GSK-3 (Glycogen synthase kinase 3) is a serine/threonine protein kinase, consisting of GSK-3α andβsubunit, which plays a key role in many different biological processes including tumorigenesis, cell survival, and developmental patterning. GSK-3 is constitutively active in non-stimulated cells and can negatively regulate canonical Wnt/β-catenin signaling. CHIR 99021 （CT99021）is highly selective aminopyrimidine-derivatived inhibitor of GSK-3 with IC50 of 10nM and 6.7nM for GSK-3α and GSK-3β(measured by kinase assays), respectively, and exhibits >500-fold selectivity for GSK-3 over closely related kinases, such as cdc2[8]Bennett CN, Ross SE, Longo KA, Bajnok L, Hemati N, Johnson KW, Harrison SD, MacDougald OA. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004. doi: 10.1074/jbc.M204527200. Epub 2002 Jun 7. PMID: 12055200. https://pubmed.ncbi.nlm.nih.gov/12055200/. CHIR-99021 can mimic Wnt signaling in preadipocytes. Analysis of cytosolic and membranous fractions show that the free cytosolic -catenin was evaluated in 3T3-L1 preadipocytes treated with 3uM CHIR-99021 for 4 h [4]. Chir-99021 can activate glycogen synthase in cells. The concentrations of CHIR-99021 causing half-maximal glycogen synthase stimulation (EC50) were 76 nmol/l for CHO-IR cells. A single oral dose of CHIR 99021 (30mg/kg) rapidly lowered plasma glucose in ZDF rats[8]. In combination with different small molecules, CHIR-99021 can facilitate cardiomyocyte differentiation from human embryonic stem cells and iPS cells[10][9], reprogramming, like generation of functional astrocytes from mammalian fibroblasts[10] and maintaining undifferentiated mouse ES cells in the absence of LIF[11].
作用机制
CHIR 99021 can inhibit GSK3 by competing for their ATP-binding sites.
CHIR 99021 细胞实验
Cell Line
Concentration
Treated Time
Description
References
Cos7 cells
8 nM
20 minutes
GSK3 inhibition increases macropinocytosis
Cell Rep. 2020 Jul 28;32(4):107973
MLO-Y4
5 µM
24 hours
Activate Wnt signaling pathway, promote osteogenic differentiation and mineralization of ST2 cells
Int J Mol Sci. 2023 Mar 22;24(6):6008
HaCaT cells
8 nM
20 minutes
GSK3 inhibition increases macropinocytosis
Cell Rep. 2020 Jul 28;32(4):107973
mouse embryonic stem cells (mESCs)
3 μM
48 hours
To investigate the effect of GSK-3 inhibition on Xist RNA expression, results showed that GSK-3 inhibition caused loss of Xist RNA expression.
Nat Commun. 2022 May 6;13(1):2516
HEK293T cells
5 or 10 μM
Activate Wnt/β-catenin signaling to a level similar to Wnt plus R-spondin1
Cell Discov. 2018 Sep 4;4:49.
B cells
10 µM
48 h
Induced IL-10+ Bregs and suppressed TNF-α expression
Mol Ther. 2024 Dec 4;32(12):4372-4382.
Human induced pluripotent stem cells (hiPSCs)
8 μM
3 days
By activating the Wnt signaling pathway, hiPSCs were induced to differentiate into cardiac lateral plate mesoderm cells (hiCLPM).
Circulation. 2024 Apr 30;149(18):1435-1456.
Lgr5+ intestinal stem cells
10 μM
7 days
Maintain self-renewal and proliferation of Lgr5+ intestinal stem cells
Cell Discov. 2018 Sep 4;4:49.
primary murine colonic epithelial cells
2.5 μM
48 h
To investigate the impact of Wnt signaling on intracellular Ca2+ oscillation patterns, results showed that CHIR-99021 significantly increased Ca2+ spike frequency and decreased pulse width
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