The 38-Amino-Acid Form of Pituitary Adenylate Cyclase-Activating Polypeptide Induces Neurite Outgrowth in PC12 Cells that Is Dependent on Protein Kinase C and Extracellular Signal-Regulated Kinase but not on Protein Kinase A, Nerve Growth Factor Receptor Tyrosine Kinase, p21ras G protein, and pp60c-srcCytoplasmic Tyrosine Kinase
- 1Department of Pharmacology, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91010, Israel (P.L.), 2Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health (H.J.), and3Division of Cytokine Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 (D.F.)
Abstract
The 38-amino-acid isoform of pituitary adenylate cyclase-activating polypeptide (PACAP38) elicits a robust outgrowth of neurites in cultured PC12 cells. Initiation of neurite outgrowth occurs within 4–8 hr after the addition of PACAP38. Treatment with PACAP38 does not elicit collateral activation of p140trk nerve growth factor receptor tyrosine kinase activity, nor is it associated with tyrosine phosphorylation of suc1-associated neurotrophic factor target, a selective target of neurotrophin tyrosine kinase receptors. Coadministration of epidermal growth factor with PACAP38 elicits an enhanced response. Induction of neurites is also observed on the addition of PACAP38 to dominant negative Src and Ras PC12 cell variants. PACAP38 stimulates extracellular signal-regulated kinase (Erk) activity >10-fold within 5 min, and the effect is augmented by cotreatment with epidermal growth factor. Pretreatment with the cAMP-dependent protein kinase-selective inhibitor, H-89, is ineffective as an antagonist of PACAP38-induced neurite outgrowth, whereas down-regulation of protein kinase C (PKC) by phorbol ester or incubation with PKC-selective inhibitors GF109203X and calphostin C effectively blocks PACAP38-stimulated neurite formation. Stimulation of Erk activity is inhibited by incubation with PD90859, a pharmacological antagonist of the threonine/tyrosine dual-specificity Erk. Inhibition of ligand-stimulated Erk activation prevents PACAP38-induced neurite outgrowth. Collectively, these findings indicate that PACAP38-stimulated neuritogenesis requires PKC and Erk activation but is independent of cAMP-dependent protein kinase, nerve growth factor receptor tyrosine kinase, p21ras G protein, and pp60c-src cytoplasmic tyrosine kinase.
Footnotes
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Send reprint requests to: Donald W. Fink, Jr., Ph.D., FDA/CBER-Division of Cytokine Biology, 1401 Rockville Pike, Suite 200 N/HFM-505, Rockville, MD 20852-1448. E-mail:finkd{at}al.cber.fda.gov
- Abbreviations:
- NGF
- nerve growth factor
- PACAP38
- pituitary adenylate cyclase-activating polypeptide with 38 residues and an amidated carboxyl terminus
- EGF
- epidermal growth factor
- Src
- pp60c-src cytoplasmic tyrosine kinase
- Trk
- p140trk nerve growth factor receptor tyrosine kinase
- Ras
- p21ras G protein
- Erk
- extracellular signal-regulated kinase
- MEK
- threonine/tyrosine dual-specificity extracellular signal-regulated kinase
- PVDF
- polyvinylidene difluoride
- SNT
- suc1-associated neurotrophic factor target
- PMA
- phorbol-12-myristate-13-acetate
- PKA
- cAMP-dependent protein kinase, PKC, calcium- and phospholipid-dependent protein kinase C
- MBP
- myelin basic protein
- PC12nnr5
- a pheochromocytoma PC12 clone nonresponsive to NGF
- PC12–6.24
- a PC12 clone overexpressing human p140trk
- GsRasDN6
- dexamethasone-inducible, dominant-negative Ras PC12 clone
- M-M17–26
- stable, dominant-negative Ras PC12 clone
- SDS
- sodium dodecyl sulfate
- PAGE
- polyacrylamide gel electrophoresis
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- Received March 9, 1998.
- Accepted June 3, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
