JCS ePress online publication date 9 June 2009 Research Article
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Distinct roles for isoforms of the catalytic subunit of class-IA PI3K in the regulation of behaviour of murine embryonic stem cells Emmajayne Kingham and Melanie Welham* Department of Pharmacy and Pharmacology and The Centre for Regenerative Medicine, University of Bath, Claverton Down, Bath BA2 7AY, UK *Author for correspondence (e-mail:
[email protected])
Journal of Cell Science
Accepted 3 April 2009 Journal of Cell Science 122, 2311-2321 Published by The Company of Biologists 2009 doi:10.1242/jcs.046557
Summary Self-renewal of embryonic stem cells (ESCs) is essential for maintenance of pluripotency, which is defined as the ability to differentiate into any specialised cell type comprising the adult organism. Understanding the mechanisms that regulate ESC self-renewal and proliferation is required before ESCs can fulfil their potential in regenerative therapies, and murine ESCs (mESCs) have been widely used as a model. Members of the class-IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses, including cell migration, proliferation and survival. PI3Ks have been reported to regulate both proliferation and self-renewal of mESCs. Here we investigate the contribution of specific class-IA PI3K isoforms to the regulation of mESC fate using small-molecule inhibitors with selectivity for particular class-IA PI3K catalytic isoforms, and siRNA-mediated knockdown. Pharmacological inhibition or knockdown of p110β promoted mESC
Introduction Murine embryonic stem cells (mESCs) are derived from the inner cell mass (ICM) of the preimplantation blastocyst at embryonic day 3.5 (E3.5) (Evans and Kaufman, 1981). When grown in culture in the presence of serum, leukaemia inhibitory factor (LIF) is required to maintain mESCs in an undifferentiated, pluripotent state (Smith, 2001). Pluripotency is defined as the ability to produce all the differentiated cell types that comprise an adult organism. So that pluripotency is maintained, ESCs in culture need to self-renew, which in essence is proliferation accompanied by the suppression of differentiation. LIF promotes an array of intracellular signalling pathways that are important for promoting self-renewal, including the activation of Stat3 (Boeuf et al., 1997; Cartwright et al., 2005; Niwa et al., 1998) and phosphoinositide 3-kinase (PI3K) signalling (Paling et al., 2004). The MAPK pathway is also activated by LIF and FGF4 but, rather than contribute to self-renewal, activation of this pathway promotes differentiation (Burdon et al., 1999; Kunath et al., 2007; Stavridis et al., 2007). The PI3K family of lipid kinases catalyse the addition of a phosphate group to the D3 position of the inositol ring of phosphoinositides, generating PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which subsequently act as secondary messengers for signal transduction (Vanhaesebroeck and Waterfield, 1999). Mammalian PI3Ks are categorised into three classes, with the class-IA PI3Ks comprising a 110-kDa catalytic subunit (p110α, p110β or p110δ, encoded by the genes Pik3ca, Pik3cb and Pik3cd, respectively) coupled to a regulatory subunit (p85α, p55α or p50α,
differentiation, accompanied by a decrease in expression of Nanog. By comparison, pharmacological inhibition or siRNAmediated knockdown of p110α had no effect on mESC selfrenewal per se, but instead appeared to reduce proliferation, which was accompanied by inhibition of leukaemia inhibitory factor (LIF) and insulin-induced PI3K signalling. Our results suggest that PI3Ks contribute to the regulation of both mESC pluripotency and proliferation by differential coupling to selected p110 catalytic isoforms.
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/13/2311/DC1 Key words: Embryonic stem cells, Pluripotency, Cell proliferation, PI3 kinase, Signal transduction
encoded by Pik3r1; p85β, encoded by Pik3r2; or p55γ, encoded by Pik3r3) (Cantrell, 2001; Vanhaesebroeck and Waterfield, 1999). The phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome ten) dephosphorylates PtdIns(3,4,5)P3, the key product of class-IA PI3K activity and so acts as a negative regulator of this pathway. In mESCs, PI3Ks were first implicated in the regulation of cell proliferation, initially owing to the fact that PTEN-null mESCs proliferate in reduced levels of serum and show a modestly enhanced growth rate (Sun et al., 1999). Direct inhibition of PI3K signalling with LY294002 (at a dose of 25 μM) was reported to lead to a decrease in mESC proliferation (Jirmanova et al., 2002), whereas deletion or pharmacological inhibition of mammalian target of rapamycin (mTOR), a downstream effector of PI3K signalling, also resulted in proliferative defects (Murakami et al., 2004). Furthermore, deletion of the mESC-specific Ras protein, ERas, leads to a reduction in proliferation of ESCs, which was rescued by expression of an activated form of p110α (Takahashi et al., 2003). By contrast, ESCs lacking the key PI3K target phosphoinositidedependent kinase-1 exhibit no changes in proliferation compared with wild-type cells (Williams et al., 2000). We, and others, have also implicated PI3Ks in the regulation of ESC self-renewal. We demonstrated that inhibition of PI3Ks with the broad-selectivity PI3K inhibitor LY294002 (at a dose of 5 μM) leads to a loss of self-renewal, without demonstrating appreciable effects on proliferation, and implicated class-IA PI3Ks in this response (Paling et al., 2004). Furthermore, we have shown that
Journal of Cell Science
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Journal of Cell Science 122 (13)
both LIF and BMP4 activate PI3K signalling in mESCs (Welham et al., 2007). Consistent with our observations, expression of a myristoylated, active form of protein kinase B (PKB, also known as Akt) promotes self-renewal of mESCs in the absence of LIF (Watanabe et al., 2006). PKB normally inhibits glycogen-synthase kinase-3 (GSK-3) activity; consequently, it is interesting to note that inhibition of GSK-3 with 6-bromoindirubin-3⬘-oxime (BIO) (Sato et al., 2004), or a series of bisindolylmaleimides (Bone et al., 2009), enhances mESC self-renewal. In addition, PKB was identified in a screen for activators of self-renewal (Pritsker et al., 2006), whereas a short-interfering RNA (siRNA)-based investigation demonstrated a requirement for the PKB-binding protein Tcl1 in the maintenance of mESC pluripotency (Ivanova et al., 2006). Mechanistically, we have recently reported that PI3K activity appears to be coupled with the regulation of expression of the homeodomain transcription factor Nanog (Storm et al., 2007), which, along with Oct4 and Sox2, forms the core transcriptional circuitry that is involved in the maintenance of pluripotency (Niwa, 2007). A requirement for PI3K signalling in the maintenance of pluripotency of human ESCs has also been reported (Armstrong et al., 2006; Pyle et al., 2006). Class-IA PI3Ks are expressed from the single-cell stage of mammalian development and their activity is required in order for development to proceed normally (Riley et al., 2005; Riley et al., 2006). Evidence supports the notion that individual isoforms play specific physiological roles in vivo. For example, mouse embryos lacking p110α exhibit proliferative defects and die at E9.5-E10.5 (Bi et al., 1999). Replacement of endogenous p110α, by knock-in of a kinase-dead version of p110α, also results in embryonic lethality of homozygous embryos, whereas heterozygotes are viable and fertile but exhibit disrupted insulin signalling (Foukas et al., 2006). Interestingly, embryos that are deficient in p110β perish at a very early stage of development, with a failure in blastocyst implantation (Bi et al., 2002). By contrast, mice that are deficient in active p110δ are viable and fertile but are subject to impaired immune responses (Okkenhaug et al., 2002). In addition to distinct physiological roles, there is evidence emerging that class-IA PI3K isoforms are also differentially regulated in distinct cellular contexts (Papakonstanti et al., 2008). To develop a greater understanding of the role of PI3Ks in the regulation of ESC behaviour, we investigated the contribution of individual isoforms to the control of mESC fate. Using inhibitors that are selective for different isoforms, complemented by siRNAmediated knockdown, our findings suggest that p110β plays the predominant role in the regulation of ESC self-renewal, whereas p110α appears to regulate ESC proliferation. Results PI3Ks have previously been implicated in the regulation of both ESC proliferation and self-renewal (see Introduction), and, in this study, we sought to investigate whether individual isoforms of the class-IA PI3K catalytic subunit are responsible for regulating distinct aspects of mESC behaviour. Recently, a number of cell-permeable, smallmolecule inhibitors have been characterised regarding their selectivity for the class-I PI3K family (Condliffe et al., 2005; Hayakawa et al., 2006; Hennessy et al., 2005; Jackson et al., 2005; Knight et al., 2006; Sadhu et al., 2003). We used a range of these small molecules, complemented with siRNA-mediated knockdown of individual isoforms, to investigate the roles of different isoforms of the classIA PI3K catalytic subunit in the regulation of ESC self-renewal.
PI3K p110β-mediated signalling regulates self-renewal
Mouse embryos homozygous for a partial deletion of Pik3cb, the gene encoding the p110β subunit, exhibit early embryonic lethality (E3.5) and cells cannot be derived from these blastocysts (Bi et al., 2002). mESCs are isolated from blastocysts at E3.5; hence, the role of p110β in mESCs was of particular interest. Treatment of mESCs, cultured in the presence LIF and serum, with the p110β-isoform selective inhibitor TGX-221 (Condliffe et al., 2005; Hennessy et al., 2005; Jackson et al., 2005) generated colonies displaying a more differentiated morphology and reduced staining for alkaline phosphatase, a marker of undifferentiated mESCs, compared with controls (Fig. 1A). Treatment with 25 nM or 50 nM TGX-221 led to significant decreases in the proportion of alkaline-phosphatasepositive colonies, as was observed following incubation with LY294002 (Fig. 1B), whereas higher doses did not lead to further decreases (supplementary material Fig. S1A). No significant change in the total number of colonies generated was observed. A lesspotent p110β inhibitor, TGX-121, had similar effects to TGX-221 (supplementary material Fig. S1B). These results, using two different p110β selective inhibitors, suggest that this isoform is involved in the regulation of mESC self-renewal. To further characterise the involvement of p110β in the regulation of mESC self-renewal, mESCs were pre-treated with TGX-221 prior to acute stimulation with LIF (Fig. 1C). Surprisingly, inhibition of p110β with TGX-221 did not affect basal or LIF-stimulated levels of phosphorylation of Ser473 of PKB (Fig. 1C, pPKB). Similarly, basal levels of both GSK-3α/β phosphorylation and ribosomal protein S6 phosphorylation (sites Ser20/9 and Ser 235/236, respectively) were unchanged by TGX-221 treatment. Whereas LIFstimulated phosphorylation of the MAPKs Erk1 and Erk2 was unaffected by TGX-221, basal levels of phosphorylated Erk2 were modestly enhanced in unstimulated cells following incubation with TGX-221. Pre-treatment with TGX-121 demonstrated similar effects on basal signalling and negligible consistent changes on LIFstimulated signalling (supplementary material Fig. S1C). We also examined the longer-term effects of inhibiting p110β on downstream signalling in mESCs cultured in the presence of LIF. As shown in Fig. 1D, treatment for 4 days with TGX-221 led to a decrease in PKB phosphorylation and a small decline in S6 phosphorylation, whereas levels of Erk phosphorylation were unaltered. p110β is required to maintain optimal mESC self-renewal
To complement the use of TGX-221, we further investigated the role of p110β in mESC self-renewal using siRNAs targeting Pik3cb to knockdown the expression of p110β. Both Smartpool siRNAs (Dharmacon), supplied as a pool of four single siRNAs, and individual Silencer Select siRNAs (Ambion, Applied Biosystems) were used to decrease Pik3cb gene expression. As a positive control, we used Smartpool siRNAs targeting Nanog, a homeodomain transcription factor known to play a key role in controlling ESC fate (Chambers et al., 2003; Chambers et al., 2007; Mitsui et al., 2003) that we have shown is downregulated following inhibition of PI3Ks over a 72-hour timecourse (Storm et al., 2007). Quantitative reverse transcriptase (RT)-PCR revealed significant knockdown of p110β expression using Smartpool Pik3cb siRNAs (Fig. 2A), with 50 nM being most effective. No effect on expression of Pik3ca or Pik3cd RNAs observed (supplementary material Fig. S2A,B). Owing to antibody-sensitivity issues and low expression levels, we were unable to consistently detect catalytic-isoform protein expression, so cannot entirely rule out compensation at the protein level. However, levels of p85 subunit were either unchanged
Distinct PI3K isoforms regulate ESC fate
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Journal of Cell Science
or slightly decreased following knockdown of p110β (Fig. 2Eii), the latter being consistent with the overall stoichiometry of p85:p110 being preserved. No decrease in the level of mRNA encoding p110β was detected following Nanog knockdown (Fig. 2A), whereas expression of Nanog mRNA was decreased following knockdown of p110β (Fig. 2B), consistent with our previous observation that PI3K inhibition leads to the downregulation of Nanog expression (Storm et al., 2007). On assessment of self-renewal, knockdown of p110β led to a reduction of approximately 50% in the proportion of alkaline-phosphatase-positive colonies (Fig. 2C). Furthermore, a significant reduction in the proportion of pure self-renewing alkaline-phosphatase-positive colonies was observed using 50 nM of p110β-targeting siRNA (Fig. 2D). Importantly, the levels of selfrenewal following siRNA-mediated knockdown of p110β were comparable to pharmacological inhibition of p110β with TGX-221 (Fig. 1B) and siRNA-mediated knockdown of Nanog (Fig. 2C). Consistent with a decrease in self-renewal, the level of Nanog protein was also reduced following knockdown of p110β expression (Fig. 2Ei); however, the effect of targeting p110β on Oct4 expression was not as clear, despite cells appearing morphologically differentiated. This is reminiscent of the effects we observed upon inhibition of PI3Ks with LY294002, in which Oct4 levels were maintained even though cells appear differentiated, possibly owing to the slower decline in Oct4 protein expression upon loss of pluripotency (Paling et al., 2004). For subsequent analyses, we used Nanog expression as an indicator of ESC pluripotency. We further verified the role of p110β in the regulation of mESC pluripotency using siRNAs that target different Pi3kcb exons to those targeted by the Smartpool siRNAs. As shown in Fig. 3A, the siRNAs Pik3cb s93107 and Pik3cb s93108 (which reduced p110β RNA expression between 40 and 60%, Fig. 3B, but did not affect levels of Pik3ca or Pik3cd RNAs) (supplementary material Fig. S2C,D) reduced the ability of mESCs to generate alkalinephosphatase-positive, self-renewing colonies (Fig. 3A). Consistent with the loss of alkaline-phosphatase staining, Nanog RNA (Fig. 3C) and protein (Fig. 3D) expression were also reduced following p110β knockdown, whereas levels of the p85 regulatory subunit were unchanged (Fig. 3D). Taken together, these results indicate that p110β plays a role in the maintenance of pluripotency of mESCs. When expression of p110β is reduced or when p110β is selectively inhibited, mESCs lose their alkaline-phosphatase expression and exhibit a decrease in Nanog RNA and protein expression, features that are consistent with a loss of self-renewal. p110δ and the control of mESC fate
Fig. 1. Pharmacological inhibition of p110β leads to a decrease in self-renewal of murine ESCs. (A) Murine ESCs (mESCs), cultured in the presence of LIF and serum, were treated for 4 days with LY294002 or TGX-221 at the doses shown or with vehicle alone (DMSO). Self-renewal was assessed by staining for alkaline-phosphatase activity and representative colonies are shown. Scale bars: 100 μm. (B) The average percentage of alkaline-phosphatase-positive, selfrenewing colonies in each condition are shown with s.e.m. (n=4). ANOVA and Dunnett’s post-hoc test were applied; ***P
