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.
Subscribe to Journal
Get full journal access for 1 year
only $33.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hockberger PE, Yousif L, Nam SC. Identification of acutely isolated cells from developing rat cerebellum. Neuroimage. 1994;1:276–87.
Ziai R, Pan YC, Hulmes JD, Sangameswaran L, Morgan JI. Isolation, sequence, and developmental profile of a brain-specific polypeptide, PEP-19. Proc Natl Acad Sci USA. 1986;83:8420–3.
Wei P, Blundon JA, Rong Y, Zakharenko SS, Morgan JI. Impaired locomotor learning and altered cerebellar synaptic plasticity in pep-19/PCP4-null mice. Mol Cell Biol. 2011;31:2838–344.
Mouton-Liger F, Thomas S, Rattenbach R, Magnol L, Larigaldie V, Ledru A, et al. PCP4 (PEP19) overexpression induces premature neuronal differentiation associated with Ca(2+)/calmodulin-dependent kinase II-δ activation in mouse models of Down syndrome. J Comp Neurol. 2011;519:2779–802.
Utal AK, Stopka AL, Roy M, Coleman PD. PEP-19 immunohistochemistry defines the basal ganglia and associated structures in the adult human brain, and is dramatically reduced in Huntington’s disease. Neuroscience. 1998;86:1055–63.
Iwamoto K, Bundo M, Yamamoto M, Ozawa H, Saito T, Kato T. Decreased expression of NEFH and PCP4/PEP19 in the prefrontal cortex of alcoholics. Neurosci Res. 2004;49:379–85.
Bourdeau V, Deschênes J, Laperrière D, Aid M, White JH, Mader S. Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells. Nucleic Acids Res. 2008;36:76–93.
Hamada T, Souda M, Yoshimura T, Sasaguri S, Hatanaka K, Tasaki T, et al. Anti-apoptotic effects of PCP4/PEP19 in human breast cancer cell lines: a novel oncotarget. Oncotarget. 2014;5:6076–86.
Wang T, Satoh F, Morimoto R, Nakamura Y, Sasano H, Auchus RJ, et al. Gene expression profiles in aldosterone-producing adenomas and adjacent adrenal glands. Eur J Endocrinol. 2011;164:613–9.
Kanamori T, Takakura K, Mandai M, Kariya M, Fukuhara K, Kusakari T, et al. PEP-19 overexpression in human uterine leiomyoma. Mol Hum Reprod. 2003;9:709–17.
Yoshimura T, Hamada T, Hijioka H, Souda M, Hatanaka K, Yoshioka T, et al. PCP4/PEP19 promotes migration, invasion and adhesion in human breast cancer MCF-7 and T47D cells. Oncotarget. 2016;7:49065–74.
Honjo K, Hamada T, Yoshimura T, Yokoyama S, Yamada S, Tan YQ, et al. PCP4/PEP19 upregulates aromatase gene expression via CYP19A1 promoter I.1 in human breast cancer SK-BR-3 cells. Oncotarget. 2018;9:29619–33.
Harashima S, Wang Y, Horiuchi T, Seino Y, inagaki N. Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release. J Neurosci Res. 2011;89:1519–30.
Erhardt JA, Legos JJ, Johanson RA, Slemmon JR, Wang X. Expression of PEP-19 inhibits apoptosis in PC12 cells. Neuroreport. 2000;11:3719–23.
Bowen RL, Verdile G, Liu T, Parlow AF, Perry G, Smith MA, et al. Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition. J Biol Chem. 2004;279:20539–45.
Janesick A, Wu SC, Blumberg B. Retinoic acid signaling and neuronal differentiation. Cell Mol Life Sci. 2015;72:1559–76.
Devanna P, Middelbeek J, Vernes SC. FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways. Front Cell Neurosci. 2014;8:305.
Oikari LE, Okolicsanyi RK, Griffiths LR, Haupt LM. Data defining markers of human neural stem cell lineage potential. Data Brief. 2016;7:206–15.
Hollenhorst PC, Jones DA, Graves BJ. Expression profiles frame the promoter specificity dilemma of the ETS family of transcription factors. Nucleic Acids Res. 2004;32:5693–702.
Yoshimatsu Y, Yamazaki T, Mihira H, Itoh T, Suehiro J, Yuki K, et al. Ets family members induce lymphangiogenesis through physical and functional interaction with Prox1. J Cell Sci. 2011;124:2753–62.
Shimada H, DeLellis RA, Tissier F. Neuroblastic tumours of the adrenal gland. In: Lloid RV, Osamura RY, Kloppel G, Rosai J, editors. WHO classification of tumours of endocrine organs. 4th ed. Lyon, France: IARC; 2016. p. 196–203.
Andres D, Keyser BM, Petrali J, Benton B, Hubbard KS, McNutt PM, et al. Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with trans-retinoic acid. BMC Neurosci. 2013;14:49.
Filograna R, Civiero L, Ferrari V, Codolo G, Greggio E, Bubacco L, et al. Analysis of the catecholaminergic phenotype in human SH-SY5Y and BE(2)-M17 neuroblastoma cell lines upon differentiation. PLoS ONE. 2015;10:e0136769.
Sharif S, Ghahremani MH, Soleimani M. Induction of morphological and functional differentiation of human neuroblastoma cells by miR-124. J Biosci. 2017;42:555–63.
Kim EJ, Ables JL, Dickel LK, Eisch AJ, Johnson JE. Ascl1 (Mash1) defines cells with long-term neurogenic potential in subgranular and subventricular zones in adult mouse brain. PLoS ONE. 2011;6:e18472.
Castro DS, Martynoga B, Parras C, Ramesh V, Pacary E, Johnston C, et al. A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. Genes Dev. 2011;25:930–45.
von Bohlen und Halbach O. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 2011;345:1–19.
Borromeo MD, Savage TK, Kollipara RK, He M, Augustyn A, Osborne JK, et al. ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroendocrine tumors and regulate distinct genetic programs. Cell Rep. 2016;16:1259–72.
Kawamata H, Tachibana M, Fujimori T, Imai Y. Differentiation-inducing therapy for solid tumors. Curr Pharm Des. 2006;12:379–85.
Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B, et al. The International Neuroblastoma Pathology Classification (the Shimada system). Cancer. 1999;86:364–72.
Jögi A, Vaapil M, Johansson M, Påhlman S. Cancer cell differentiation heterogeneity and aggressive behavior in solid tumors. Ups J Med Sci. 2012;117:217–24.
Bernhardt EB, Jalal SI. Small cell lung cancer. Cancer Treat Res. 2016;170:301–22.
Gazdar AF, Bunn PA, Minna JD. Small-cell lung cancer: what we know, what we need to know and the path forward. Nat Rev Cancer. 2017;17:725–37.
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).
Conflict of interest
The authors declare that they have no conflict of interest.
The IHC studies using human tissues were approved by the ethics committees for clinical and epidemiologic research at Kagoshima University.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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