3. Results
4.4 Pelota is required for self-renewal and differentiation of embryonic stem cells
Numerous studies have attempted to uncover the basic biology of the ESC and delineate mechanisms of pluripotency. However, many interesting challenges must be met in order to further understand the basic regulation of these cells. The current study introduced Pelo as a master regulator for a coordinated network of signaling pathways in stem cell pluripotency and differentiation. Here we report that Pelo-null ESCs maintain the pluripotency state under differentiating culture conditions.
Previous studies in our group showed that Pelo deficiency did not markedly affect the self-renewal of ESCs or their capacity to differentiate in teratoma assays. However, their differentiation into ExEn in the embryoid bodies was severely compromised. The failure of Pelo-null ESCs to silence the pluripotency-related genes during the EB formation was attributed to failed signals from surrounding ExEn that induce the differentiation program (Nyamsuren et al.
2014). That was also the case in Dido3-deficient EB where the loss of Dido3 expression delays primitive endoderm formation and maintains pluripotency (Fütterer et al. 2012).
Using defined monolayer culture conditions, we demonstrated that Pelo-null ESCs retain their capacity to form undifferentiated dome shaped colonies and maintain expression of pluripotent markers under differentiation conditions. Our analysis of PeloΔ/− ESCs revealed high levels of pAKT compared to control PeloF/−. Because previous studies have demonstrated the critical roles of PI3K/AKT in the self-renewal of ESCs (Takahashi et al. 2005;McLean 2007), we assumed that activation of AKT signaling supports the pluripotency of mutant ES cells and hereby affect the differentiation. However, addition of Wortaminin, a PI3K inhibitor, in cultures did not induce differentiation of the mutant ESCs judged by the ESCs morphology and with the persistent
4 Discussion 61 expression of the pluripotent marker Oct4. These findings led us to determine other factors apart from PI3K/AKT that can maintain the ESCs pluripotency.
On the other hand addition of Retonic acid induced complete differentiation of Pelo-mutant ESCs.RA is known in the literature as a potent differentiation agent. In contrast, retinol, which is the precursor of RA, was found to maintain the ESC self-renewal by elevating the expression of Nanog (Chen et al. 2007). Later, it was described that retinol maintains the pluripotency of ES cells through activation of PI3K/AKT in an insulin-like growth factor I (IGF-1) receptor/insulin receptor substrate 1(IRS-1)-dependent manner (Chen and Khillan 2010). Analysis of ESCs revealed that these cells are unable to metabolize retinol to retinoic acid due to lack of retinol metabolizing machinery in the ESCs. This result was further supported by the observations that addition of retinoic acid leads to their complete differentiation (Chen and Khillan 2008). In absence of LIF, ESCs are capable of synthesizing active retinoids (hydroxyretinol and 4-oxoretinol) from retinol. These retinoids can activate transcription through the retinoic acid receptors (RARs), leading to ESCs differentiation without forming retinoic acid (Lane et al.
1999). Based on these information, we speculate that PeloΔ/− ESCs fail to metabolize retinol to active retinoids in differentiating culture conditions. Therefore, accumulation of retinol in Pelo-mutant ESCs may be responsible for maintaining the ESCs pluripotency. To prove this speculation we checked the expression of stra8 gene, as its promoter region contains RA response elements, suggesting that RA could be turning on this gene directly (Zhou et al. 2008).
Expression analysis of stra8 mRNA in PeloΔ/− ESCs were significantly lower than that in control ESCs in undifferentiated state as well as after inducing differentiation. These results confirm our assumption that retinol metabolism in Pelo-mutant ESCs is affected.
4 Discussion 62 Recent study by Obrochta et al. (2015) linked the activity of Foxo1to retinol metabolism.
Thus, cis-acting elements of Foxo1 have been identified in the promoters of genes associated with retinoid metabolism, including retinol dehydrogenases (RDH). The authors showed that enhancing the activity of PI3K/AKT signaling pathway decreases the transcription activity of Foxo1, followed by repression of RDH transcription, leading to decrease in retinoic acid synthesis from retinol.
Although AKT activity enhance the nuclear export of phosphorylated FOXO proteins and hereby inhibit their transcriptional activity (Figure 20) (Van den Berg & Burgering 2011), our protein blot analysis revealed a significant increase in the levels of nuclear Foxo1in Pelo-mutant ESCs. This result corresponds to Zhang et al. (2011) findings, which highlighted the essential role of Foxo1 in maintaining ESCs pluripotency through direct control of Oct4 and Sox2 expression. Despite enhanced activity of PI3K\AKT signaling pathway, Foxo1 was nuclear and transcriptionally active concluding that PI3K\AKT signaling pathway is not the predominant regulator of Foxo1 in ESCs. Other study presumed that Foxo1 inhibition by pAKT is overridden by other signaling pathways (Yamagata et al. 2008).
Figure 20. Model of FOXO regulation by PI3K\AKT signaling pathway. Phosphorylated AKT phosphorylates FOXO transcription factors leading to nuclear export and inactivation of transcriptional activity by FOXO.
4 Discussion 63 Therefore, we were interested to have a more detailed insight about the effect of increased transcription activity of Foxo1 in Pelo-null ESCs. The association of Foxo proteins with β-catenin was elucidated by Almeida et al. (2007). The same group in 2013 provided genetic evidence that FOXOs do indeed bind with β-catenin diverting the limited pool of β-catenin from Wnt/TCF to FOXO mediated transcription resulting in attenuation of β-catenin/TCF transcription in murine osteoblast progenitors (Figure 21). Generally Wnt binding to the Frizzled-LRP5/6 receptor complex halting the proteolytic destruction of β-catenin, which then moves into the nucleus where it binds to and activates the T cell factor (TCF) lymphoid-enhancer binding factor (LEF) transcription factors and regulates the expression of Wnt-target genes (Figure 21) (Rodda and McMahon 2006). The binding of FOXOs to β-catenin mechanism, restrain the proliferative effects of Wnt signaling.Suppression of the proliferative β-catenin/TCF transcriptional signal by FOXOs is probably a defense mechanism against diverse stress stimuli in many tissues, as evidenced by the increased association of β-catenin with FOXOs in H2O2-treated bone and colon cancer cells (Hoogeboom et al. 2008).
4 Discussion 64
Figure 21. Model for binding of FOXOs to β-catenin in ESCs. In presence of Pelo (right panel) binding of Wnt ligand to a Frizzled/LRP-5/6 receptor complex (right panel) leads to stabilization of hypophosphorylated β-catenin, which interacts with TCF/LEF proteins in the nucleus and regulates Wnt-target genes transcription. In the absence of Pelo (left panel), β-catenin tends to bind to nuclear Foxo1 diverting β-catenin from β-catenin/TCF target genes transcription (Adapted from Sun 2010).
In Regards to these literatures and to our results that showed increased levels of Foxo1 in Pelo-null ESCs. We hypothesized that in ESCs upon Pelo deletion Foxo1 diverts β-catenin from TCF/LEF leading to attenuation of β-catenin/TCF transcription activity and altering Foxo-mediated transcription (Figure 22). This hypothesis is supported by our pervious results that related the decreased proliferative capacity of Pelo-mutant ESCs to low levels of c-Myc, a candidate for β-catenin/TCF transcription activity, as well as by the significant decrease of TCF/LEF transcription activity in Pelo-mutant ESCs as judged by TOP/FOP-FlASH reporter assay.
4 Discussion 65
Figure 22. Model of Mechanisms that contribute to Pelo-null ESCs pluripotency. Pelo deletion increases AKT activity, Foxo1escapes the inhibitory effect of pAKT and remain nuclear. Activity of Foxo1 maintains the ESCs pleuripotency and decreases β-catenin/TCF-mediated transcription. This disrupts the RDH expression and thereby blocks the metabolism of retinol to active retinoids, which induce ESCs differentiation in absence of LIF. Retinol also contributes to ESCs pluripotency and lack of differentiation. (Adapted from Obrochta et al. 2015).