**3. Functional role of NFκB signaling in stem cells**

**2.2. The non-canonical NFκB signaling pathway**

6 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

pathway requires TRAF2 recruitment [68].

**2.4. NFκB subunit functions**

NFκB proteins like p65 [77-81].

**2.3. CD40 stimulates both NFκB pathway components**

Non-canonical NFκB signaling is stimulated via the NFκB-inducing kinase (NIK), which leads to phosphorylation of the p100 precursor protein and generation of the p52:RelB dimer that translocates to the nucleus to activate gene transcription. This pathway is uniquely dependent on steady state levels of *NIK* expression, which are controlled under normal conditions through TRAF3-directed ubiquitination and proteasomal degradation. Non-canonical NFκB signaling is slow but persistent and requires de novo NIK protein synthesis and NIK stabilization [58]. It is activated by receptors that belong to the TNFR (tumor necrosis factor receptor) super‐ family like BAFF (B-cell-activating factor), CD40 or lymphotoxin β-receptor (LTβR) [59-62].

The common feature of these receptors is the possession of a TRAF-binding motif, which recruits TRAF members (e.g., TRAF2 and TRAF3) during ligand ligation [63, 64]. Receptor recruitment of TRAF members triggers their degradation, and leads to NIK activation and p100 processing [65]. Additionally, BAFF is an important component of pluripotency-supporting growth media for the culture of ESC and a regulator of B-cell maturation [66]. It predominantly activates non-canonical NFκB signaling due to its possession of an atypical TRAF-binding sequence, which interacts only with TRAF3 but not with TRAF2 [67]. TRAF3 degradation is sufficient to trigger non-canonical NFκB signaling, whereby activation of the canonical NFκB

Another receptor associated with NFκB signaling is CD40, which is expressed on various cell types including B cells and monocytes. The CD40 receptor interacts with its ligand CD40L, which is primarily expressed on activated T cells. This signaling is majorly involved in B-cell activation, dendritic cell maturation, antigen presentation and acts as a co-stimulatory pathway of T-cells [69]. Upon ligation by CD40L, CD40 targets both the canonical and noncanonical NFκB pathways via proteolysis of TRAF2 and TRAF3 [70-72]. Non-canonical NFκB signaling regulates hematopoietic stem cell self-renewal via regulating their interactions with the microenvironment [73]. The deregulation of non-canonical hematopoietic NFκB signaling

is associated with auto-immunity, inflammation and lymphoid malignancies [58, 74].

A third NFκB signaling pathway is activated following response to DNA damage that results in IκB degradation independent of IKK. This results in dimerization of free NFκB subunits that are mobilized similarly to canonical NFκB signaling [47]. Unlike RelA, RelB, and c-Rel, the p50 and p52 NFκB subunits do not contain transactivation domains in their C-terminus. Never‐ theless, the p50 and p52 NFκB members play critical roles in modulating the specificity of NFκB functions and form heterodimers with RelA, RelB, or c-Rel [75]. Cell contact-dependent signals are crucial during immune responses and can be mediated through NFκB signaling [76]. This can be augmented by co-stimulatory signals like CD40 or CD28 that directly bind to

#### **3.1. Differential roles of canonical and non-canonical NFκB signaling in embryonic stem cells**

TLR activation is not only important for mediating innate immune responses, but also for stem cell differentiation. For example, hESC are characterized by the expression of pluripotency genes and markers such as OCT4, NANOG, alkaline phosphatase (AP) and telomerase [82-86]. NFκB signaling has been demonstrated to be crucial for maintaining ESC pluripotency and viability, and drives lineage-specific differentiation [87, 88]. A balance of canonical and noncanonical NFκB signaling regulates these opposing functions; non-canonical pathway signaling maintains hESC pluripotency, and canonical pathway signaling regulates hESC viability and differentiation [89, 90]. For example, non-canonical NFκB signaling has to be silenced during cell differentiation, which allows this pathway to act like a switch between hESC self-renewal and differentiation. RelB positively regulates several key pluripotency markers and represses lineage markers by direct binding to their regulatory units. RelB downregulation reduces the expression of pluripotency genes like *SOX2* and induces differentiationassociated genes like *BRACHYURY* (mesodermal marker), *CDX2* (trophoectodermal marker) and *GATA6* (endodermal marker) [89].

#### **3.2. Canonical NFκB signaling in hematopoietic stem cells**

RelB/p52 signaling also positively regulates hematopoietic stem-progenitor cell (HSPC) selfrenewal in response to cytokines (e.g., TPO and SCF) and maintains osteoblast niches and the bone marrow stromal cell population. It negatively regulates HSPC lineage commitment through cytokine down-regulation in the bone marrow microenvironment, although it is able to direct early HSC commitment to the myeloid lineage [73, 91].

Canonical p65 signaling also regulates hematopoietic stem cell functions and lineage commit‐ ment by controlling key factors involved in hematopoietic cell fate [92-94]. Canonical NFκB signaling is positively regulated by Notch1, which facilitates nuclear retention of NFκB proteins and promotes self-renewal [95-98]. FGF2 (fibroblast growth factor 2) is important for hESC self-renewal and preserves the long-term repopulating ability of HSPC through NFκB activation [99-102]. Deletion of p65, p52 and RelB dramatically decreases HSC differentiation, function and leads to extramedullary hematopoiesis [103]. NFκB pathway components and FGF4 are highly expressed in CD34+ HSPC from cord blood, where they regulate clonogenicity. Nuclear p65 can be detected in 90% CB-derived CD34+ cells but only in 50% BM-derived CD34+ cells [104]. The important role of NFκB in regulating myeloid cell lineage development has been most potently revealed via genetic deletion of IKKβ, IκBα, and RelB, which resulted in granulocytosis, splenomegaly and impaired immune responses [73, 103].

#### **3.3. Canonical NFκB signaling during ESC differentiation**

Canonical NFκB signaling is very low in the undifferentiated pluripotent state, where it maintains hESC viability. However, it strongly increases during lineage-specific differentia‐ tion of pluripotent stem cells. p65 binds to the regulatory regions of similar differentiation genes as RelB with opposing effects on their activation or silencing. It regulates cell prolifera‐ tion by direct binding to the CYCLIN D1 promoter [89]. There are different levels of inhibiting canonical NFκB signaling: first, p65 translational repression by the microRNA cluster miR-290 to maintain low p65 protein amounts and second, the inhibition of translated p65 by physical interaction with NANOG. Similarly, *OCT4* expression is reversely correlated with canonical NFκB signaling [105]. In contrast to most observations in mouse ESC, NFκB probably plays a more important role in the maintenance of human ESC pluripotency [106]. Finally, active TLRs are expressed on embryonic, hematopoietic and mesenchymal stem cells (MSC), thus impli‐ cating their roles in a variety of stem cell types [107-110].

not observed with TLR3 activation alone but only in the presence of the reprogramming factors. Although TLR3 by itself promotes an open chromatin configuration, the reprogram‐ ming proteins are likely necessary to direct the epigenetic modifiers to the appropriate promoter sequences [137]. Lee *et al.* described the potential of inflammatory pathways to

The Role of an NFκB-STAT3 Signaling Axis in Regulating the Induction and Maintenance of the Pluripotent State

http://dx.doi.org/10.5772/57602

9

The JAK/STAT pathway (Janus kinase/signal transducer and activator of transcription) integrates a complex network of exterior signals into the cell, and can be activated by a variety of ligands and their receptors [139]. These receptors are associated with a JAK tyrosine kinase at their cytoplasmic domain. The JAK family consists of the four members JAK1, JAK2, JAK3 and TYK2 [140, 141]. Many cytokines and growth factors signal through this pathway to regulate immune responses, cell proliferation, differentiation and apoptosis [142-146]. Ligand binding induces the multimerization of gp130 receptor subunits, which brings two JAKs close to each other inducing trans-phosphorylation. Such activated JAKs phosphorylate their receptor at the C-terminus and the transcription factor STAT at tyrosine residues. This allows STAT dimerization and their nuclear translocation to induce target gene transcription. [147, 148] STAT3 acetylation is critical for stable dimer formation and DNA binding [149]. From the 7 mammalian STATs, STAT3 and STAT5 are expressed in many cell types, are activated by a plethora of cytokines and growth factors, and integrate complex biological signals [150, 151]. The other STAT proteins mainly play specific roles in the immune response to bacterial and viral infections. STAT3 is an acute phase protein with important functions during immediate immune reactions [152-154]. STAT3 can be recruited by receptor tyrosine kinases that harbor a common STAT3 binding motif in their cytoplasmic domain (e.g., GCSF (granulocyte colonystimulating factor), LIF (leukemia inhibitory factor), EGF (epidermal growth factor), PDGF (platelet-derived growth factor), interferons (IFNγ) and interleukins (IL-6, IL-10)) [155-158]. Many cytokines signal through IL-10/STAT3 to achieve an immunosuppressive function or anti-apoptotic effect [159, 160]. IL-10 is also required during terminal differentiation of immunoglobulins [161]. STAT3 can be phosphorylated at tyrosine or serine residues. The phosphorylation site can play distinct roles in the regulation of downstream gene transcription [162]. Stat3-deficient mice die during early embryogenesis due to Stat3 requirement for the

Negative feedback regulation of the JAK/STAT circuitry is mediated by the SOCS family of target genes (suppressors of cytokine signaling) in a way that activated STAT induces SOCS transcription [164, 165]. SOCS proteins can bind to phosphorylated JAKs as a pseudo-substrate to inhibit JAK kinase activity and turn off the pathway [166, 167]. SOCS are negative regulators of the immune response [168, 169]. A small peptide antagonist of SOCS1 was shown to bind to the activation loop of JAK2 leading to constitutive STAT activation and TLR3 induction. This boosts the immune system to exert broad antiviral activities [170]. The JAK/STAT pathway also interacts with many other signaling pathways in a complex manner to regulate cell

facilitate the induction of pluripotency as 'transflammation' [40, 138].

**5. Overview of the JAK/STAT pathway**

self-renewal of ESC [163].

homeostasis and immune reactions [149, 171].
