Cellular models for the analysis of signaling by protein kinase B and the forkhead transcription factor FKHR (Foxo1a).
The transcription factor FKHR (FOXO1a) is regulated by protein kinase B (PKB) and insulin controls the expression of hepatic genes like glucose-6-phosphatase (G6Pase) at least in part via these proteins. However, insulin is known to activate several pathways and it is therefore difficult to establish which effects of the hormone are attributed to PKB and FKHR signaling. The aim of the present study was the generation of cellular models which allow the specific analysis of molecular events controlled by PKB and FKHR, respectively. We generated two H4IIEC3 rat hepatoma cell lines stably expressing either a hydroxytamoxifen-regulatable form of PKB (myristoylated PKB estrogen receptor chimera; MER-PKB) or FKHR (FKHR estrogen receptor chimera; FKHR-ER) by retroviral infection and determined the regulation of the G6Pase transcript by Northern blotting and enzyme assays. Activation of the regulatable PKB fusion protein almost completely reduced the dexamethasone/cAMP-stimulated G6Pase mRNA levels comparable to the effect of insulin. In contrast, stimulation of FKHR-ER with tamoxifen increased the expression of the dexamethasone/cAMP-induced G6Pase mRNA and the G6Pase enzymatic activity about 2.5- to 3-fold. The present data demonstrate that activation of PKB is sufficient to mimic the effect of insulin on the expression of G6Pase and that FKHR acts as an activator of the G6Pase gene indicating that the established cellular models are suitable for the specific analysis of downstream targets of these signaling molecules. Therefore, these cell systems might serve as useful tools for the development of anti-diabetic drugs.
Orth
Hans-Martin
HM
Institut für Pharmakologie und Toxikologie, RWTH Aachen, D-52057 Aachen, Germany.
Krüger
Klaus-Dieter
KD
Schmoll
Dieter
D
Grempler
Rolf
R
Scherbaum
Werner A
WA
Joost
Hans-Georg
HG
Bornstein
Stefan R
SR
Barthel
Andreas
A
D000818:Animals; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation; D051858:Forkhead Transcription Factors; D005786:Gene Expression Regulation / Q000187:drug effects; D005952:Glucose-6-Phosphatase / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D000071161:Forkhead Box Protein O1
D000242:Cyclic AMP; D004268:DNA-Binding Proteins; D003907:Dexamethasone; D051858:Forkhead Transcription Factors; D005952:Glucose-6-Phosphatase; D009419:Nerve Tissue Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1