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Forskolin promotes neuronal differentiation of PC12 cells via the PKA-CREB-dependent signaling pathway. Activation of PKA by forskolin phosphorylates CREB, which then binds to CRE sites in numerous gene promoters. However, it is unclear which gene contains the CRE sites responsible for forskolin-induced neuronal differentiation. In this study, we investigated how an immediate early gene,  nur77 , which has CRE sites in the promoter region, contributes to the early stage of differentiation of forskolin-treated PC12 cells. After treatment with forskolin, expression of Nur77 was upregulated within 1 hr. In addition, knockdown of  nur77  inhibited neurite outgrowth induced by forskolin. We also revealed that the specific four CRE sites near the transcriptional start site (TSS) of  nur77  were strongly associated with phosphorylated CREB within 1 hr after treatment with forskolin. To analyze the roles of these four sites, reporter assays using the  nur77  promoter region were performed. The results showed that  nur77  expression was mediated through three of the CRE sites, −242, −222, and −78, and that −78, the nearest of the three to the TSS of  nur77 , was particularly important. An analysis of neuronal markers controlled by Nur77 after A-CREB-Nur77-Synapsin1 signaling pathway plays a pivotal role in differentiation of forskolin-induced PC12 cells.

Molecular mechanism of nur77 gene expression and downstream target genes in the early stage of forskolin-induced differentiation in PC12 cells