Pituitary adenylate cyclase-activating polyp eptide-induced differentiation of embryonic neural stem cells into astrocytes is mediated via the β 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 4 alpha-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 beta II PKC, but lacked PKC gamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the beta II isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKC alpha increased linearly up to Day 6. Overexpression of PKC beta II with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominantnegative mutant of PKC beta II proved inhibitory. These results indicate that the p isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs intoastrocytes. (c) 2006Wiley-Liss, Inc.