**8. Interactions between NFκB and STAT3 signaling**

#### **8.1. Synergistic NFκB and STAT3 signaling**

The NFκB and STAT3 pathways are closely interconnected in regulating immune responses [247, 248]. STAT3 activation itself induces further STAT3 phosphorylation. Un-phosphorylat‐ ed STAT3 that accumulates in the cell can bind to un-phosphorylated NFκB in competition with IκB. The resulting STAT3/NFκB dimer localizes to the nucleus to induce NFκB-dependent gene expression [249]. STAT3 associates with the p300/CBP (CREB-binding protein) coactivator enabling its histone acetyltranferase activity to open chromatin structures, which allows chromatin-modifying proteins to bind the DNA and activate gene transcription. [250, 251] Tyrosine-phosphorylated and acetylated STAT3 additionally binds to the NFκB precursor protein p100 and induces its processing to p52 by activation of IKKα. STAT3 then binds to the DNA-binding p52 complex to assist in the activation of target genes [252]. Both, the NFκB and STAT3 pathway synergize during terminal B-cell differentiation [253]. Phosho-p65/STAT3 dimers and phospho-STAT3/NFκB dimer complexes can bind to κB motifs. Also, phospho-STAT3 and phosho-p50 interact with each other. Soluble CD40L rapidly activates NFκB p65 and up-regulates IL10 receptors on the cell surface. This renders STAT3 more susceptible to IL-10 induced phosphorylation [161]. Macrophage activation is regulated by Toll-like recep‐ tors, JAK/STAT signaling and immunoreceptors that signal via ITAM motifs [254, 255]. These pathways have low activity levels under homeostatic conditions but are strongly activated during innate immune responses. ITAM-coupled receptors cooperate with TLRs in driving NFκB signaling and inflammation during infections, whereas extensive ITAM activation inhibits JAK/STAT signaling to limit the immune reaction [256, 257]. Pleiotropic cytokines like interferons and IL-6 regulate the balance of pro-and anti-inflammatory functions by activating variable levels of STAT1 and STAT3 [258].

### **8.2. NFκB and STAT3 synergies in stem cells**

**7.2. STAT3 is an epigenetic regulator**

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

stage [243-246].

Stat3 activation regulates major epigenetic events that induce an open-chromatin state during late-stage reprogramming to establish pluripotency [236-238]. For example, Stat3 signaling stimulates DNA methylations to silence lineage commitment genes and facilitates DNA demethylations to activate pluripotency-related genes [106, 239, 240]. Other chromatin modifications include histone acetylation and deacetylation, which are catalyzed by enzymes with histone acetyltransferase (HAT) or histone deacetylase (HDAC) activities. Histone acetylation is associated with an open chromatin state that allows active gene transcription. HDAC inhibitors are known to significantly improve the efficiency of iPSC generation by allowing promoter accessibility [128, 241, 242]. STAT3 suppresses HDAC expression and repressive chromatin regulators to establish an open-chromatin structure giving full access to transcriptional machineries. The key pluripotency factor Nanog cooperates with Stat3 to maintain ESC pluripotency [173]. Interestingly, HDAC inhibitors but not *NANOG* over-

expression rescues complete reprogramming in the presence of STAT3 inhibition.

**8. Interactions between NFκB and STAT3 signaling**

**8.1. Synergistic NFκB and STAT3 signaling**

Finally, DNA demethylation is regulated in mammalian cells by Tet proteins (tet methylcyto‐ sine dioxygenase), which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet1 suppresses ESC differentiation and Tet1 knockdown leads to defects in ESC selfrenewal. Tet1 up-regulation is positively regulated by Stat3 during the late-reprogramming

The NFκB and STAT3 pathways are closely interconnected in regulating immune responses [247, 248]. STAT3 activation itself induces further STAT3 phosphorylation. Un-phosphorylat‐ ed STAT3 that accumulates in the cell can bind to un-phosphorylated NFκB in competition with IκB. The resulting STAT3/NFκB dimer localizes to the nucleus to induce NFκB-dependent gene expression [249]. STAT3 associates with the p300/CBP (CREB-binding protein) coactivator enabling its histone acetyltranferase activity to open chromatin structures, which allows chromatin-modifying proteins to bind the DNA and activate gene transcription. [250, 251] Tyrosine-phosphorylated and acetylated STAT3 additionally binds to the NFκB precursor protein p100 and induces its processing to p52 by activation of IKKα. STAT3 then binds to the DNA-binding p52 complex to assist in the activation of target genes [252]. Both, the NFκB and STAT3 pathway synergize during terminal B-cell differentiation [253]. Phosho-p65/STAT3 dimers and phospho-STAT3/NFκB dimer complexes can bind to κB motifs. Also, phospho-STAT3 and phosho-p50 interact with each other. Soluble CD40L rapidly activates NFκB p65 and up-regulates IL10 receptors on the cell surface. This renders STAT3 more susceptible to IL-10 induced phosphorylation [161]. Macrophage activation is regulated by Toll-like recep‐ tors, JAK/STAT signaling and immunoreceptors that signal via ITAM motifs [254, 255]. These pathways have low activity levels under homeostatic conditions but are strongly activated during innate immune responses. ITAM-coupled receptors cooperate with TLRs in driving

NFκB and STAT3 are also part of an important stem cell pathway axis [259, 260]. A functional link between NANOG, NFκB and LIF/STAT3 signaling was shown in the maintenance of pluripotency [228]. Non-canonical NFκB signaling is activated by STAT3 through activation of IKKα and p100 processing [58]. Conversely, STAT3 inhibits TLR-induced canonical NFκB activity probably through up-regulated SOCS3. C-terminal binding of NANOG inhibits the pro-differentiation activities of canonical NFκB signaling and directly cooperates with STAT3 to maintain ESC pluripotency. NANOG and STAT3 bind to each other and synergistically activate STAT3-dependent promoters [106, 261].

The STAT3 pathway also interacts with many signaling pathways that are critically involved in the reprogramming process. For example, STAT3 signaling activates the MYC transcriptome and signals in loop with LIN28 [229]. LIN28 is expressed in undifferentiated hESC and is able to enhance the reprogramming efficiency of fibroblasts. It is down-regulated upon ESC differentiation [262-265]. Proto-oncogene tyrosine-protein kinase Src activation triggers an inflammatory response mediated by NFκB that directly activates *IL6* and *Lin28B* expression through a binding site in the first intron. IL6-mediated activation of STAT3 transcription is necessary for monocyte activation and tumorigenesis. IL6 itself further activates NFκB, thereby completing a positive NFκB-STAT3-IL6 feedback loop that links inflammation to cell trans‐ formation [229]. Constitutive STAT3 signaling maintains constitutive NFκB activity in tumors by inhibiting its nuclear export through p65 acetylation, although STAT3 signaling inhibits NFκB activation during normal immune responses [52].
