Abstract
The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone α 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 α 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 α 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 α subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the α 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 α 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 α subunit. Thus, synergistic activation of the α subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex.
ACKNOWLEDGMENTS
We thank Richard A. Maurer (Oregon Health Sciences University, Portland), Colin Clay (Colorado State University, Fort Collins), Roger Davis (University of Massachusetts, Worcester), Linda VanAelst (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), David Ginty (Johns Hopkins University, Baltimore, Md.), Paul Dobner (University of Massachusetts, Worcester), Michael Greenberg (Harvard Medical School, Boston, Mass.), and Charles Vinson (National Cancer Institute, Bethesda, Md.) for generously sharing valuable reagents. Special thanks go to Jane Reusch (University of Colorado, Denver) for helpful discussions during the course of these studies. We thank Joanne Fortune (Cornell University, Ithaca, N.Y.) and Colin Clay (Colorado State University, Fort Collins) for helpful criticisms during the preparation of the manuscript.
This work was supported by a Consolidated Research Grant from the College of Veterinary Medicine, Cornell University.