The cAMP Pathway Regulates Both Transcription and Activity of the Paired Homeobox Transcription Factor Phox2a Required for Development of Neural Crest-derived and Central Nervous System-derived Catecholaminergic Neurons

Abstract

Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, bone morphogenetic protein 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor Phox2a. In NC cultures inhibition of cAMPresponse element-binding protein (CREB)-mediated transcription by expression of dominant-negative CREB suppresses Phox2a transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREB DIEDML , despite inducing Phox2a transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREB-DIEDML mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent protein kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-10 nM okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated Phox2a dephosphorylation, cAMP-dependent Phox2a DNA binding in vitro, and cAMP-and Phox2a-dependent dopamine-␤-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent Phox2a dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both Phox2a transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development. The neural crest (NC)3 is a pluripotent cell population derived from the lateral ridges of the neuroepithelium during closure of the neural tube (1). NC cells migrate along defined paths in the developing embryo, generating diverse cell types (2). The sympathoadrenal (SA) lineage originating from the trunk region of the neural tube (1) in response to instructive, microenvironmental signals develops to sympathetic neurons and chromaffin cells of the adrenal medulla (3). Cells committed to the SA lineage are characterized by expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, and dopamine-␤-hydroxylase (DBH), catalyzing the conversion of dopamine to norepinephrine (3).Bone morphogenetic proteins (BMPs) produced by the developing aorta (4 -9) and unidentified signals originating from the notochord and ventral neural tube (10 -14) are required for SA cell development. However, in ...

The cAMP Pathway in Combination with BMP2 Regulates Phox2a Transcription via cAMP Response Element Binding Sites

“…cAMP signaling in synergy with BMP2 induces development of the SA lineage by increasing the expression of the homeodomain transcription factor Phox2a (7) in a CREB-mediated mechanism (8). cAMP signaling also regulates the transactivation potential of Phox2a (8), consistent with studies by Lo et al (6).…”

“…1A). In our previous studies we have shown that cotreatment of NC cells with BMP2 and cAMP synergistically induces expression of the endogenous Phox2a gene (7,8), as well as of TH mRNA (Fig. 1B).…”

“…The sorted NC cells were then grown either under control conditions or with BMP2ϩIBMX cotreatment. After 4 days of culture the NC cells were immunostained with TH antibody to monitor TH protein expression, a marker of SA cell development (7,8). We hypothesized, if the transcriptional activity of the transiently transfected hPhox2a were authentic, these hPhox2a-fluorescence-positive NC cells would be expressing the endogenous Phox2a gene.…”

Timing the Phox-Trot: Duration of Phox2a-Dependent Transcription Is Controlled by an Intramolecular Dephosphorylation/Phosphorylation Clock

“…The S153-phosphorylated, S202/S206/S208-dephosphorylated form of Phox2a is a molecule no longer capable of association with DNA and therefore no longer able to transactivate the p27 kip1 promoter. This finding is consistent with previous work indicating that cAMP induces Phox2a binding to the p27 kip1 promoter, transcription of the p27 kip1 gene, and CAD and neural crest cell differentiation (4,14). Mapping of cAMP, protein kinase A (PKA), and protein phosphatase regulation of Phox2a activation and p27 kip1 gene transcription to specific serine residues on Phox2a has allowed a processive model for sequential activation and built-in deactivation of Phox2a regulation of the p27 kip1 gene promoter to emerge (Fig.…”

The Phox2 pathway is differentially expressed in neuroblastoma tumors, but no mutations were found in the candidate tumor suppressor gene PHOX2A

“…Although independently expressed, in sympathetic ganglia it seems like Phox2b is necessary for maintained expression of ASCL1 (12,15). Recent findings suggest that cAMP signaling works synergistically with BMPs in promoting expression of these genes (25) (Fig. 4).…”