Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.
BACKGROUND
Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized.


METHODS
Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated.


RESULTS
CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T.


CONCLUSIONS
Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.
Institute of Clinical Biochemistry, Hannover Medical School, Carl-Neuberg-Str, 1, Hannover 30625, Germany. naujok.ortwin@mh-hannover.de.
Ortwin
O
Naujok
Jana
J
Lentes
Ulf
U
Diekmann
Claudia
C
Davenport
Sigurd
S
Lenzen
D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D005326:Fetal Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000633:toxicity*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020825:T-Box Domain Proteins / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects* / Q000235:genetics; D000098908:Brachyury Protein
D000631:Aminopyridines; D005326:Fetal Proteins; D038362:Glycogen Synthase Kinase 3; D007211:Indoles; D008301:Maleimides; D050814:Octamer Transcription Factor-3; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein; C473710:Chir 98014; C473711:Chir 99021; C494174:Pou5f1 protein, mouse; C417521:SB 216763