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Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells

The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACA...

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Autores principales: Shibato, Junko, Takenoya, Fumiko, Hirabayashi, Takahiro, Kimura, Ai, Yamashita, Michio, Takasaki, Ichiro, Rakwal, Randeep, Shioda, Seiji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371652/
https://www.ncbi.nlm.nih.gov/pubmed/34422037
http://dx.doi.org/10.1155/2021/2522454
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author Shibato, Junko
Takenoya, Fumiko
Hirabayashi, Takahiro
Kimura, Ai
Yamashita, Michio
Takasaki, Ichiro
Rakwal, Randeep
Shioda, Seiji
author_facet Shibato, Junko
Takenoya, Fumiko
Hirabayashi, Takahiro
Kimura, Ai
Yamashita, Michio
Takasaki, Ichiro
Rakwal, Randeep
Shioda, Seiji
author_sort Shibato, Junko
collection PubMed
description The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10(−7) M, 10(−8) M, and 10(−9) M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10(−7) M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.
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spelling pubmed-83716522021-08-19 Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells Shibato, Junko Takenoya, Fumiko Hirabayashi, Takahiro Kimura, Ai Yamashita, Michio Takasaki, Ichiro Rakwal, Randeep Shioda, Seiji Neural Plast Research Article The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10(−7) M, 10(−8) M, and 10(−9) M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10(−7) M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further. Hindawi 2021-08-07 /pmc/articles/PMC8371652/ /pubmed/34422037 http://dx.doi.org/10.1155/2021/2522454 Text en Copyright © 2021 Junko Shibato et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Shibato, Junko
Takenoya, Fumiko
Hirabayashi, Takahiro
Kimura, Ai
Yamashita, Michio
Takasaki, Ichiro
Rakwal, Randeep
Shioda, Seiji
Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title_full Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title_fullStr Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title_full_unstemmed Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title_short Molecular Mechanism for PACAP 38-Induced Neurite Outgrowth in PC12 Cells
title_sort molecular mechanism for pacap 38-induced neurite outgrowth in pc12 cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371652/
https://www.ncbi.nlm.nih.gov/pubmed/34422037
http://dx.doi.org/10.1155/2021/2522454
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