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GFRA4 improves the neurogenic potential of enteric neural crest stem cells via hedgehog pathway

Pediatric Research (2024)Cite this article

Abstract

Background

Hirschsprung disease (HSCR) is a congenital intestinal disease characterised by functional obstruction of the colon. Herein, we investigated the role and mechanism of the gene GFRA4 in HSCR.

Methods

GFRA4 expression in the ganglionic and aganglionic segment tissues in patients with HSCR and healthy colon tissues were detected using qRT-PCR, western blot, and immunohistochemistry. Cell proliferation, cycle distribution, apoptosis, changes in mitochondrial membrane potential, and differentiation were assessed in mouse enteric neural crest stem cells (ENCSCs) using the CCK-8 assay, EdU staining, flow cytometry, JC-1 probe, and immunofluorescence, respectively. GSEA analysis was performed to screen the signaling pathways regulated by GFRA4.

Results

GFRA4 was downregulated in aganglionic segment tissues compared to control and ganglionic segment tissues. GFRA4 overexpression promoted proliferation and differentiation, and inhibited apoptosis in ENCSCs, while GFRA4 down-regulation had the opposite result. GFRA4 activated the hedgehog pathway. GFRA4 overexpression enhanced the expression of key factors of the hedgehog pathway, including SMO, SHH, and GLI1. However, GFRA4 down-regulation reduced their expression. An antagonist of hedgehog pathway, cyclopamine, attenuated the effect of GFRA4 overexpression on proliferation, differentiation, and apoptosis of ENCSCs.

Conclusion

GFRA4 promotes proliferation and differentiation but inhibits apoptosis of ENCSCs via the hedgehog pathway in HSCR.

Impact

  • This study confirms that GFRA4 improves the proliferation and differentiation of ENCSCs via modulation of the hedgehog pathway.

  • This study for the first time revealed the role and the mechanism of the action of GFRA4 in HSCR, which indicates that GFRA4 may play a role in the pathological development of HSCR.

  • Our findings may lay the foundation for further investigation of the mechanisms underlying HSCR development and into targets of HSCR treatment.

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Fig. 1: Low expression of GFRA4 in human HSCR specimens.
Fig. 2: GFRA4 improved the proliferation of enteric neural crest stem cells (ENCSCs).
Fig. 3: GFRA4 inhibited mitochondria-dependent apoptosis in enteric neural crest stem cells (ENCSCs).
Fig. 4: GFRA4 improved the differentiation of enteric neural crest stem cells (ENCSCs).
Fig. 5: GFRA4 activated the hedgehog signaling pathway in enteric neural crest stem cells (ENCSCs).
Fig. 6: GFRA4 improved the proliferation and differentiation of enteric neural crest stem cells (ENCSCs) via the hedgehog signaling pathway.

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Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

Project ZR2020MH048 supported by Shandong Provincial Natural Science Foundation and the Natural Science Foundation of Tibet Autonomous Region (No.XZ202101ZR0004G)

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Author notes

  1. These authors contributed equally: Fangfang Zhang, Mingyu Cui.

Authors and Affiliations

  1. Department of Pediatric Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China

    Fangfang Zhang, Mingyu Cui, Lijuan Zhang, Bangzhen Ma, Feng Guo & Gang Wang

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  1. Fangfang Zhang
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Contributions

Conception and design: F.F.Z.; Perform research: F.F.Z., L.J.Z., and M.Y.C.; Data analysis and interpretation: B.Z.M., F.G., and G.W.; Manuscript writing: All authors; Final approval of manuscript: All authors.

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Correspondence to Gang Wang.

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Zhang, F., Cui, M., Zhang, L. et al. GFRA4 improves the neurogenic potential of enteric neural crest stem cells via hedgehog pathway. Pediatr Res (2024). https://doi.org/10.1038/s41390-024-03158-8

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  • DOI: https://doi.org/10.1038/s41390-024-03158-8

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