Molecular and Cellular Biology, May 2000, p. 3331-3344, Vol. 20, No. 10
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Subunit Gene by
Epidermal Growth Factor and Forskolin
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Received 25 October 1999/Returned for modification 10 December 1999/Accepted 17 February 2000
The aim of these studies was to elucidate a role for epidermal
growth factor (EGF) signaling in the transcriptional regulation of the
glycoprotein hormone
subunit gene, a subunit of
chorionic gonadotropin. Studies examined the effects of EGF and the
adenylate cyclase activator forskolin on the expression of a
transfected
subunit reporter gene in a human choriocarcinoma cell
line (JEG3). At maximal doses, administration of EGF resulted in a 50%
increase in a subunit reporter activity; forskolin administration
induced a fivefold activation; the combined actions of EGF and
forskolin resulted in synergistic activation (greater than eightfold)
of the
subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to
mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of
extracellular signal-regulated kinase (ERK) enzyme activity compared
with EGF alone. Specific blockade of ERK activation was sufficient to
block EGF-forskolin-induced synergistic activation of the
subunit
reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated
protein kinase inhibitor did not influence activation of the
reporter. However, overexpression of c-Jun N-terminal kinase
(JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the
subunit reporter. CRE binding studies suggested that the CRE complex
consisted of CRE binding protein and EGF-ERK-dependent recruitment of
c-Jun-c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos
(A-Fos) that specifically disrupts c-Jun-c-Fos DNA binding inhibited
synergistic activation of the
subunit. Thus, synergistic activation
of the
subunit gene induced by EGF-forskolin requires the ERK and
JNK cascades and the recruitment of AP-1 to the CRE binding complex.
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