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PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells

Laboratory Investigation volume 100, pages1551–1563(2020)Cite this article

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Abstract

Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is 7.6 kDa peptide originally found in Purkinje cells. PCP4/PEP19 is a differentiation maker of Purkinje cells, where it functions as an antiapoptotic factor. Cerebral neuronal cells also express PCP4/PEP19, which may be related to neuronal cell survival. However, evidence suggests that PCP4/PEP19 may also be involved in neuronal differentiation. Here, we investigated the effects of PCP4/PEP19 expression on neuronal differentiation by analyzing neurite outgrowth, and expression of neuronal differentiation markers in cultured human neuroblastoma M17 cells. When PCP4/PEP19 expression was reduced by siRNA-mediated knockdown, neurite outgrowth was significantly increased. Among many differentiation markers tested, expression of NeuroD1 was increased, while that of Ascl1 was decreased upon PCP4/PEP19 knockdown. Furthermore, luciferase reporter assays revealed that PCP4/PEP19 knockdown upregulated NeuroD1 and downregulated Ascl1 expression, at the transcriptional level. These results suggest a new function of PCP4/PEP19, which suppresses neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1 expression in M17 cells. Furthermore, immunohistochemical studies showed that PCP4/PEP19 localizes in the nuclei of human neuroblastoma cells. Therefore, PCP4/PEP19 may also be an intranuclear negative regulator of neuronal differentiation and may thus be a potential therapeutic target to promote cellular differentiation in human neuroblastoma.

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Fig. 1: Anti-PCP4/PEP19 antibody preparation, and effects of AtRA and siRNA effects on PCP4/PEP19 expression and proliferation of M17 cells.
Fig. 2: Neurite outgrowth and neuronal differentiation after AtRA treatment and PCP4/PEP19 knockdown monitored by tubulin immunostaining.
Fig. 3: Neurite outgrowth and neuronal differentiation after AtRA treatment and PCP4/PEP19 knockdown monitored by actin distribution.
Fig. 4: Morphometric analysis of neurite outgrowth.
Fig. 5: Effects of PCP4/PEP19 and AtRA on Ascl1 and NeuroD1 expression.
Fig. 6: Relation of PCP4/PEP19, Ascl1, and NeuroD1 expression in M17 and SH-SY5Y cells.
Fig. 7: Subcellular localization of PCP4/PEP19 in human neuroblastoma tissues and M17 cells.
Fig. 8: Schematic presentation of PCP4/PEP19 regulation of neural differentiation in neuroblastoma M17 cells.

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Acknowledgements

The authors greatly appreciate the excellent technical assistance of Ms. Orie Iwaya and Ms. Mai Tokudome (Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences).

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Affiliations

  1. Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

    Ikumi Kitazono, Taiji Hamada, Mari Kirishima, Seiya Yokoyama, Toshiaki Akahane & Akihide Tanimoto

  2. Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

    Takuya Yoshimura

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  1. Ikumi Kitazono
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Correspondence to Akihide Tanimoto.

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The IHC studies using human tissues were approved by the ethics committees for clinical and epidemiologic research at Kagoshima University.

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Kitazono, I., Hamada, T., Yoshimura, T. et al. PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells. Lab Invest 100, 1551–1563 (2020). https://doi.org/10.1038/s41374-020-0462-z

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