Pituitary adenylate cyclase-activating polypeptide-induced differentiation of embryonic neural stem cells into astrocytes is mediated via the beta isoform of protein kinase C.
We have found previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases the number of astrocytes generated from cultured mouse neural stem cells (NSCs) via a mechanism that is independent of the cyclic AMP/protein kinase A pathway (Ohno et al., 2005). In the present study, the signaling pathway involved in the differentiation process was further investigated. PACAP-induced differentiation was inhibited by the phospholipase C inhibitor, U73122, the protein kinase C (PKC) inhibitor, chelerythrine, and the intracellular calcium chelator, BAPTA-AM, and was mimicked by phorbol 12-myristate 13-acetate (PMA), but not by 4alpha-PMA. These results suggest that the PACAP-generated signal was mediated via the PACAP receptor, PAC1 stimulated heterotrimeric G-protein, resulting in activation of phospholipase C, followed by calcium- and phospholipid-dependent protein kinase C (cPKC). To elucidate the involvement of the different isoforms of cPKC, their gene and protein expression were examined. Embryonic NSCs expressed alpha and betaII PKC, but lacked PKCgamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the betaII isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKCalpha increased linearly up to Day 6. Overexpression of PKCbetaII with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominant-negative mutant of PKCbetaII proved inhibitory. These results indicate that the beta isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs into astrocytes.
Watanabe
Jun
J
Department of Anatomy, School of Medicine, Showa University, Tokyo, Japan.
Ohba
Motoi
M
Ohno
Fusako
F
Kikuyama
Sakae
S
Nakamura
Masahisa
M
Nakaya
Kazuyasu
K
Arimura
Akira
A
Shioda
Seiji
S
Nakajo
Shigeo
S
D000256:Adenoviridae / Q000502:physiology; D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D002452:Cell Count / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013234:Stem Cells / Q000187:drug effects* / Q000502:physiology; D014162:Transfection / Q000379:methods
D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D020033:Protein Isoforms; D012333:RNA, Messenger; D011493:Protein Kinase C; D064546:Protein Kinase C beta