CtBP2 Downregulation during Neural Crest Specification Induces Expression of Mitf and REST, Resulting in Melanocyte Differentiation and Sympathoadrenal Lineage Suppression

Abstract

Trunk neural crest (NC) cells differentiate to neurons, melanocytes, and glia. In NC cultures, cyclic AMP (cAMP) induces melanocyte differentiation while suppressing the neuronal sympathoadrenal lineage, depending on the signal intensity. Melanocyte differentiation requires activation of CREB and cAMP-dependent protein kinase A (PKA), but the role of PKA is not understood. We have demonstrated, in NC cultures, cAMP-induced transcription of the microphthalmia-associated transcription factor gene ( Mitf ) and the RE-1 silencing transcription factor gene ( REST ), both Wnt-regulated genes. In NC cultures and zebrafish, knockdown of the corepressor of Wnt-mediated transcription C-terminal binding protein 2 (CtBP2) but not CtBP1 derepressed Mitf and REST expression and enhanced melanocyte differentiation. cAMP in NC and B16 melanoma cells decreased CtBP2 protein levels, while inhibition of PKA or proteasome rescued CtBP2 degradation. Interestingly, knockdown of homeodomain-interacting protein kinase 2 (HIPK2), a CtBP stability modulator, increased CtBP2 levels, suppressed expression of Mitf, REST, and melanocyte differentiation, and increased neuronal gene expression and sympathoadrenal lineage differentiation. We conclude that cAMP/PKA via HIPK2 promotes CtBP2 degradation, leading to Mitf and REST expression. Mitf induces melanocyte specification, and REST suppresses neuron-specific gene expression and the sympathoadrenal lineage. Our studies identify a novel role for REST in NC cell differentiation and suggest cross talk between cAMP and Wnt signaling in NC lineage specification.

Down-regulation of RE-1 silencing transcription factor (REST) in advanced prostate cancer by hypoxia-induced miR-106b~25

“…1A). The enhanced number of TH-positive cells and the reduced number of melanocytes in the same culture, due to hypoxia treatment, supports our previous results of a dynamic balance between the neuronal vs. melanocyte lineages (34). …”

“…We employed primary cultures of avian NC cells grown in hypoxia for 24hrs, from day 0 to day 1 after replating (34), followed by incubation in normoxia. Under these conditions, we observed enhanced number of cells immunostaining for tyrosine hydroxylase (TH), a marker of sympathoadrenal neurons, and significantly reduced number of pigmented melanocytes (Fig.…”

“…We have shown earlier that expression levels of REST control the switch between neuronal and non-neuronal cell fates, and down-regulation of REST is essential for neuronal differentiation (34). Also, REST is a tumor suppressor involved in various types of cancers (32).…”

“…Primary cultures of trunk neural crest (NC) cells were prepared from Japanese quail (Coturnix coturnix) embryos as described (34). LNCaP cells were purchased from ATCC and cultured in RPMI 1640 (Invitrogen) containing 2.0g/L NaHCO 3 and 10% fetal bovine serum (Atlanta Biologicals).…”

Interaction with Cyclin H/Cyclin-dependent Kinase 7 (CCNH/CDK7) Stabilizes C-terminal Binding Protein 2 (CtBP2) and Promotes Cancer Cell Migration

“…Several phosphorylation sites have been mapped for CtBP2 that are correlated with accelerated degradation. For instance, HIPK2 phosphorylates CtBP2 at serine 428 whereas PKA phosphorylates CtBP2 at 164 although it is likely that these two enzymes may act coordinately (27). Our data indicate that CCNH/CDK7 regulates CtBP2 stability by at least in part influencing its phosphorylation status (Fig.5), suggesting that cellular CtBP2 levels lie in the balance between CCNH/CDK7 and the kinases that phosphorylate and target CtBP2 for destruction.…”