A Novel Cyclic AMP/Epac1/CaMKI Signaling Cascade Promotes GCM1 Desumoylation and Placental Cell Fusion

Abstract

Cyclic AMP (cAMP) signaling and the placental transcription factor glial cell missing 1 (GCM1) regulate expression of syncytin-1 and -2 fusogenic proteins, which are critical for syncytiotrophoblast formation by trophoblast fusion. We recently revealed a cAMP/protein kinase A (PKA)/CBP signaling pathway that activates GCM1 by coordinating GCM1 phosphorylation and acetylation. In contrast, GCM1 activity is downregulated by sumoylation of Lys156. How GCM1 sumoylation is regulated was unknown. Here, we identify a novel PKA-independent cAMP signaling pathway as the critical regulator of GCM1 sumoylation. We show that Epac1 and Rap1, in response to cAMP, activate CaMKI to phosphorylate Ser47 in GCM1. This phosphorylation facilitates the interaction between GCM1 and the desumoylating enzyme SENP1 and thereby leads to GCM1 desumoylation and activation. Using RNA interference (RNAi), we further demonstrate that 8-(4-chlorophenylthio)-2-O-Me-cAMP-AM (8-CPT-AM), an Epac activator, stimulates syncytin-1 and -2 gene expression and cell fusion of placental BeWo cells in a GCM1-dependent manner. Importantly, the cell fusion defect in GCM1-knockdown BeWo cells can be reversed and enhanced by the RNAi-resistant phosphomimetic GCM1(S47D) mutant. Our study has identified a novel cAMP/Epac1/CaMKI/GCM1 signaling cascade that stimulates trophoblast fusion through promoting GCM1 phosphorylation and desumoylation.The syncytiotrophoblast in human placental villus is a multinucleated cell layer that mediates gas and nutrient transport between mother and fetus. Differentiation of the syncytiotrophoblast involves cell fusion of subjacent mononucleated cytotrophoblast cells. As the syncytiotrophoblast layer undergoes apoptosis and sheds into circulation, replenishment with a new syncytiotrophoblast layer is required to maintain its structural and functional integrity. In this regard, cyclic AMP (cAMP) is a key player for syncytiotrophoblast differentiation by stimulating placental cell fusion and syncytin gene expression (4,19,24,33). Stimulation of placental cell fusion by cAMP is suppressed by the protein kinase A (PKA) inhibitor H89, indicating that PKA is a critical downstream effector of cAMP signaling in the regulation of placental cell fusion (19).The placental transcription factor GCM1 (also known as GCMa) controls trophoblast fusion via transcriptional activation of two membrane fusogenic proteins, syncytin-1 and -2 (22, 38). An RNA interference (RNAi) study by Baczyk et al.(2) has shown that GCM1 is required for the cAMP stimulant forskolin to promote placental BeWo cell fusion. Indeed, GCM1 activity can be regulated by multiple posttranslational modifications, which may be critical to fine-tune its activity in the regulation of trophoblast fusion and syncytiotrophoblast differentiation. GCM1 is a labile protein subject to ubiquitination via the F-box protein FBW2 when the Ser322 residue in GCM1 is phosphorylated by glycogen synthase kinase 3␤ (GSK-3␤) (7,31,36). Our recent studies have demonstrated that PKA enhances the interact...

Epac1-dependent phospholamban phosphorylation mediates the cardiac response to stresses

“…We found that 10 μM 8-CPT-AM was necessary to obtain a significant PLN phosphorylation signal with our detection system in neonatal rat cardiac myocytes ( Figure 2D), and thus this concentration was used. This concentration of 8-CPT-AM is in line with those used in previous studies to examine EPAC-mediated signaling, i.e., 10 μM for adult rat cardiac myocytes (34) or 50 μM for 293T, JAR, and BeWo cells (35). Furthermore, Brette et al reported very recently that 8-CPT-AM at 10 μM did not exert an inhibitory effect on phosphodiesterase (PDE) activity (34).…”

PMA-induced GCMa phosphorylation stimulates its transcriptional activity and degradation

“…These data suggest that in JEG-3 cells the PMA-induced PKC-and MEK/ ERK-dependent pathway stimulates hCG production but much less so than the FSK-induced cAMPdependent pathway. The cAMP-dependent pathway has been shown to increase the expression level and transcriptional activity of GCMa in trophoblast cells (6,7,27), leading to trophoblast differentiation. In contrast, the PKC-and MEK/ERK-dependent pathway enhances GCMa degradation as well as transcriptional activity.…”

“…Recent studies have shown that the expression level and transcriptional activity of GCMa are regulated by its post-translational modifications. The acetylation and deacetylation by CRE-binding protein-binding protein (CBP) and histone deacetylase 3 (HDAC3) regulate the protein stability of GCMa (7,13), and the sumoylation destabilizes the interaction between GCMa and the specific DNA sequence called GCM motif (6,12). The phosphorylation of GCMa on serine 322 results in its ubiquitination and degradation (11).…”

ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion

“…5,6 PKA directs multiple pathways important in trophoblast fusion, including the expression of the transcription factor glial cell missing 1 (GCM1). 7,8 Coordination and propagation of the fusion signal is controlled via gap junctions, 9,10 cytoskeletal reorganization 11,12 and remodeling of intercellular adhesion complexes. 13,14 Intercellular adherens junctions, composed of members of the cadherin family of cell adhesion molecules, are dynamically regulated during trophoblast syncytialization.…”