogênese/fisiologia , Neuroglia/citologia , Receptores Patched , Receptor Patched-1 , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Notch/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: The rearranged during transfection (RET) gene encodes a single-pass receptor whose proper expression and function are essential for the development of enteric nervous system. Mutations in RET regulatory regions are also associated with Hirschsprung disease (HSCR) (aganglionosis of the colon). We previously showed that 2 polymorphisms in RET promoter are associated with the increased risk of HSCR. These single nucleotide polymorphisms overlap with the NK2 homeobox 1 (Nkx2-1) binding motif interrupting the physical interaction of NKX2-1 with the RET promoter and result in reduced RET transcription. In this study, we further delineated Nkx2-1-mediated RET Transcription. METHODS AND RESULTS: First, we demonstrated that PHOX2B, like SOX10 and NKX2-1, is expressed in the mature enteric ganglions of human gut by immunohistochemistry. Second, subsequent dual-luciferase-reporter studies indicated that Nkx2-1 indeed works coordinately with Phox2b and Sox10, but not Pax3, to mediate RET transcription. In addition, identification of Phox2b responsive region in RET promoter further provides solid evidence of the potential functional interaction between Phox2b and RET. CONCLUSION: In sum, Phox2b and Sox10 act together with Nkx2.1 to modify RET signaling and this interaction may also contribute to HSCR susceptibility.
Assuntos
Doença de Hirschsprung/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas c-ret/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral/metabolismo , Sistema Nervoso Entérico/metabolismo , Epistasia Genética/genética , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Genótipo , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Transdução de Sinais/genética , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Transfecção/estatística & dados numéricosRESUMO
Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.