Insulin inhibits the estrogen-dependent expression of the chicken very low density apolipoprotein II gene in Leghorn male hepatoma cells.
Expression of the very low density apolipoprotein II (apoVLDLII) gene in the chicken is absolutely dependent on estrogen. ApoVLDLII mRNA is expressed in the Leghorn male hepatoma (LMH) cell line in response to estrogen in completely defined medium. Addition of serum to these cultures results in a decrease in apoVLDLII mRNA. Data in this report demonstrate that 1 nM insulin has the same inhibitory effect as 10% serum. Insulin inhibits apoVLDLII mRNA in a dose-dependent manner; 100 fM insulin inhibits the estrogen-dependent response by 76%. After transfection of LMH cells with apoVLDLII sequences from an 8.9-kilobase (kb) genomic clone (pApo107) that contains the entire 2.9-kb coding sequence along with approximately 3 kb each of 5'- and 3'-flanking DNA, the estrogen-dependent expression of apoVLDLII mRNA from both the endogenous gene and transfected DNA is reduced by insulin. Furthermore, insulin reduces by more than 90% the estrogen-dependent expression from a chimeric construct, pApoCAT, which contains apoVLDLII sequences -900/+1455 cloned 5' of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. To determine the specificity of the response, expression of the pApoCAT construct was tested with insulin-like growth factor-I and insulin. Three hundred picomolar insulin inhibits the estrogen-mediated CAT activity by 50%. Insulin-like growth factor-I at this concentration has no effect or slightly increases the estrogen-dependent expression of pApoCAT, suggesting that the observed inhibitory action is mediated by the insulin receptor. Consequently, the LMH cells provide an excellent model system in which to study the molecular mechanism of insulin and estrogen interaction in the regulation of gene expression.
Department of Biochemistry, School of Medicine, West Virginia University, Morgantown 26506.
E A
EA
Berkowitz
W W
WW
Chu
M I
MI
Evans
D000818:Animals; D001053:Apolipoproteins / Q000235:genetics*; D001483:Base Sequence; D001769:Blood; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004967:Estrogens / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D007328:Insulin / Q000008:administration & dosage / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008079:Lipoproteins, VLDL / Q000235:genetics*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D001053:Apolipoproteins; D004967:Estrogens; D007328:Insulin; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C027610:apolipoprotein VLDL II; D007334:Insulin-Like Growth Factor I