Progesterone decreases DNA binding factor activity and the expression in Xenopus oocytes of a cAMP responsive gene from rat liver.
Stage VI Xenopus oocytes were injected with a plasmid (pBB0.6-CAT) which contains the cAMP regulatory element (CRE) from the rat liver phosphoenolpyruvate carboxykinase (PEPCK) gene fused upstream from a reporter gene [chloramphenicol acetyltransferase (CAT)]. Inhibition of the expression of the reporter gene (average = 51%) was observed in the presence of 10 microM progesterone, which is known to lead to inactivation of the oocyte cAMP dependent protein kinase (A kinase). In contrast, oocytes injected with a control plasmid (pSV2CAT), which contains no CRE, exhibited a variable increase (average = 31%) in CAT activity after progesterone treatment. Injection of the purified bovine cardiac A kinase catalytic subunit prior to exposure of oocytes injected with pBB0.6 CAT to progesterone prevents the loss of CAT activity generated by incubation with the steroid. Gel retardation analyses with oocyte lysates and a labeled synthetic oligonucleotide fragment containing the CRE from the PEPCK gene showed the existence of a complex with the same Rf and specificity as that formed with rat liver extracts. Subsequent exposure to progesterone, however, led to a rapid and extensive decrease in this binding activity. Taken together, these results are consistent with but do not prove the hypothesis that progesterone treatment and A kinase inactivation lead to a decrease in pBB0.6 CAT expression by virtue of a decline in the binding activity of an oocyte factor(s) to the CRE of the PEPCK fragment in pBB0.6-CAT, thereby decreasing transcription of the CAT gene.
Williams
J A
JA
Department of Biochemistry, LSU Medical Center, New Orleans.
Schlichter
D
D
Wicks
W D
WD
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005809:Genes, Regulator; D009865:Oocytes / Q000187:drug effects; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D010957:Plasmids; D011374:Progesterone / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014982:Xenopus laevis
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)