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Messenger Ribonucleic Acid Expression of Term Villous Trophoblasts1
Department of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Martin Knöfler, Department of Obstetrics and Gynecology, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna. E-mail: martin. knoefler{at}akh-wien.ac.at
Differentiation of primary villous cytotrophoblasts into syncytia is
associated with increasing production of 
and ß human CG subunits,
which is predominantly governed at the level of messenger RNA
expression. Here, we present a detailed study on the mechanisms
involved in the differentiation-dependent regulation of the
trophoblast-specific CG gene promoter. Site-directed mutations in
each of the five DNA-elements of the composite enhancer were performed
to investigate the contribution of the individual regulatory sequences
to the overall transcriptional activity of the promoter at two
different stages of trophoblast in vitro
differentiation. We show that deletion of one cyclic AMP response
element (CRE) did not affect CG promoter activity in
cytotrophoblasts; however, it reduced transcription by 33% in
differentiating cultures. Removal of both CREs almost abolished
transcription at early and later stages of in vitro
differentiation. Upon mutation the enhancer elements ACT, JRE, and
CCAAT significantly decreased luciferase reporter transcription;
however their contribution to the total promoter activity did not
change during in vitro differentiation. Contrary to
that, mutated TSE diminished promoter activity by 19% during 12 and
48 h of cultivation but reduced luciferase expression by 78%
between 48 and 84 h of differentiation. In electrophoretic
mobility shift assay, the TSE interacted with activating protein
(AP)-2 in both primary trophoblasts and choriocarcinoma cells. While
CRE-interacting proteins were detectable 12 h after isolation, the
TSE-binding complex did not appear before 36 h of in
vitro differentiation. During syncytium formation increasing
protein expression of activating transcription factor (ATF)-1, cAMP
response element-binding protein (CREB)-1, and AP-2 was observed on
Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated
between 24 and 78 h of trophoblast cultivation. By fluorescence
immunohistochemistry, we show that CREB-1 was predominantly expressed
in syncytiotrophoblasts, whereas ATF-1 and AP-2 localized to the
syncytium and some cytototrophoblasts as well as to stromal and
endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1
and the coactivator protein CBP were primarily detected in syncytial
nuclei, suggesting the presence of functional, cAMP-dependent
transcriptional complexes in the differentiated tissue. In agreement to
the in vivo situation, phosphorylated CREB-1/ATF-1 were
observed in nuclei of the differentiated trophoblast cultures. The
activity of the CG promoter as well as CREB-1/ATF-1 phosphorylation
increased upon elevation of cAMP levels and overexpression of the
catalytic subunit of protein kinase A. Additionally, we demonstrate
that overproduction of the enzyme enhanced protein expression and
binding of AP-2 to the TSE. We conclude that
differentiation-dependent transcription of the CG gene in villous
trophoblasts is mainly governed by increasing expression of AP-2 and
PKA-dependent phosphorylation of CREB-1 and ATF-1.
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