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OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

ABSTRACT: The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3β, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

SUBMITTER: Shibato J

PROVIDER: S-EPMC9964364 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

Shibato Junko J Takenoya Fumiko F Yamashita Michio M Gupta Ravi R Min Cheol Woo CW Kim Sun Tae ST Kimura Ai A Takasaki Ichiro I Hori Motohide M Shioda Seiji S Rakwal Randeep R

International journal of molecular sciences 20230220 4

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3β, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify ...[more]

PMID: 36835581

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Project 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.

Dataset Information

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

Publications

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

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