1. Introduction
The pioneering landmark, established by Takahashi and Yamanaka (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) in reprogramming somatic cells into induced pluripotent stem (iPS) cells using the four transcriptional factors of Oct4, Sox2, Klf4, and c-Myc, represents one of the most important paradigm shifts in current stem cell biology. This unprecedented discovery could potentially revolutionize regenerative medicine, cell-based therapy and personalized medicine. Despite recent great advancement in cell reprogramming, there are still considerable technical challenges to circumvent restrictions of applications of reprogramming technology (Kawamura et al., 2009; Saha and Jaenisch, 2009). The utilization of over-expressed transcriptional factors, which of many play oncogenic roles, during somatic reprogramming posts the risk of malignant transformation, thus, limiting its clinical applications. Moreover, the reprogramming process using these factors is still inefficient in some of cell types, and is not always successful in other kinds of cells (Kawamura et al., 2009; Marion et al., 2009; Menendez et al., 2012). Therefore, the underlying mechanisms for signaling control of these factors still need to be further explored.
Somatic cell reprogramming is a complicated cellular process that is controlled by many signaling networks. Accumulated evidence indicated that stemness transition can be detected in some tumor cells following the introduction of relevant signal stimulation, and cancer cells or differentiated cells can be changed into stem cell-like cells that go through less-differentiated stages (Chen et al., 2008; Fodde and Brabletz, 2007; Huang et al., 2009; Liu et al., 2009a). However, stemness transition may not lead to a full reprogramming of treated cells, which is determined by the delicate controls of signaling network activities in living cells. Interestingly, stemness transition may accompany epithelial-mesenchymal transition (EMT) events in cancer cells, and both programs are closely linked to the core stem cell gene network activities. Not surprisingly, multiple signaling pathways have been reported to be involved in EMT events and generation of stem cell-like cells. Wnt/β-catenin and TGF-β signaling are two potent inducers of EMT during embryonic development and cancer progression (Li et al., 2010; Mani et al., 2008; Morel et al., 2008; Scheel et al., 2011). Other involved pathways in these cellular activities may include BMP/Activin/Nodal, Notch, Hedgehog, Fibroblast growth factor signaling, and others (Chen et al., 2008; Huang et al., 2009; Kang and Massague, 2004; Natalwala et al., 2008; Thiery, 2002, 2003; Wu and Zhou, 2008).
The Wnt/β-catenin signaling pathway, highly conserved among various species and composed of a large family of proteins that control many biological properties (Fodde and Brabletz, 2007; Kikuchi et al., 2009; ten Berge et al., 2008b), may play a central role in the control of reprogramming and stemness process. This pathway includes more than two hundred genes and plays a critical role in modulating the delicate balance among stemness, proliferation, and differentiation in certain stem cell niches and tumor cells (Gu et al., 2010; Katoh, 2007; Lowry et al., 2005; Reya and Clevers, 2005). The established evidence reveals that various levels of Wnt/β-catenin signaling are likely to contribute to distinct cellular activities such as stemness transition, differentiation, carcinogenesis, and the EMT program. Therefore, the cellular activities and fate decisions are determined by this signaling activity in both dosage-dependent and tissue-dependent fashions (Anton et al., 2007; Kikuchi et al., 2009; Lluis et al., 2008; Reya and Clevers, 2005; Slack et al., 1995; Tapia and Scholer, 2010a; ten Berge et al., 2008a; Vermeulen et al., 2010). However, whether and how this signaling pathway has its direct influence on pluripotency gene networks and EMT events is largely unexplored.
As mentioned previously, cell fate decisions are controlled by both positive and negative forces in human cells. It has been well-established that tumor suppressor genes (TSGs) are important regulators to control cell proliferation, differentiation and cell death. Not surprisingly, these genes also play important roles in programming, reprogramming, and stemness transition in human cells. The well-studied TSGs, such as
Epigenetic regulation is one of the important mechanisms in the regulation of TSG activities. Recently, epigenetic modification has been shown to influence the reprogramming process, suggesting that many known TSGs may be involved in these cellular activities. Some reports illustrated that a dedifferentiation process of somatic cells to iPS cells involves dynamic epigenetic remodeling. In addition, there seem to be interactions between reprogramming transcription factors and epigenetic modifiers during these cellular activities (Takahashi, 2010).
In this chapter, the role of TSGs in cell reprogramming and stemness process, and regulation of these genes during stem cell renewal will be discussed, as described in Figure 1. We will review the role of TSG-mediated pathways and epigenetics as a barrier in cell fate determinations.
1.1. CDKN2A (p16INK4A and p14ARF) gene
The
Concurrently, Li et al. also worked on the role of
The epidermis is a tissue that undergoes continual and rapid self-renewal, and which is dependent on the presence of stem cells and transient amplifying keratinocytes. In primary human keratinocytes,
1.2. RB1 gene
Humans and other mammalians are unable to regenerate large portions of lost limbs or other internal organs after traumatic injury or surgical excisions. In contrast, lower vertebrates are able to regenerate entire limbs, the lens of the eye, and portions of the heart (Brockes and Kumar, 2008; Poss et al., 2002; Tanaka and Weidinger, 2008). The difference can be explained in part by the observation that inactivation of
The
The
1.3. p53 gene
Researchers have newly identified roles played by
During somatic cell reprogramming, the
The
The function of
Cicalese et al. suggested that the function of
The suppression of the reprogramming efficiency of iPS cells by
Another barrier affecting the reprogramming is the
In a recent report by Lee K.H. et al.,
Recently, researchers demonstrated that the p53-miR34-Wnt network is a determinant factor of dichotomy between stem cell properties and tumor progression. miR34, one of the direct downstream targets of
Chang et al suggested that
2. Signaling pathways involved in the reprogramming and stemness transition
2.1. TGF–β signaling pathway
TGF-β signaling pathways play multiple roles in regulating tumorigenesis and other cellular processes, including reprogramming, stemness transition, and EMT events. Many components in this signaling pathway were defined to participate in both oncogenic and tumor suppressive pathways in various tumors. This provides a complicated story for researchers to study the function of TGF-β signaling pathways in stem cells or reprogrammed cells. The ligands of the TGF-β family have multiple functions and can cause opposite effects in different cell types. The TGF-β can regulate cell proliferation, growth arrest, differentiation, survival, cell migration, and also the pluripotency of cells. In cancer, over-expression of TGFβ1 and deregulation of the TGF-β receptor type II (TGFBRII) were reported to associate with skin cancer tumorigenesis and invasiveness (Cui et al., 1996). However, the role of TGF-β signaling in regulating reprogramming is still not well-defined. In a previous report, TGF-β family ligands play an important role in reprogramming of somatic cells into iPS cells, regulating ESCs self-renewal, pluripotency maintenance, and controlling differentiation.
TGF-β signalling may have the ability to induce reprogramming of somatic cells into iPS cells. Treatment of TGF-β/activin inhibitor in partially reprogrammed iPS cells can induce Nanog expression (Ichida et al., 2009; Maherali and Hochedlinger, 2009). Furthermore, the functional role of TGF-β in regulating the reprogramming was defined by utilizing chemical TGF-β inhibitors. Interestingly, inhibition of TGF-β signaling can enhance the mouse fibroblast reprogramming efficiency. A substitute of Sox2 (E-616452) and TGFBR1 kinase (SB-431542) inhibitor, were reported to replace the function of Sox2 in MEFs with Oct4, Klf4, and c-Myc expression (Ichida et al., 2009; Maherali and Hochedlinger, 2009). These results suggest the important roles TGF-β plays in the controlling reprogramming process.
Maintenance of the pluripotencies and self renewal properties are important for both ESCs and iPS cells.The canonical TGF-β signaling pathway may play important regulatory roles in ESCs maintenance and generation of pluripotency. BMP4 together with the LIF protein can induce Oct4 expression (Ying et al., 2003). The BMP activated Smad signaling to support self-renewal properties of stem cells. The inhibition of Smad activities by the Smad6 and Smad7 in the ES cells induced smaller and fewer ES cell colon formation (Ying et al., 2003). Secretion of BMP4 by the feeder cells is necessary for ES cell self-renewal (Ying et al., 2003). Inhibition of the Erk and p38 MAPK pathways can further enhance the BMP4-associated effect on self-renewal of mouse ESCs (Qi et al., 2004). Besides this, bFGF (basic fibroblast growth factor) and activin are also important to maintain the pluripotency in human ESCs (Greber et al., 2010; James et al., 2005). The TGF-β signalling may play multifunctional roles in regulating pluripotency of cells. Smad1 was reported to suppress the expression of Nanog by inhibiting its promoter activities (Jiang and Ng, 2008; Xu et al., 2008). The Smad proteins were reported to bind directly to Nanog promoter (Xu et al., 2008) and this is the major mechanism for Smad proteins to regulate Nanog expression. These results suggest the multiple roles of TGF-β signaling in the regulation of stem cell renewal.
Furthermore, TGF-β also plays a role to control the differentiation of ESCs. One of the TGF-β family members, BMP4, was reported to associate with induction of inhibitor of differentiation (
Collaborating with Wnt signaling, TGF-β signaling is also involved in the EMT program and both pathways are regarded as the axis of EMT in breast cells (Scheel et al., 2011). The hypothesis of these two pathways linked to the stem cell networks and TSG pathways is presented in Figure 2.
2.2.Wnt pathway
Cellular reprogramming can be achieved by overexpression of defined transcription factors in somatic cells (Ichida et al., 2009; Takahashi et al., 2007). However, the underlying mechanism of signaling activities that regulated these factors are not fully understood now. Overexpression for certain genes may not be suitable for all pathways, such as
The lower levels of Wnt signaling play a critical role in the control of development of several types of tissues through a dosage-dependent manner, as reported in crypt progenitor cells (Batlle et al., 2002; Korinek et al., 1998), hair follicles (Lowry et al., 2005), and hematopoietic stem cells (Luis et al., 2011). Taken together, observations from both
More and more evidence revealed that Wnt signaling plays important roles in maintenance of pluripotency in ESCs and cell self-renewal (Cole et al., 2008; Lluis et al., 2008; Macarthur et al., 2009; Marson et al., 2008; Takao et al., 2007). For example, expression of
Interestingly, previous study demonstrated that Wnt3a can also stimulate human ES cell differentiation, rather than only regulate human ES cell proliferation. The canonical Wnt signaling levels are minimal in the undifferentiated human ES cells but greatly increase after Wnt3a treatment and induce differentiation (Dravid et al., 2005). Dramatic increase of reprogrammed cell numbers can be observed when ES cells, which have a low level of nuclear β-catenin, are fused with neural stem cells. This is mainly due to the low nuclear β-catenin level being able to protect fused cells from apoptosis (Lluis et al., 2010), suggests the importance of β-catenin levels in the regulation of stem cell reprogramming. This finding may help to explain the balance between the maintenance of pluripotency of stem cells and apoptosis, as excess β-catenin can induce p53 expression (Damalas et al., 1999), which was found to induce apoptosis in stem cells to maintain genome integrity. The p53 protein was reported to be a transcription regulator of the Wnt signaling and it bound on the promoter regions of some Wnt signaling members for a general stress response in the mouse ES cells (Lee et al., 2010), which may provide a feedback mechanism to control the deregulation of the β-catenin during the reprogramming process.
It should be noted that inappropriate activation of components of this signaling pathway have been observed in many human cancers and differentiated stem cells, in which the high levels of
3. Possible mechanisms to regulate TSGs expression in reprogramming
It is well-accepted now that epigenetic regulations are important events to control gene expression in human cells. Promoter hypermethylation and histone modification are two major events to regulate gene expression in various human tumors. The
4. Conclusions
The known and unknown TSGs are the important participators in the regulation of cell reprogramming and stemness transition. These genes are components of various signaling pathways, and play different roles in maintaining cell pluripotency, regulating cell differentiation and proliferation, cell cycle control, apoptosis, and other cell fate decisions. These genes controlling cellular activities act in a time-dependent or a dosage-dependent manner in various tissues. Although detailed underlying mechanisms are not fully clear now, more and more evidence indicates that some TSG signaling activities are determinant forces in important cellular processes, including cell reprogramming. A proposed hypothesis illustrates this in Figure 3. Understanding of the delicate control of these signaling networks in living cells will provide more insight in reprogramming studies and regenerative medicine.
Acknowledgments
The University of Hong Kong Seed Funding Program for Basic Research (Project Codes: 201007159005 and 201111159142) to YC.References
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