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Atypical TG2 expression activates the NF-κB pathway
inflammatory proteins, which were expressed in a TG2- quantity-dependent manner (Figure 5A). Secretory pro- teins not targeting NF-κB genes but expressed in a TG2- dependent manner also included NC9 sensitive secretory proteins (Figure 5C).
and considerably increases cytosolic levels (Figure 6D2, upper left, red bar). In addition, both phospho-p65/RelA and p50 showed similar distribution patterns upon NC9 treatment (Figure 6D2, lower left and right, black and red bars).
Discussion
Treatment of APL principally comprises ATRA-based therapy, which leads to terminal differentiation of APL cells to neutrophil granulocytes. In adverse events, this treatment is frequently associated with severe hyper- inflammatory reactions, leading to organ infiltration of differentiating APL cells.
The pathogenic processes of the hyper-inflammatory cascade in DS are not completely understood. At least two different mechanisms appear to play important roles in DS development: differentiation of APL cells with cytokine release and adhesion, and migration of differentiated APL cells to different organs. ATRA-induced differentiation of APL cells is associated with elevated expression of inflam- matory cytokines and adhesion molecules called integrins. Activation of neutrophils is elicited by mediators, includ- ing chemokines, selectins, and integrin-mediated outside- in signaling. Locally produced chemokines mediate a sequence of events leading to extravasation of leukocytes at the inflammatory site. Neutrophils express different chemokine receptors, including CXCR2, whose most potent ligand is IL8 (CXCL8).31 Shibakura et al. first demonstrated that ATRA could induce synthesis and secretion of IL8 in NB4 cells. Thereafter, it was shown that not only do ATRA-treated APL cells express IL8 mRNA in ex vivo cell cultures, but also IL8, as well as chemokines such as MCP-1 (CCL2), MIP-1a (CCL3), and MIP-1b (CCL4), are present in the serum of APL patients who developed DS during ATRA treatment.32 It has also become obvious that NB4 cells secrete IL8 constitutively, which is further enhanced during ATRA-induced differen- tiation, and cannot be inhibited by dexamethasone.33 In the presence of IL8, neutrophil granulocytes increase the amounts of CD11b integrin receptor along with its bind- ing activity and the amounts of CD11c on their cell sur- faces.34,35 Simultaneously, secreted TNF-α also stimulates neutrophils, triggering activation of CD11c.36 Since 1998, CD11b has been used as a surface marker of granulocytic cell differentiation of NB4 cells in published research, in spite of the fact that CD11b is mainly stored intracellular- ly in both specific and gelatinase granules and secretory vesicles under normal physiological conditions.37-39 Our observations confirm that during ATRA-induced differen- tiation of NB4 cell lines, CD11b and CD11c receptors were translocated in large quantities to the cell surface, and show that the amount of surface CD11b cannot be further increased by chemoattractant or phorbol-esters (Figure 3A-C and Online Supplementary Figure S5). Nevertheless, Nupponen et al. found that while “neu- trophil CD11b expression and circulating interleukin-8 represent a diagnostic marker for early-onset neonatal sep- sis,” CD11c is also a potential diagnostic biomarker for sepsis and systemic inflammation.40 An antibody detecting activation-associated CD11c revealed gradually increasing cell-surface receptor expression in an activated state, with a high affinity for ligands on ATRA-treated NB4 cell lines (Figure 2C). In this manner, inflammatory cytokines could
TG2 contributes to expression and nuclear translocation of NF-κB, which is significantly reduced by the TG2 inhibitor NC9
NF-κB promoter-driven luciferase reporter activity and the extent of the expression of several NF-κB-controlled inflammatory biomarkers suggested that NF-κB transcrip- tional activity might depend on the magnitude of the expression of TG2 (Figures 4D and 5A). Our starting observation was that NB4 cells treated with ATRA plus NC9 expressed lower amounts of TG2 protein than cells treated only with ATRA (Figures 1B and 6B). First, we examined the impact of NC9 on the transcription level of TG2 mRNA and found that it did not change. The amount of TG2 protein was affected by NC9, while TG2 mRNA levels remained unaffected suggesting that the proteolytic degradation of TG2 might be increased by NC9. To test this hypothesis, 11-day differentiated NB4 cells were treated for 3 h with MG132 proteasome inhibitor. Western blot analysis of TG2 expression confirmed the enhanced proteolytic degradation of TG2 during the NC9 treatment. In the presence of NC9, the amount of TG2 decreased to approximately one-tenth that measured in cells treated with ATRA alone (Figure 6E1 and E2).
Next, we investigated the effect of the lower TG2 pro- tein levels on the expression of p65/RelA, p50, and phos- pho(Ser536)-p65/RelA components of NF-κB and found that the absence, or reduced gene expression, or suppres- sion of TG2 by NC9 significantly restricted the expression of p65/RelA, phospho-p65/RelA and p50 proteins in NB4- WT, TG2-C, TG2-KD, TG2-ha, and TG2-KO cells (Figure 6C1 and C2, upper and lower, left and right panels). However, we have long been aware that in the ATRA-dif- ferentiated NB4-WT cells, significant amounts of TG2 protein translocate to the nucleus during differentiation.20 To demonstrate the role of nuclear TG2, after 11 days of differentiation, the cytosolic and nuclear fractions of NB4- WT were separated and analyzed for the expression of TG2, p65/RelA, phospho-p65/RelA, and p50 (Figure 6D1 and D2). In the differentiated cells, NC9 treatment was associated with significantly increased cytosolic TG2 lev- els and lower levels in the nucleus compared to cells treat- ed only with ATRA (Figure 6D2, upper left). Concurrently, the amount of total and nuclear p65/RelA also decreased with NC9 treatment (Figure 6D2, upper right). Distribution patterns of phospho(Ser536)-p65/RelA, which is the transcriptionally active form of p65/RelA, were very similar to the TG2 protein detected in the total, cytosolic, and nuclear fractions (Figure 6D2, upper and lower right, black and red bars). Both total and nuclear phospho-p65/RelA protein contents were significantly reduced in the presence of NC9 inhibitor (Figure 6D2, lower left, black and red bars). The p50 subunit of NF-B showed patterns of cellular distribution very similar to p65/RelA (Figure 6D2, lower right and upper right, black bars), with a distinctive difference in the amount of nuclear p50 protein remaining low, around the detection limit, in NC9-treated NB4-WT cells (Figure 6D2, lower right, black and red bars). The cellular distribution of TG2 indicated that NC9 inhibits nuclear translocation of TG2
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