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

Recently, our group has shown that the pituitary adenylate cyclase-activating polypeptide (PACAP)-induced neurite projection elongation in rat adrenal-derived pheochromocytoma cell line (PC12) is mediated via PAC1 receptor-mediated dephosphorylation of CRMP2 majorly through PI3K/AKT and MEK/ERK pathways. However, the mechanism of neuronal outgrowth by PACAP, including CRMP2 dephosphorylation, remains unclear. In our previous report, we found that GSK-3β, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 hours after the addition of PACAP, but the early dephosphorylation of CRMP2 by PACAP remains unclear. Thus, we considered it important to identify early factors in PACAP-induced neurite projection elongation and performed omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeling in conjunction with liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 minutes after PACAP addition. Results surprisingly revealed a number of key regulators involved in neurite outgrowth, including known ones. We also identified factors that may be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways. The results of this comprehensive analysis may provide important new information on the molecular mechanisms of neuronal differentiation induced by PACAP. Whole genome DNA microarray was carried out on the PC12 cells treated with PACAP38 (10⁻⁷ M) for the respective time points (5 – 120 minutes) and washed cells were stored at -80°C until RNA isolation. Total RNA was extracted from PC12 cells cultured in a 10 cm culture dish using the RNeasy Mini Kit (74104, Qiagen). Total RNA extracted from PC12 cells for each control and treatment (n=6) was pooled in each group, prior to DNA microarray analysis.