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K. Jambrovics et al.
result in rolling, stable adhesion, microvascular sequestra- tion, and infiltration of differentiated APL cells, with increasing potential for the APL-mediated organ damage observed in DS.38,41
rium of TG2 shifts from the closed GTP-binding form to the open conformation characterized by disorganized GTP-binding sites.25 According to our studies, NC9- induced conformational changes in TG2 significantly affect the NF-κB signaling pathway. In the presence of NC9, total TG2 is reduced, as is the nuclear translocation of the decreased amount of TG2, significantly increasing the amount of cytosolic TG2. In turn, the reduced amount of nuclear TG2 correlates with reduced total lev- els of nuclear p50, p65/RelA, and phospho-p65/RelA pro- teins in NB4-WT cells, while the level of the transcrip- tionally active form of p65 and phospho-p65 is increased significantly in the cytosol. This raises the possibility that TG2 in its GTP-bound closed conformation can readily translocate to the nucleus and assist the nuclear transloca- tion of p65/RelA. When TG2 is no longer capable of adopting its GTP-bound closed conformation, due to modification by NC9, the accumulation of both TG2 and p65/RelA in the cytosol results in low NF-κB transcription activity, and, consequently, in the significantly reduced production of inflammatory cytokines and chemokines. Finally, the balance of TG2 synthesis and degradation is changed by degradation triggered by NC9.
TNFα is among the most effective physiological induc- ers of NF-κB activity. In NB4-WT cells, TNFα did not enhance further NF-κB activity already highly elevated by ATRA, but it increased NF-κB transcriptional activity from 5.2x102±1.4x102 to 3.2x104±7.2x103 RLU in NB4 TG2-KO cells (Figure 4D). This may indicate that NF-κB maintains a low basal transcriptional activity in the absence of TG2 in NB4 TG2-KO cells, and adding exogenous TNFα could trigger more NF-κB transcription activity from the IκB:p65:p50 complex. In NB4-WT cells, ATRA-induced atypical expression of TG2 can exacerbate low-level inflammation (IL8 expression, see above) as its expression is associated with both upregulation and activation of NF- κB, resulting in increased endogenous TNFα synthesis and secretion, which may become a sort of self-accelerating process (Figure 4A2-D and Figure 6C1 and 2).
Our findings strongly indicate that ATRA-induced TG2 expression is associated with translocation of NF-κB into the nucleus, upregulation of numerous inflammatory genes, and secretion of their products. These include TG2-quantity-dependent-regulated NF-κB transcription factor target genes: TNFα, I-309 (CCL-1), IP-10 (CXCL10), MIP-3α (CCL20), IL10, ICAM-1, MCSF, IL- 1ra, MDC (CCL22), and PAI-1, whose amounts were sig- nificantly reduced in the absence of TG2 in NB4 TG2-KO cells, with the exception of IL-1ra (Figure 5A). Similarly, among TG2-modulated NF-κB transcription factor target genes and TG2 inhibitor NC9-insensitive secretory pro- teins [MCP-1 (CCL2), MIP-1a (CCL3), MIP-1b (CCL4), cytokines IL-1b, IL-8, IL-9, CCL-28, and OPN (SPP1)], only MIP-1a (CCL3) did not show a significant decrease in TG2 deficiency (Figure 5B). In addition, of the remain- ing 15 TG2 expression-dependent secretory proteins, only one was not significantly changed in the absence of TG2 expression. Together, these results suggest that expression of TG2 in ATRA-induced differentiating APL cells is a crucial factor in the developing inflammatory process.
The suppression of TG2 parallels the considerably low levels of p50 and phospho (Ser536) p65/RelA (Figure 6C2), suggesting that ATRA-induced expression of TG2 reprograms APL cells to be inflammatory neutrophils and induces NF-κB nuclear translocation. Destruction of IκB is stimulated by several signals such as lipopolysaccharide, ROS, TNFα, and Il-b. One possibility for TG2-dependent NF-κB activation is that TG2 interacts with IκB to initiate its non-proteasomal degradation, causing both activation and nuclear translocation of NF-κB-s.42 Alternatively, TG2-mediated cross-linking of IκBα and activation of NF- κB has been previously described, yet we could not detect any forms of IκBα polymer in differentiated NB4 cells (K Jambrovics, 2018, unpublished observation). It was demon- strated that TG2 can form a complex in cytosol as well as in nuclei, with p65 binding to the promoter of the HIF-1α transcription factor, and in this sense, TG2 could become a transcriptional co-regulator in the nucleus.42
In the presence of NC9, an irreversible transamidase site-specific inhibitor of TG2, the conformational equilib-
Among the secreted cytokines and chemokines, TNFα and IL-1b are the most powerful inflammatory agents. At the highest concentrations of TNFα (0.8-1.2 ng/L in DS patients), they cause capillary leakage and reduced car- diac, lung, and renal function.38,43,44 Our data indicate that TG2 is a critical component of this process, and NC9- induced inhibition of TG2 may prevent development of this dangerous side effect of retinoid therapy.
Overall, our study revealed a novel, active role of TG2 in expression and activation of the components of NF-κB and thus in the development of atypical response to con- ventional ATRA treatment of APL. Targeted suppression of the TG2-dependent process may alleviate the common and potentially fatal toxicity of retinoid treatment in APL, representing an important potential therapeutic strategy.
Funding
This work was supported by Hungarian grants from the National Research Found OTKA NK105046, and in part by both OTKA K 129139 and TÁMOP-4.2.2.D-15/1/KONV- 2015-0016 project implemented through the New Széchenyi Plan, co-financed by the European Social Fund. The work was in part also supported by the GINOP-2.3.2-15-2016-00020 | 2.3.2-15-2016-00020 MolMedEx TUMORDNS grant and the EFOP-3.6.1-16-2016-00022|3.6.1-16-2016-00022 “Debrecen Venture Catapult Program”. The work is supported by the GINOP-2.3.2-15-2016-00006|2.3.2-15-2016-00006 project co-financed by the European Union and the European Regional Development Fund. KJ received a fellowship from the DOTE Apoptosis Research Foundation. The research was partly financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Biotechnology thematic programme of the University of Debrecen.
Acknowledgments
The authors would like to thank Dr. István Szatmári and Pál Botó for assistance with cell sorting, Orsolya Molnár for clone selection and Western blot analyses of the KO cell lines.
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haematologica | 2019; 104(3)