The human placental trophoblast cell can be classified as either a cytotrophoblast or a syncytiotrophoblast. Cytotrophoblasts can function as stem cells for the development of the syncytiotrophoblast layer via cell fusion. An envelope gene of the human endogenous retrovirus family W (HERV-W) called syncytin is specifically expressed in the syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein; therefore, it can mediate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this process by regulating syncytin gene expression. In this report, we demonstrate that GCMa was able to regulate syncytin gene expression via two GCMa-binding sites upstream of the 5-long terminal repeat of the syncytin-harboring HERV-W family member in BeWo and JEG3 cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhanced syncytin gene expression and syncytinmediated cell fusion in BeWo and JEG3 cells but not in HeLa cells. Therefore, the integration site of the syncytin-harboring HERV-W family member in the human genome is close to the functional GCMa-binding sites by which GCMa can specifically transactivate syncytin gene expression in trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.The human placenta contains a specialized cell type called a trophoblast, which is the first lineage to differentiate in embryo development and plays key roles during implantation and placentation. The human trophoblast cell can be further classified as cytotrophoblasts and syncytiotrophoblasts. In the early gestation stage, cytotrophoblast stem cells facing the maternal decidua proliferate and fuse to form a syncytium, i.e. the syncytiotrophoblast. Later on, vascular spaces called trophoblastic lacunae appear in the syncytium around day 8 -9. The cytotrophoblast layer under the syncytium can rapidly proliferate into these spaces, which results in the formation of the primary chorionic villi. Subsequently, proliferation of the cytotrophoblasts, growth of chorionic mesoderm (under the cytotrophoblast layer), and blood vessel development transform the primary villi into secondary and tertiary villi, which are composed of a core of mesenchyme cells surrounded by an inner layer of cytotrophoblasts and an outer layer of multinucleate syncytiotrophoblasts (1, 2). The syncytiotrophoblast layer (syncytium) transports nutrients and gases and produces hormones such as placental lactogen and chorionic gonadotrophin, which are indispensable for the further progression of pregnancy (1).Recently, a membrane protein termed syncytin has been demonstrated to mediate cell fusion of the human BeWo trophoblastic cell line (3). Syncytin is an envelope protein of the newly identified human endogenous ...

Human GCMa is a zinc-containing transcription factor primarily expressed in placenta. GCMa regulates expression of syncytin gene, which encodes for a placenta-specific membrane protein that mediates trophoblastic fusion and the formation of syncytiotrophoblast layer required for efficient fetal-maternal exchange of nutrients and oxygen. The adenylate cyclase activator, forskolin, stimulates syncytin gene expression and cell fusion in cultured placental cells. Here we present evidence that cyclic AMP (cAMP) signaling pathway activates the syncytin gene expression by regulating GCMa activity. We found that forskolin and protein kinase A (PKA) enhances GCMa-mediated transcriptional activation. Furthermore, PKA treatment stimulates the association of GCMa with CBP and increases GCMa acetylation. CBP primarily acetylates GCMa at lysine 367 , lysine 406 , and lysine 409 in the transactivation domain (TAD). We found that acetylation of these residues is required to protect GCMa from ubiquitination and increases the TAD stability with a concomitant increase in transcriptional activity, supporting the importance of acetylation in PKA-dependent GCMa activation. Our results reveal a novel regulation of GCMa activity by cAMP-dependent protein acetylation and provide a molecular mechanism by which cAMP signaling regulates trophoblastic fusion.

The GCM proteins GCMa/1 and GCMb/2 are novel zinccontaining transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulsechase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from ϳ30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774 -4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCF hFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system.