Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I (original) (raw)

We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-l (IGF-I), or agents which alter intracellular CAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-CAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ERmediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with lo-' M EP, CT (1 Fg/ml) + IBMX (lo-" M), 8-Br-CAMP (10m4 M), or IGF-I (20 rig/ml) resulted in an 8-to lofold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 184,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-CAMP was more readily suppressed by ICI than that induced by EP, indicating that ER in cells exposed to 8-Br-CAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3-to 5-fold upon exposure to EP, CT