Award details

Glycogen synthase kinase 3: a critical regulator of embryonic stem cell self-renewal

Reference	BB/C516528/1
Principal Investigator / Supervisor	Professor Melanie Welham
Co-Investigators / Co-Supervisors
Institution	University of Bath
Department	Pharmacy and Pharmacology
Funding type	Research
Value (£)	175,116
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	31/12/2008
Duration	43 months

Abstract

Stem cells and their progeny have tremendous therapeutic potential in regenerative medicine as a source of cells for treating a wide variety of chronic diseases. Understanding the molecular signals that regulate self-renewal and control differentiation is critical if this potential is to be realised. Leukaemia inhibitory factor, LIF, plays a role in maintaining self-renewal of murine ES cells through activation of STAT3. LIF-independent mechanisms that contribute to maintenance of self-renewal include the homeodomain protein Nanog, bone morphogenic proteins 2 and 4 and Wnt signalling, with latter of particular relevance to this proposal. A newly described GSK-3beta inhibitor, 6-bromo-indirubin-3¿-oxime (BIO), has been used to mimic activation of the Wnt pathway in ES cells. Strikingly, BIO maintained self-renewal of mouse ES cells in the absence of LIF. Intriguingly, these results are in contrast to an earlier report that described a GSK-3 inhibitor that facilitated neuronal differentiation of EC and ES cells. We have synthesised a novel bis-indolylmaleimide, SB378, which significantly increases self-renewal of murine ES cells. In vitro kinase assays demonstrate that SB378 preferentially targets GSK-3beta, with an estimated Ki of 100 nM or lower. However, unlike BIO, SB378 has only limited capacity to maintain self-renewal in the absence of LIF and does not lead to significant changes in the levels of beta-catenin or its phosphorylation, whereas BIO has been reported to increase transcription of beta-catenin-dependent genes, presumably by increasing beta-catenin levels. These results raise two important questions. First, are GSK-3 isoforms the sole targets of BIO and SB378 and if not, what are the critical kinases? Second, do GSK-3 isoforms play an essential role in regulation of self-renewal? If GSK-3 does regulate self-renewal then this would be highly significant. Therefore, the main objectives of this research proposal are to conclusively identify the kinase(s) targeted by SB378 and to increase our understanding of the role of GSK-3 in ES cell biology. The following specific objectives will be addressed and represent a series of complimentary investigations. (1) To determine whether structurally unrelated GSK-3 inhibitors have similar effects on the maintenance of ES cell self-renewal. (2) To identify the molecular target(s) of SB378 using a chemical genetic approach. (3) To characterise the biochemistry of GSK-3alpha and beta in ES cells. (4) To determine the role of GSK-3 in self-renewal of murine ES cells. (5) To synthesise analogues of SB378 with greater potency for increasing self-renewal of murine ES cells. (6) To examine the consequences of SB378 treatment and GSK-3 on genes known to be involved in control of self-renewal. (7) To use microarray expression profiling to define genes regulated by SB378 and GSK-3 in ES cells. In determining whether GSK-3 is the molecular target of SB378, we would use a chemical genetic approach allowing us to work from phenotype to genotype. A biotinylated analogue of SB378 would be synthesised and used as an affinity tool to purify its target kinase(s) from ES cells. We would also synthesise analogues of SB378 with the aim of increasing selectivity and potency. To directly determine whether GSK-3 isoforms regulate self-renewal we would initially use loss of function approaches including RNA interference. Each isoform would be knocked-down independently or together. Tet-regulated expression would be used to express dominant negative and constitutively active mutants of GSK-3 and the consequences on self-renewal and signalling examined. To gain insight into the genetic programmes regulated by GSK-3 dependent signalling in ES cells, we would use quantitative PCR to examine effects of SB378 and GSK-3 knock-down on genes whose expression is known to be associated with self-renewal, e.g. Nanog, Oct-4 and Rex-1. More global analyses would be carried out using affymetrix profiling. Joint with BB/C51390X

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/C51390X/1 Glycogen synthase kinase 3: a critical regulator of embryonic stem cell self-renewal?

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