Construction and characterization of a conditionally active construct of the insulin-regulated forkhead transcription factor FKHR.
Summary. Insulin is known to inhibit glucose-6-phosphatase gene expression through PI 3-kinase/PKB mediated phosphorylation and inactivation of the forkhead transcription factor FKHR, which is a potent transactivator of the glucose-6-phosphatase gene. To study the function and regulation of the transcription factor FKHR in hepatic cells, we constructed a hydroxytamoxifen-inducible version of FKHR by fusing a part of the hormone binding domain of the estrogen receptor (ER) to the C-terminus of FKHR (FKHR-ER). In HepG2-cells transiently transfected with plasmids encoding the FKHR-ER fusion protein and a glucose-6-phosphatase reporter construct, hydroxytamoxifen induced a marked induction of glucose-6-phosphatase promoter activity, whereas no effect was observed in control cells. We next generated a H4IIEC3 rat hepatoma cell line stably expressing both FKHR-ER and a glucose-6-phosphatase promoter-based reporter construct. After 2h stimulation with hydroxytamoxifen, the promoter activity was stimulated 3-5 fold, and continued to increase up to 100-fold after 15 h. The response was half maximal at 0.5 microM hydroxytamoxifen. Insulin (1 nM) decreased the hydroxytamoxifen induced promoter activity by about 70% of the maximal response. This cell system can be used for (1) the identification of FKHR dependent genes and for (2) high throughput screening (HTS) of agents affecting the activity of FKHR and its regulation by insulin. Abbreviations used: FKHR, forkhead in rhabdomyosarcoma; G6Pase, glucose-6-phosphatase; PKB, protein kinase B; PI 3-kinase, phosphatidyl-inositol 3-kinase; IRU, insulin-responsive unit; Tx, 4-hydroxytamoxifen, ER, estrogen receptor; HBD, hormone binding domain
Barthel
Andreas
A
Institute of Pharmacology and Toxicology, Technical University of Aachen, Wendlingweg 2, D-52057 Aachen, Germany. Andreas.Barthel@post.rwth-aachen.de
Orth
Hans-Martin
HM
Krüger
Klaus-Dieter
KD
Schmoll
Dieter
D
Joost
Hans-Georg
HG
D004268:DNA-Binding Proteins / Q000235:genetics*; D051858:Forkhead Transcription Factors; D005786:Gene Expression Regulation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D009419:Nerve Tissue Proteins; D011401:Promoter Regions, Genetic / Q000187:drug effects; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D000071161:Forkhead Box Protein O1
D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D007328:Insulin; D009419:Nerve Tissue Proteins; D013629:Tamoxifen; D014157:Transcription Factors; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1; C016601:afimoxifene