Possible role of the exchange protein directly activated by cyclic AMP (Epac) in the cyclic AMP-dependent functional differentiation and syncytialization of human placental BeWo cells.
BACKGROUND
The mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells.


METHODS
Epac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N(6)-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated.


RESULTS
Epac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCG alpha/beta mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion.


CONCLUSIONS
The Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.
Yoshie
Mikihiro
M
Department of Endocrine Pharmacology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. yoshie@toyaku.ac.jp
Kaneyama
Kei
K
Kusama
Kazuya
K
Higuma
Chinatsu
C
Nishi
Hirotaka
H
Isaka
Keiichi
K
Tamura
Kazuhiro
K
D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002460:Cell Line; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009928:Organ Specificity; D010920:Placenta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D011264:Pregnancy Trimesters; D011374:Progesterone / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects; D014327:Trophoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000502:physiology
D006063:Chorionic Gonadotropin; D020662:Guanine Nucleotide Exchange Factors; D047428:Protein Kinase Inhibitors; C116122:RAPGEF3 protein, human; C442864:RAPGEF4 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011374:Progesterone; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins