Sox2 is an important regulator of ES cell and NPC self-renewal [27
], and it is regulated as a part of the core transcriptional circuitry controlling ES cell self-renewal [35
]. However, very little is known about how it is regulated by upstream signaling pathways. One upstream pathway important for the maintenance of numerous stem cell populations is the PI3K/Akt pathway [14
]; however, it is unknown how or even whether these two critical elements of stem cell regulatory machinery interact. Here we demonstrate that Akt promotes Sox2 protein expression; however, increased Sox2 expression did not increase NPC proliferation. Given our previous work showing that Akt drives NPC proliferation and inhibits differentiation [21
], this indicates that Akt is an important regulator of both proliferation and self-renewal in NPCs, but via different downstream pathways.
Akt overexpression completely rescued differentiation-induced loss of Sox2 expression (). Additionally, Akt slightly increased Sox2 expression in cells cultured under proliferative conditions, though this was not a large effect, likely because the cells are already exposed to a strong, FGF-2-mediated mitogenic signal. Interestingly, though Akt inhibition led to a statistically significant reduction in Sox2 expression in cells cultured with FGF-2, the effect was not strong, indicating that Akt is not the only mediator of FGF-2-induced Sox2 expression. There is some evidence that Wnt/β-catenin signaling is important for NPC self-renewal [49
]; however, a link between the Wnt signal and Sox2 expression was not established in either of these studies. Additional studies may reveal interesting links between Sox2 and other signaling pathways.
It is well known that Akt promotes protein translation by activating its downstream effector mTORC1 [51
]. However, one interesting result of our work is that Akt rescues Sox2 transcript levels in cells cultured under differentiating conditions (). Additionally, Akt inhibition did not affect Sox2 protein levels in a constitutively expressing Sox2 mutant (), where the exogenous Sox2 cDNA did not contain untranslated regions, indicating that Akt inhibition did not modify protein levels by increasing protein degradation. Overall, these results indicate that Akt activation may modulate sox2
transcription. One potential mechanism for this is through stabilization of c-Myc [52
], a transcription factor shown to modulate Sox2 expression in mouse ES cells [48
]. Further experimentation is required to determine the precise effects of Akt on Sox2 transcription and translation in addition to any potential intermediate steps between Akt activity and Sox2 expression.
In addition to its role in NPCs, Akt is also known to promote the proliferation and self-renewal of ES cells [14
]; therefore, investigating the effects of Akt on Sox2 expression in ES cells could have important implications for the development of more efficient ES cell culture systems and perhaps eventually ES cell-based therapies. Further, enhancement of Akt signaling may conceivably improve the efficiency of reprogramming and the generation of iPS cells (for a review of other modulators of reprogramming, see [54
]). Recent work has demonstrated that pharmacological inhibition of ERK and GSK3β can generate iPS cells from neural stem cells transduced with only two of the four Yamanaka factors [55
]. Other studies have used GSK3β inhibition along with ERK and ALK5 inhibition to promote reprogramming [56
]. Because GSK3β activity is directly inhibited by Akt, these findings, taken together with our results, may indicate that modulation of the Akt pathway could potentially improve reprogramming efficiencies by upregulating expression of endogenous Sox2.
Our Sox2-overexpressing NPCs had a decreased ability to differentiate as measured by quantitative RT-PCR (). In particular, upon exposing cells to conditions strongly favoring astrocytic and neuronal differentiation, Sox2-overexpressing cells had ~10
lower expression of the astrocytic marker GFAP and ~2
lower expression of the neuronal marker β-tubulin III. These results, similar to those seen in NPCs overexpressing Akt [21
], further support observations in chick embryos constitutively expressing Sox2, which also experienced impaired neuronal differentiation [28
]. Notably, however, Sox2 is still required for proper neuronal differentiation and development. Cells derived from Sox2 hypomorphic mice and cultured in vitro generated abundant β-tubulin III-positive cells, but those cells failed to mature [31
]. Therefore, a minimal threshold amount of Sox2 appears to be required for proper neuronal development.
Although Sox2 overexpression inhibited differentiation, it did not increase the number of viable NPCs or their proliferation (). This is consistent with the observation that Sox2-positive cells within the subgranular zone of the hippocampus are able to remain quiescent for extended periods [27
]. Additionally, it indicates that although Sox2 is important for NPC self-renewal, it is not sufficient for their proliferation. This is not the case in other cell types. Sox2 promotes proliferation of tracheal and airway stem cells [57
], and as an oncogene it is necessary for the proliferation and transformation of lung or esophageal squamous cell carcinomas [58
In summary, this work demonstrates that in addition to being important for NPC proliferation [21
], Akt is also an important promoter of Sox2 expression, thereby maintaining NPC multipotency. Importantly, however, Sox2 itself does not promote adult NPC proliferation, but it does inhibit neuronal and glial differentiation. Therefore, Akt is a key, parallel regulator of NPC proliferation and self-renewal. It may also play these roles in maintaining or even generating other stem cell types, such as ES and iPS cells.