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

Abstract

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. Low molecular weight natural products may be useful therapeutic agents for neuronal injury 1-6 , and some of these compounds possess neurotrophic and neuroprotective properties 7. Natural products also enhance neurite outgrowth activity of nerve growth factors in experimental models 8 , but the detailed molecular mechanisms underlying the neurotrophic and neuroprotective effects of natural products have not been clearly defined. One such natural product, forskolin, is a cell-permeable diterpenoid extracted from the plant Coleus forskohlii. Forskolin also has blood-brain barrier (BBB) permeability 9. Therefore, forskolin is a potential therapeutic agent for nerve injury, and several studies have shown that forskolin increases the differentiation and survival of dopaminergic neurons in vitro 10-14. Forskolin also induces expression of tyrosine hydroxylase in human fetal brain cortex 15. In addition, forskolin increases the intracellular cAMP level by stimulation of adenylate cyclase, and cAMP-dependent signaling pathways play important roles in neuronal differentiation and neuroplasticity 16,17. Increased cAMP in cells promotes axonal regeneration and neurite outgrowth 18-20 , and it is well-known that cAMP promotes neurite outgrowth by binding to and activating protein kinase A (PKA) 21-23. Activated PKA phosphorylates cAMP response element-binding protein (CREB), which then binds to CRE (cAMP response element) sites in various gene promotor regions 24-26. Some CREB target genes have been identified as immediate early genes (IEGs) that are induced in a CRE-dependent manner. Expression of IEGs can be induced within 1 hr in response to stimuli such as neurotrophin/Trk-or cAMP/PKA-depend...

FOXA3, a Negative Regulator of Nur77 Expression and Activity in Testicular Steroidogenesis

“…is was evidenced by the decreased activity of Nur77 and its target steroidogenic gene promoters, following cAMP induction, when FOXA3 was overexpressed. CREB is a key transcription factor that regulates the expression of numerous genes including Nur77, during cellular development and differentiation [27,28]. e decreased expression of steroidogenic genes following overexpression of FOXA3 may be due to inhibition of CREB transactivation as well as expression.…”

The Heterogeneity of Response of PC12 Cells from Different Laboratories to Nerve Growth Factor and Pituitary Adenylate Cyclase-Activating Polypeptide Questions the Reproducibility of Studies Carried Out with Tumor Cell Lines

“…For more information regarding PACAP and its mechanisms of action, refer to the following review [5]. Because PC12 cells are easy to grow and exhibit obvious morphological modifications, they have been used over the last 40 years in >20,000 studies referenced in PubMed to decipher the intracellular signaling cascades involved in the effects of some growth factors and neurotransmitters on proliferation and differentiation of neuroendocrine cells [6][7][8][9][10]. Other researchers have used PC12 cells to address the ability of some molecules to act as anticancer drugs [11].…”