In this examine, we investigated the activation and in volvement of different signaling pathways in synergistic neurite outgrowth making use of three combinations of ligands. NGF PACAP, FGFb PACAP and EGF PACAP, As anticipated, all three programs showed a synergistic phosphorylation of Erk concomitant with neurite out development. Interestingly, JNK, but not Akt or P38, was pan Chk inhibitor also synergistically activated in all three methods. Unexpect edly, inhibition of JNK blocked neurite outgrowth from the NP and FP, but not EP, methods. This differential in volvement of JNK was identified for being dependent over the regulation of P90RSK action. Hence, a JNK P90RSK link was identified like a hitherto unrecognized mechanism mediating the synergistic result in neurite outgrowth. Our results thus demonstrate the involvement of distinct signaling pathways in regulating neurite out development in response to different synergistic growth issue PACAP stimulation.
Approaches Components Mouse recombinant NGF was purchased from Pepro tech, Mouse recombinant EGF was pur chased from Shenandoah Biotechnology, CP690550 PACAP was bought from American Peptide Corporation, MEK inhibitor U0126, JNK inhibitor SP600125, PI3K inhibitor LY294002, and P38 inhibitor SB203580 were obtained from LC Laboratories, P90RSK inhibitor BRD7389 was obtained from Santa Cruz Biotechnology, Primary anti bodies towards phospho particular Erk1 2, pan Erk1 two, phospho exact JNK, pan JNK, phospho exact P38, phospho specific Akt, phospho precise P90RSK, and pan RSK were obtained from Cell Signaling Technologies, An antibody against phospho unique c Jun was obtained from Abnova, Human recombinant FGFb and an antibody against actin had been purchased from EMD Millipore, Horseradish peroxidase conjugated sec ondary antibodies, Imperial Protein Stain and Hoechst have been obtained from Thermo Scientific, Cell culture Rat pheochromocytoma PC12 cells have been cultured in Dulbeccos minimal critical medium supplemented with 10% heat inactivated fetal bovine serum and 5% Horse Serum, Cells had been cultured with 100U ml peni cillin and one hundred mg ml streptomycin, and maintained inside a hu midified incubator with 5% CO2 at 37 C.