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K. Jambrovics et al.
inflammatory differentiating leukemic cells in the blood- stream, releasing chemokines and cytokines in a so-called cytokine storm, which shifts endothelial cell function from normal toward inflammatory processes. DS is also characterized by manifestation of unexplained fever, res- piratory distress, pleural and pericardial effusions, pul- monary edema, episodic hypotension, and vascular capil- lary leakage, which may lead to acute renal failure.13,14 Although glucocorticoid treatment leads to recovery in most patients within 12 hours (h) and resolution of symp- toms within 24 h, the condition is fatal in 1-5% of patients. Dexamethasone treatment will not inhibit the induction of chemokines in differentiating APL cells.15,16
ATRA-induced differentiation can be modeled to a cer- tain extent using NB4 APL cells.17-19 The differentiation process involves modulation of thousands of genes to pro- duce functional neutrophil granulocytes. The most highly up-regulated gene in ATRA-activated maturation of NB4 cells is tissue transglutaminase (TG2). TG2 expression silencing in NB4 cells has revealed functional TG2 partici- pation in modulation of gene expression, reactive oxygen species (ROS) generation, cytokine expression, adhesion, and migration, and phagocytic capacity of differentiated neutrophil granulocytes.20,21
TG2 is a Ca2+-dependent protein cross-linking enzyme that also adds amines to proteins and is capable of deami- dating γ-carboxamide groups of particular protein-bound glutamines.22,23 In addition, TG2 has several enzymatic activities that do not require Ca2+; it can hydrolyze guano- sine triphosphate (GTP) and adenosine triphosphate (ATP), can mediate signal transduction via G-protein-cou- pled receptors, and has protein kinase and protein disul- fide isomerase activities. Recent evidence shows that TG2 in the GTP-bound/closed (signaling) conformation drives cancer cell survival.24,25
To provide firm evidence for the critical involvement of TG2 in ATRA-induced differentiation of promyelocytic leukemia cells to inflammatory neutrophils, we generated TG2-deleted NB4 cells and applied a cell-penetrable, irre- versible TG2 inhibitor to observe how TG2 influences the development of inflammatory states. Our results demon- strate that ATRA-induced atypical TG2 expression enhances NF-κB gene expression, nuclear translocation, and transcriptional activation of NF-κB target genes, lead- ing to unregulated production of inflammatory cytokines and chemokines.
Methods
Cell lines, treatments and measurements
The cell culture conditions of the NB4 APL cell line have been described previously.18
The NB4 TG2-KO cell line was generated from the wild-type cell line by TALEN which is described in detail in the Online Supplementary Appendix. NB4 cell lines were treated with 1 μM ATRA (Sigma-Aldrich) or 1 mM ATRA + 30 mM NC9 (30 mM stock solution) and collected at the indicated time points. Phorbol- myristate acetate (PMA) in 10 nM or tumor necrosis factor alpha (TNF-α) at a concentration of 2 ng/mL were used.
luciferase construct was used, stably integrated into the genomic DNA of NB4 cell lines (CLS-013-L8-QIAGEN). The assay was per- formed according to the manufacturer’s protocol. The transfected cells were selected by administration of puromycin (Sigma- Aldrich) at a final concentration of 10 mg/mL. Measurement of luciferase activity was performed using a Bright-GloTM Luciferase Assay System (Promega). The data were validated by GraphPad Prism 7.0 using a parallel normalizing method based on cell num- bers and protein concentration.
The preparation of the RNA samples has been published previ- ously.20,21 Real-time Q-PCR reaction utilized the following probes (ABI, Applied Biosystems): TGM2, MCP-1, TNF-α, IL-1b, GP91PHOX, NCF2, GAPDH, and CYCLO-D. The analysis was performed using an ABI Prism 7900 (ABI, Applied Biosystems). Relative expression levels were normalized to the level of cyclophilin-D and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Secreted cytokine concentration was measured using an ELISA Kit (BioLegend/RayBiotech) according to the manufacturer’s instructions.
NB4-WT cells were treated as described above for 11 days. At day 11, cells were treated with 5 mM MG132 for 3 h. Samples were harvested after 30 minutes, 1 h, 2 h, and 3 h, and handled as described previously.20,21
A more detailed description of the methods used are available in the Online Supplementary Appendix.
Results
TG2 accelerates phagocytotic and antimicrobial functions of differentiating NB4 cell lines
The NBT test is a simple method for examining phago- cytic and oxygen-dependent antimicrobial ability of neu- trophil granulocytes. We previously reported that NB4 TG2-KD (TG2 knockdown) cells reduced NBT but to a lesser extent than wild-type NB4 (NB4-WT) cells after three days of ATRA treatment.21 To determine the contri- bution and correlation of TG2 expression levels to differ- entiated neutrophil granulocyte status, NB4 human acute promyelocytic leukemia cells (NB4-WT) and sublines NB4 TG2-C (virus control), NB4 TG2-KD, NB4 TG2-ha (het- erozygous allele), NB4 TG2-KO (knockout) (see Online Supplementary Figures S1-S3) were treated with ATRA. First, the level of TG2 mRNA transcription and protein expression was determined in cell lines; it was found that while the TG2-KO cell line did not express TG2, the NB4- WT and NB4 TG2-C cell lines expressed TG2 abundantly and comparably. However, the NB4 TG2-KD and NB4 TG2-ha cell lines expressed TG2 at intermediate levels between the total and TG2-deficient level (Figure 1A and B). TG2-deficient conditions (TG2-KO) led to a rightward shift in the NBT-positivity response curve compared to NB4-WT cells, indicating that the moderate or total expression of TG2 accelerated the differentiation process in NB4 TG2-KD or TG2-ha and NB4 TG2-C or NB4-WT cells, respectively, resulting in an early increase in NBT- positivity and a saturation curve over the 11-day time scale (Figure 1C and Online Supplementary Figure S4).
ATRA induces expression of leukocyte b2 integrin receptors MAC-1 and p150,95 and their high-affinity state on the cell surface of NB4 cell lines
Analysis of the cell-surface expression of L-selectin, CD11b, and CD11c, which are indicators of the degree of
Western blot analysis (preparation of cell lysate and subcellular fractions), cell preparation, and staining methods for fluorescence- activated cell sorting (FACS) analysis and the nitro-blue-tetrazoli- um test (NBT) have been described previously.20,21
To evaluate NF-κB pathway activity, an NF-κB promoter-driven
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haematologica | 2019; 104(3)