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Originally published online as doi:10.1189/jlb.0103004 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:223-232.)
© 2003 by Society for Leukocyte Biology

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-{alpha} promote the NF-{kappa}B-dependent maturation of normal and leukemic myeloid cells

Paola Secchiero*, Daniela Milani*, Arianna Gonelli*, Elisabetta Melloni{dagger}, Diana Campioni{ddagger}, Davide Gibellini§, Silvano Capitani*,** and Giorgio Zauli{dagger}

* Departments of Morphology and Embryology, Human Anatomy Section, and
{ddagger} Biomedical Sciences and Advanced Therapies, Hematology Section, St. Anna Hospital, University of Ferrara, Italy;
{dagger} Department of Human Normal Morphology, University of Trieste, Italy;
§ Department of Experimental and Clinical Medicine, Microbiology Section, University of Bologna, St. Orsola Hospital, Italy; and
** Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Italy

Correspondence: Paola Secchiero, Ph.D., Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy; E-mail: secchier{at}mail.umbi.umd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-{alpha} induced monocytic maturation of primary normal CD34-derived myeloid precursors and of the M2/M3-type acute myeloid leukemia HL-60 cell line, associated to increased nuclear factor (NF)-{kappa}B activity and nuclear translocation of p75, p65, and p50 NF-{kappa}B family members. Consistently, both cytokines also induced the degradation of the NF-{kappa}B inhibitors, I{kappa}B{alpha} and I{kappa}B{varepsilon}, and up-regulated the surface expression of TRAIL-R3, a known NF-{kappa}B target. However, NF-{kappa}B activation and I{kappa}B degradation occurred with different time-courses, since TNF-{alpha} was more potent, rapid, and transient than TRAIL. Of the two TRAIL receptors constitutively expressed by HL-60 (TRAIL-R1 and TRAIL-R2), only the former was involved in I{kappa}B degradation, as demonstrated by using agonistic anti-TRAIL receptor antibodies. Moreover, NF-{kappa}B nuclear translocation induced by TRAIL but not by TNF-{alpha} was abrogated by z-IETD-fmk, a caspase-8-specific inhibitor. The key role of NF-{kappa}B in mediating the biological effects of TNF-{alpha} and TRAIL was demonstrated by the ability of unrelated pharmacological inhibitors of the NF-{kappa}B pathway (parthenolide and MG-132) to abrogate TNF-{alpha}- and TRAIL-induced monocytic maturation. These findings demonstrate that NF-{kappa}B is essential for monocytic maturation and is activated via distinct pathways, involving or not involving caspases, by the related cytokines TRAIL and TNF-{alpha}.

Key Words: TRAIL • TNF-{alpha} • NF-{kappa}B • myeloid cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor {alpha} (TNF-{alpha}) is the prototypical member of the structurally related TNF family of cytokines, which plays an important role in diverse cellular events, such as inflammation, septic shock, and apoptosis, by interacting with two high-affinity receptors: TNF-receptor (TNF-R)1 and TNF-R2 [1 ]. This family of cytokines includes TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand [2 , 3 ], which specifically binds to four transmembrane TRAIL receptors belonging to the TNF-R family. Although TRAIL-R1 and TRAIL-R2 transduce apoptotic signals, TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) are homologous to TRAIL-R1 and TRAIL-R2 in their cysteine-rich, extracellular domain but lack intracellular death domain and apoptosis-inducing capability [4 ]. The unique feature of TRAIL, with respect to TNF-{alpha} or other cytokines of this family, is considered its ability to induce apoptosis in a variety of neoplastic cells, including several hematological malignancies [5 6 7 8 9 10 11 12 ], displaying minimal toxicity on most normal cells. Besides activating the caspase-dependent apoptosis of cancer cells, TRAIL also impairs erythropoiesis in normal and pathological conditions [13 , 14 ], and it promotes monocytic maturation [15 ] and up-regulates prostanoid production in the HL-60 leukemic cell line and primary CD34-derived myeloid cells [16 ]. It is therefore possible that TRAIL plays different lineage-specific roles in the regulation of normal and leukemic hemopoiesis.

In this context, some studies have described increased nuclear factor (NF)-{kappa}B activity during monocytic differentiation of leukemic cell lines and normal monocytic precursors in response to phorbol esters or TNF-{alpha} [17 , 18 ]. In contrast, other studies have shown that HL-60 monocytic maturation was enhanced by NF-{kappa}B antagonists combined to suboptimal doses of 1{alpha},25-dihydroxyvitamin D [19 , 20 ]. The NF-{kappa}B family of transcription factors consists of binary complexes of subunits with related promoter-binding and transactivation properties. The p65/RelA, p68/RelB, and p75/c-Rel subunits stimulate transcription, whereas the p50/NF-{kappa}B1 and p52/NF-{kappa}B2 subunits serve primarily to bind to DNA [21 ]. On these bases, the experiments illustrated in the present study were designed to investigate the role of NF-{kappa}B in monocytic maturation induced by TRAIL by analyzing changes in the subcellular localization, activity, and regulation of NF-{kappa}B family members. TNF-{alpha} was used in parallel as a positive control [17 , 18 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant histidine-6-tagged TRAIL was purified as described previously [14 15 16 ]. TNF-{alpha} was purchased from Peprotec (London, UK).

Parthenolide, an inhibitor of I{kappa}B degradation and NF-{kappa}B DNA-binding activity, was purchased from Alexis Biochemicals (San Diego, CA) and was used at the final concentration of 10 µM. MG-132 (Biomol Research Laboratories, Plymouth Meeting, PA), an unrelated, pharmacological inhibitor of NF-{kappa}B affecting the proteosome pathway, was used at the final concentration of 1 µM. The broad caspase inhibitor z-VAD-fmk, the selective caspase-8 z-IETD-fmk, and caspase-9 z-LEHD-fmk inhibitors (all from Calbiochem, La Jolla, CA) were used at the final concentration of 10–20 µM.

For Western blotting analyses, the following primary antibodies were used: rabbit polyclonal anti-p65/RelA, anti-p75/c-Rel, anti-p68/RelB, anti-p50/NF-{kappa}B1, anti-p52/NF-{kappa}B2, anti-I{kappa}B{alpha}, and anti-I{kappa}B{varepsilon} (all from Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-histone H1 (Upstate Biotechnology, Lake Placid, NY); and mouse anti-tubulin (Sigma Chemical Co., St. Louis, MO).

For flow cytometry analyses, mouse monoclonal anti-human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 antibodies were from Alexis (Lausen, Switzerland), and fluorescein isothiocyanate (FITC)-conjugated anti-human CD11b, phycoerythrin (PE)-conjugated anti-human CD14 antibodies, and PE-conjugated secondary antibody were from Becton Dickinson (San José, CA).

Agonistic polyclonal anti-TRAIL-R1 and anti-TRAIL-R2 antibodies were from R&D Systems (Minneapolis, MN).

Cells and assessment of apoptosis and differentiation
HL-60 cell line, an M2/M3-type acute myeloid leukemia (AML), was grown in RPMI (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco). Cord blood (CB) specimens, collected according to institutional guidelines, were obtained during normal, full-term deliveries. CB CD34+ cells were isolated by using a magnetic cell-sorting program Mini-MACS and the CD34 isolation kit (Miltenyi Biotech, Auburn, CA), in accordance with the manufacturer’s instructions, and were cultured as described previously [15 , 16 ].

At different time-points post-treatment with TRAIL and TNF-{alpha}, cell cultures were analyzed by: counting the total number of viable cells by trypan blue dye exclusion; evaluating the degree of apoptosis by FITC-conjugated Annexin-V and propidium iodide (PI) staining followed by flow cytometry analysis; examining cell morphology by staining with May-Grunwald-Giemsa followed by light microscopy examination; examining the expression of monocytic maturative genes, such as CD14 and human monocyte-associated esterase-1 (HMSE-1), by reverse transcriptase-polymerase chain reaction (RT-PCR); and monitoring the expression of maturative cell-surface antigens by flow cytometry.

In most experiments, apoptotic cells were removed from the cultures by using the Dead Cell Removal kit (Miltenyi Biotech).

Flow cytometry analyses
The expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was analyzed by indirect staining using anti-human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 monoclonal antibodies (mAb; Alexis) followed by PE-conjugated rabbit anti-mouse immunoglobulin G (IgG; Sigma Chemical Co.). Aspecific fluorescence was assessed by using normal mouse IgG followed by a second layer as above.

For apoptosis detection and quantification, cells were stained with PI and FITC-conjugated Annexin-V (Alexis Biochemicals), according to the manufacturer’s instructions, and were analyzed as detailed previously [14 , 15 ].

To examine the presence of CD11b and CD14 surface antigens, aliquots of 0.5 x 106 cells/experimental point were subjected to single- or multiple-label staining with FITC- or PE-conjugated mAb. Aspecific fluorescence was assessed by using isotype-matched controls.

Data collected from 10,000 cells are reported as percentage of positive cells or mean fluorescence intensity (MFI) values. All analyses were performed by using a FACScan flow cytometer and the Lysis II software (Becton Dickinson).

RNA analysis
RNA purification from cell cultures was performed using the SV Total RNA Isolation System (Promega, Madison, WI), following the manufacturer’s protocol. Synthesis of first-strand cDNA and amplification were performed using the Access RT-PCR system (Promega) and specific primer sets, following the manufacturer’s protocol. The primers used for the detection of HMSE-1 and CD14 mRNA were as described previously [22 ]. For the amplification of ß-actin, reactions were performed using the following primer sets: forward, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', and reverse, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'. The resulting RT-PCR products were resolved on 2% agarose gels and visualized with ethidium bromide.

Assays for caspase activity and NF-{kappa}B DNA binding
Caspase-8 activity was measured by using the Caspase Colorimetric assay kit (Alexis Biochemicals) as described by the manufacturer. NF-{kappa}B induction was measured using the Trans-AM NF-{kappa}B p65 and p50 kits (Active Motif, Rixensart, Belgium), which measure the level of active form of NF-{kappa}B contained in cell extracts able to specifically bind to an oligonucleotide containing the NF-{kappa}B consensus site (5'-GGGACTTTCC-3') attached to a 96-well plate. Assays were performed in triplicates, according to the manufacturer’s instructions. Caspase and NF-{kappa}B DNA-binding activities were determined as absorbance values measured by using a microplate reader (Multiskan Ascent, Dasit, Milano, Italy). An increase in fluorescence was linear over extract concentration.

Transient transfections
The transcription activity of NF-{kappa}B, upon TNF-{alpha} or TRAIL treatment, was assayed by the transfection of a chloramphenicol acetyltransferase (CAT) reporter containing a specific consensus sequence of NF-{kappa}B. Plasmid pNF-{kappa}B-CAT, a generous gift of Dr. Enzo Lalli (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), was achieved by cloning four consensus sequences (5'-GGGACTTTCC-3') specific for a NF-{kappa}B transcription factor, 5' to a CAT gene of the pBL2-CAT backbone plasmid. Transient transfection experiments were performed using the diethylaminoethyl (DEAE)–dextran method, as described previously [23 ]. In single-transfection experiments, 107 HL-60 cells were maintained in RPMI + 1% FCS for 24 h and were then transfected with 10 µg pNF-{kappa}B-CAT or with pBL2CAT empty vector in 500 µg DEAE–dextran and 20 µg chloroquine/ml for 60 min. The next day, culture medium was replaced with fresh RPMI containing 10% fetal bovine serum and was exposed for 6 h to TRAIL or TNF-{alpha} before lysis for the CAT assay, performed using volumes of extract corresponding to equal protein amounts. Protein determination was performed by the Bradford assay (Bio-Rad, Richmond, CA).

Immunoblotting
Protein concentrations in total cellular lysates and nuclear extracts were estimated by the Bio-Rad protein assay, according to the manufacturer’s protocol. Equivalent amounts of proteins (50 µg) per sample were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-acrylamide gels. The gels were then electroblotted onto a nitrocellulose membrane. Equal loading of protein in each lane was confirmed by brief staining of the blot with 0.1% Ponceau S, followed by destaining before reacting with the specific primary antibodies. After incubation with peroxidase-conjugated anti-rabbit or anti-mouse IgG, specific reactions were revealed with the enhanced chemiluminescence Western blotting detection reagent (Amersham, Arlington Heights, IL).

Statistical analysis
Data were analyzed using the two-tailed, two-sample t-test (Minitab statistical analysis software, State College, PA). Values of P< 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} and TRAIL promote maturation along the monocytic lineage in primary normal monocytic precursors and HL-60 cells
In the first group of experiments, we demonstrated that TNF-{alpha} induced the progressive appearance in culture of cells with typical monocytic features when added to primary, normal CD34-derived myeloid precursors (Fig. 1A ) and HL-60 cells (Fig. 1B) , an M2/M3-type AML, which represents a convenient model to elucidate the regulation and function of factors that coordinate the differentiation along the monocytic or granulocytic lineages [15 , 16 ]. Consistent with these morphological data and in agreement with previous data of other authors [17 , 18 ], TNF-{alpha} significantly up-regulated the expression of differentiation markers, as documented by the induction of gene markers characteristic of the monocytic lineage, such as CD14 and HMSE-1, as evaluated by RT-PCR (Fig. 1C) , and by phenotypic changes characterized by the simultaneous up-regulation of CD14 and CD11b surface antigens (Fig. 1D) . Similar findings were described previously in the same cell models, using recombinant TRAIL [15 ]. In addition, as TRAIL also induced marked apoptosis when added to HL-60 cells [15 ], here, we could demonstrate that CD14-positive HL-60 cells appearing in culture after 24 h of TRAIL treatment were negative for Annexin-V staining (Fig. 1E) . This clearly demonstrates that cells maturating along the monocytic lineage were those escaping apoptosis. In the following experiments, cultures were processed with the dead removal kit, which allows removal of necrotic and apoptotic cells before performing protein analyses to avoid the possible interference of dead cells observed from 6 h of TRAIL treatment onward.



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  		Figure 1. TNF-{alpha} induces monocytic differentiation of primary CD34-derived monocytic precursors and HL-60 cells. Primary CD34-derived monocytic cultures (A) and HL-60 cells (B) were left untreated or treated with TNF-{alpha} (for 72 h) before cytocentrifugation and staining with May-Grunwald-Giemsa solution. *, Cells with monocytic morphology. Representative fields of five separate experiments are shown. Original magnification, 400x. (C) Semiquantitative RT-PCR was applied to analyze CD14 and HMSE-1 mRNA levels in TNF-{alpha}-treated HL-60 cultures as compared with untreated cells. Equivalent amounts of RNA extracted from untreated (Untr.) and TNF-{alpha}-treated cells were used for 1:5 limiting-step dilution (lanes 1–3) before RT-PCR. ß-Actin amplification was used to confirm comparability of the samples. Ethidium bromide-stained agarose gel of RT-PCR products is shown. M, 100-bp ladder. (D) Surface CD14 expression (y-axis) was analyzed in combination with surface CD11b (x-axis) in HL-60, untreated or treated with TNF-{alpha}. Representative negative control, constituted by cells stained with irrelevant (Irr.) isotye-matched antibodies, is shown. (E) Surface CD14 expression (y-axis) was analyzed in combination with Annexin-V (x-axis) in HL-60, untreated or treated with TRAIL. (D and E) Numbers within quadrants indicate the percentage of cells in the different populations. Data shown are from a single experiment representative of five independent experiments.

 
TRAIL and TNF-{alpha} activate NF-{kappa}B activity in normal myeloid precursors and HL-60 cells. To get insights into the molecular mechanisms underlining monocytic maturation induced by both cytokines, the DNA binding of p65/RelA and p50/NF-{kappa}B1 was initially checked in untreated and TNF-{alpha}- or TRAIL-treated cultures, using the Trans-AM assay, which specifically measures the level of the active form of p65/RelA and p50/NF-{kappa}B1 (Fig. 2 ). A marked (P<0.01) induction of NF-{kappa}B-DNA binding over untreated cultures was observed, peaking at 1 h of TNF-{alpha} treatment, showing a decline thereafter in primary normal precursor cultures (Fig. 2A) and HL-60 cells (Fig. 2C) . Conversely, TRAIL-treated cultures showed a delayed kinetics of NF-{kappa}B activation, which became significantly (P<0.01) greater than control cultures after 3–6 h in primary normal (Fig. 2B) and HL-60 (Fig. 2D) cells. The specificity of the binding was confirmed by competition of an excess of a wild-type consensus oligonucleotide-binding site and by using parthenolide, a pharmacological inhibitor of NF-{kappa}B. According to previous data of other authors obtained in different cell types [23 24 25 26 27 ], the maximal level of NF-{kappa}B activation induced by TRAIL was lower than that induced by TNF-{alpha}.



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  		Figure 2. TNF-{alpha} and TRAIL induce NF-{kappa}B activation in primary, CD34-derived monocytic precursors and HL-60. Primary, CD34-derived monocytic cultures (A and B) and HL-60 cells (C–D) were left untreated or exposed to TNF-{alpha} or TRAIL for the indicated times. Active NF-{kappa}B complex was detected by anti-p65 or anti-p50 antibodies and measured by enzyme-linked immunosorbent assay, as described in Materials and Methods. The levels of NF-{kappa}B activity were determined as absorbance (450 nm) values, and NF-{kappa}B activity in untreated cells was set as 100%. (C and D) HL-60 were treated with TNF-{alpha} or TRAIL in the absence or presence of parthenolide (Part.). (A–D) All samples were also tested in the presence of an excess (20 pmol/well) of competitor oligonucleotide (oligo) to confirm the specificity of the assay. (E) HL-60 cells were transiently transfected with NF-{kappa}B–CAT reporter plasmid or with the pCAT control vector. After transfection, cells were left untreated (Unt.) or stimulated with TNF-{alpha} or TRAIL before CAT assay. Results are expressed as fold activity over the control (Unt.) cells. Data represent the means ± SD of three independent experiments performed in duplicate. *, Statistically significant differences (P<0.05) between treatments.

 
We next examined the transcriptional activation of NF-{kappa}B by a NF-{kappa}B-dependent reporter gene expression assay. For this purpose, HL-60 cells were transiently transfected with {kappa}B-CAT reporter plasmid construct and stimulated with TRAIL and TNF-{alpha}, and the NF-{kappa}B transactivity was measured by the CAT assay (Fig. 2E) . NF-{kappa}B transactivation was induced over background levels represented by cells transfected but left untreated ~13-fold (P<0.01) and fivefold (P<0.01) in TNF-{alpha}-treated and TRAIL-treated HL-60, respectively. Taken together, these data suggest that TNF-{alpha} and TRAIL can induce NF-{kappa}B-DNA binding and {kappa}B-dependent transcriptional activity in HL-60 cells.

TNF-{alpha} and TRAIL modulate the surface expression of TRAIL receptors
As TRAIL receptors have been reported to be up-regulated by NF-{kappa}B family members in a cell type-specific manner [28 29 30 31 ], we next investigated whether this was also the case in HL-60 cells. Flow cytometry analysis of untreated HL-60 cells showed detectable surface levels of TRAIL-R1 and TRAIL-R2 expression coupled to undetectable surface expression of TRAIL-R3 and TRAIL-R4 (Fig. 3 ). The addition of TNF-{alpha} or TRAIL to HL-60 cells for 48 h induced a marked up-regulation of TRAIL-R3, and the effect on TRAIL-R1 and -R2 was more complex. In fact, although TRAIL addition significantly (P<0.01) decreased the levels of TRAIL-R1 and to a lesser extent of -R2, TNF-{alpha} induced a modest up-regulation of TRAIL-R1 and TRAIL-R2.



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  		Figure 3. TNF-{alpha} and TRAIL modulate the surface expression of TRAIL-receptors in HL-60 cells. Surface TRAIL-receptor expression was evaluated by flow cytometry in HL-60 cells left untreated or exposed to TNF-{alpha} or TRAIL for 48 h. Shadowed histograms represent cells stained with antibodies specific for the indicated TRAIL-receptors (TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4), and open histograms represent the background fluorescence obtained from the staining of the same cultures with isotype-matched, control Ab. A representative of four separate experiments is shown.

 
TNF-{alpha} and TRAIL induced NF-{kappa}B nuclear translocation and I{kappa}B degradation with different time courses
The effect of TNF-{alpha} and TRAIL on the cellular localization and activity of NF-{kappa}B family members (p65/RelA, p68/RelB, p75/c-Rel, p50/NF-{kappa}B1, and p52/NF-{kappa}B2) was assessed next. No significant variations in the expression of NF-{kappa}B members were detected analyzing total cell lysates from untreated and TNF-{alpha}- or TRAIL-treated cultures (Fig. 4A and 4B , and data not shown for p68/RelB and p52/NF-{kappa}B2). Conversely, a striking induction of the nuclear p65/RelA, p75/c-Rel, and p50/NF-{kappa}B1 proteins was found upon exposure to TNF-{alpha} or TRAIL (Fig. 4A and 4B) , and p68/RelB and p52/NF-{kappa}B2 did not show reproducible nuclear translocation (data not shown). Under unstimulated conditions, NF-{kappa}B is retained in the cytosol by virtue of binding to members of the I{kappa}B family. When cells are treated with agonists known to activate NF-{kappa}B, I{kappa}B kinase-ß (IKKß) phosphorylates I{kappa}B and in so doing, targets I{kappa}B to proteosomal degradation [21 ]. This liberates NF-{kappa}B to translocate to the nucleus, where it binds to regulatory elements of NF-{kappa}B-inducible genes. Based on the data illustrated above, TNF-{alpha} and TRAIL should be able to induce I{kappa}B degradation. Accordingly, the quantities of I{kappa}B were determined by Western blotting after various times of TNF-{alpha} and TRAIL treatment (Fig. 5 ). Similar to what has been previously reported in other cell models [21 ], TNF-{alpha} induced a rapid degradation of I{kappa}B{alpha} and I{kappa}B{varepsilon} starting after 5–10 min of treatment. After 3–6 h, the levels of I{kappa}B{alpha} and I{kappa}B{varepsilon} detected were progressively and significantly (P<0.05) increased, as compared with control levels (Fig. 5A) . This was an expected finding as a result of the NF-{kappa}B-dependent, transcriptional activation of I{kappa}B genes soon after I{kappa}B{alpha} and I{kappa}B{varepsilon} degradation is initiated [21 ]. Of note, TRAIL induced a time-course of I{kappa}B{alpha} and I{kappa}B{varepsilon} degradation distinct from that observed upon TNF-{alpha} treatment. In fact, the levels of I{kappa}B were similar to control up to 1 h of TRAIL treatment, and they were significantly (P<0.01) reduced after 3 h and also remained persistently reduced after 6 h (Fig. 5B) .



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  		Figure 4. TNF-{alpha} and TRAIL induce NF-{kappa}B nuclear traslocation. HL-60 cells were treated with TNF-{alpha} (A) or TRAIL (B) and incubated for 3 h before harvesting the cells for lysis. Equal amounts of total cell lysates (Tot.) and nuclear extracts (NE) were size-fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and were analyzed for the expression and localization of NF-{kappa}B members using specific antibodies. Data shown are from a single experiment representative of five independent experiments.

 


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  		Figure 5. TNF-{alpha} and TRAIL induce I{kappa}B{alpha} and -{varepsilon} degradation. HL-60 cells were treated with TNF-{alpha} (A) or TRAIL (B) and incubated for the indicated times before harvesting the cells for lysis. Equal amounts of total cell lysates were analyzed for degradation of I{kappa}B{alpha} and I{kappa}B{varepsilon} by Western blotting using specific antibodies to each. Equal loading was confirmed by tubulin staining. One of four experiments with similar results is shown.

 
The induction of I{kappa}B degradation by TRAIL is mediated by TRAIL-R1
To elucidate whether the induction of NF-{kappa}B by TRAIL in HL-60 cells was specifically mediated by TRAIL-R1 or -R2, HL-60 cells were challenged with agonistic, polyclonal anti-TRAIL-R1 and anti-TRAIL-R2 antibodies, which mimic the interaction between TRAIL and each TRAIL-R without cross-reacting among each other. Remarkably, treatment of HL-60 with anti-TRAIL-R1 but not with anti-TRAIL-R2 (final dilution for both antibodies, 1:25) induced degradation of I{kappa}B{alpha} (Fig. 6 ) and I{kappa}B{varepsilon} (data not shown) with the same kinetics of TRAIL. Consistent with a selective role of TRAIL-R1 in mediating the biological effects of TRAIL in HL-60, in a previous study, we have shown that TRAIL-R1 was responsible for TRAIL-mediated monocytic maturation and induction of apoptosis of HL-60 cells [15 ].



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  		Figure 6. TRAIL-R1 mediates I{kappa}B degradation. HL-60 cells were left untreated (Unt.) or exposed to agonistic polyclonal anti-TRAIL-R1 (anti-R1) and anti-TRAIL-R2 (anti-R2) antibodies, as well as to TRAIL and TNF-{alpha} (for comparison). Equal amounts of total cell lysates were analyzed for degradation of I{kappa}B{alpha} by Western blotting. Equal loading was confirmed by tubulin. One of three experiments with similar results is shown.

 
NF-{kappa}B activation induced by TRAIL but not by TNF-{alpha} is inhibited by the caspase-8 inhibitor z-IETD-fmk
The activation of NF-{kappa}B in response to TNF-{alpha} and TRAIL was next investigated by determining the nuclear translocation of NF-{kappa}B members and the degradation of I{kappa}B in the presence of specific pharmacological inhibitors. As expected, the NF-{kappa}B pharmacological inhibitor parthenolide blocked nuclear translocation of NF-{kappa}B family members (Fig. 7A and 7B ) as well as the I{kappa}B degradation (Fig. 7C) induced by TNF-{alpha} or TRAIL. Similar findings were obtained using MG-132, an unrelated, pharmacological inhibitor of NF-{kappa}B, which inhibits proteosomal degradation of I{kappa}B (data not shown). The selective caspase-8 inhibitor z-IETD-fmk did not affect nuclear NF-{kappa}B translocation (Fig. 7A) and I{kappa}B degradation (Fig. 7C) in TNF-{alpha}-treated cultures. Conversely, z-IETD-fmk (Fig. 7B and 7C) and the pan-caspase inhibitor z-VAD-fmk but not the caspase-9 inhibitor z-LEHD-fmk (data not shown) completely abrogated nuclear translocation of NF-{kappa}B and I{kappa}B degradation induced by TRAIL. Consistently, TRAIL but not TNF-{alpha} induced a progressive increase of caspase-8 activity in HL-60 cells, peaking at 3 h and showing a plateau thereafter (Fig. 7D) .



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  		Figure 7. Caspase-8 inhibitor (z-IETD-fmk) abrogates TRAIL- but not TNF-{alpha}-mediated NF-{kappa}B nuclear translocation and I{kappa}B degradation. HL-60 cell cultures were left untreated or treated with TNF-{alpha} and TRAIL in the absence (Vehicle) or in the presence of the indicated inhibitors. Part., Parthenolide; z-IETD, z-IETD-fmk. (A and B) Equal amount of nuclear extracts were size-fractionated by SDS-PAGE and were analyzed for the expression of the indicated NF-{kappa}B members using specific antibodies. Equal loading was confirmed by histone H1 staining. This experiment is representative of three independent experiments that gave similar results. (C) Equal amounts of total cell lysates were analyzed for degradation of I{kappa}B{alpha} by Western blotting. Data are representative of four independent experiments. (D) The levels of caspase-8 activity were determined as absorbance values (405 nm), and caspase activity in untreated cells was set as 100%. Data represent the means ± SD of three independent experiments performed in duplicate. *, Statistically significant differences (P<0.05) between treatments are shown.

 
NF-{kappa}B activation induced by TNF-{alpha} or TRAIL plays an essential role in monocytic maturation of HL-60 cells
It remained important to demonstrate whether the NF-{kappa}B pathway played a role in TNF-{alpha}- and TRAIL-mediated monocytic maturation. To address this issue, 45 min before cytokine addition, cells were preincubated with pharmacological inhibitors specific for the NF-{kappa}B (parthenolide and MG-132), caspase-8 (z-IETD-fmk), and caspase-9 (z-LEHD-fmk). The maturation markers were then analyzed after 24 h by flow cytometry (Fig. 8 ). Parthenolide and MG-132 (not shown) significantly (P<0.01) reduced CD11b (Fig. 8A and 8C) and completely abrogated CD14 (Fig. 8A and 8D) monocytic markers induced by TNF-{alpha}. Neither z-IETD-fmk nor z-LEHD-fmk affected TNF-{alpha}-mediated differentiation. Conversely, incubation of HL-60 cells with parthenolide, MG-132 (not shown), or z-IETD-fmk but not with z-LEHD-fmk completely prevented (P<0.01) the TRAIL-mediated, maturational effect (Fig. 8B 8C 8D) .



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  		Figure 8. Differential effect of pharmacological inhibitors on TNF-{alpha}- and TRAIL-mediated CD11b and CD14 induction in HL-60 cells. Cultures were left untreated or treated for 24 h with TNF-{alpha} and TRAIL in the absence (vehicle) or in the presence of the following inhibitors: partenolide (Part.), z-IETD-fmk (z-IETD), or z-LEHD-fmk (z-LEHD) before determination of surface CD11b and CD14 expression by flow cytometry. (A and B) Shadowed histograms represent cells stained with antibodies specific for the indicated antigens, and open histograms represent control cells stained with irrelevant isotype-matched antibodies. Data shown are from a single experiment representative of six independent experiments with similar results. (C and D) The expression of CD11b and CD14 in HL-60 cultures, treated as indicated, is reported as MFI (C) and percentage of positive cells (D), respectively. Data represent the means ± SD of three separate experiments performed in duplicate. *, Statistically significant differences (P<0.05) between treatments are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of NF-{kappa}B in monocytic maturation of normal and leukemic myeloid precursors has been controversial, as positive [17 , 18 , 32 , 33 ] or negative [19 20 21 ] role(s) have been attributed to NF-{kappa}B transcription factor family members on monocytopoiesis. This mainly depends on the different cell types (CD34-derived primary, normal precursors, primary leukemic blasts, U937, ML-1, and HL-60 cell lines) and different inducers of differentiation (cytokines, phorbol esters, and 1{alpha},25-dihydroxyvitamin D) used in those studies. In particular, Sokoloski et al. [19 ] and Kang et al. [20 ] have reported that inhibition of NF-{kappa}B, by using a p65/RelA antisense approach or the pharmacological inhibitor parthenolide, the same used in our study, markedly increased the degree of HL-60 monocytic differentiation when simultaneously combined with low doses of 1{alpha},25-dihydroxyvitamin D. A possible explanation for the apparent discrepancy of the findings of these authors with the results of our present study is that NF-{kappa}B might negatively interfere with the differentiation program triggered by 1{alpha},25-dihydroxyvitamin D, which is known to interact with a specific cytoplasmic receptor (vitamin D receptor) and to induce genomic and nongenomic effects leading to monocytic differentiation.

Conversely, the results of our study clearly demonstrated that activation of NF-{kappa}B was required to induce monocytic differentiation in primary, normal precursors as well as in a HL-60 cell line in response to two distinct members of the TNF family of cytokines. Although we have tried to correlate the differential kinetics and potency of NF-{kappa}B activation with the degree of differentiation induced by TRAIL and TNF-{alpha}, such correlation was not observed, at least at the end-point of our differentiation studies (48–72 h). It is possible that monocytic differentiation of HL-60 cells occurs once a threshold level of NF-{kappa}B activation is achieved.

Remarkably, TNF-{alpha} and TRAIL induced a significant modulation of the expression of TRAIL receptors, and the most prominent modification was the robust up-regulation of TRAIL-R3. Similarly, TNF-{alpha} has been shown to increase TRAIL-R3 expression in HeLa cells, in which TRAIL-R3 induction has been shown to confer resistance to TRAIL-induced apoptosis [29 ]. The effect of TNF-{alpha} and TRAIL was divergent on TRAIL-R1 and -R2, which showed a modest up-regulation upon TNF-{alpha} addition and a significant down-regulation upon TRAIL treatment. Our interpretation of these findings is that the down-regulation of TRAIL-R1 and -R2 upon TRAIL addition is likely a result of a phenomenon of ligand-receptor internalization, which masks other effects potentially induced by TRAIL, such as the moderate up-regulation observed in cells treated with TNF-{alpha}.

It is also noteworthy that the ability of TRAIL to induce I{kappa}B degradation, the initial step required for NF-{kappa}B activation, was mediated by TRAIL-R1 but not by TRAIL-R2. This was demonstrated in experiments performed with polyclonal anti-TRAIL-R1 and anti-TRAIL-R2 antibodies, thus clearly indicating that the two TRAIL receptors were not functionally redundant in mediating NF-{kappa}B activation. Similarly, Santini et al. [32 ] have shown that agonistic antibodies against TNF-R1 induced monocytic maturation of primary leukemic blasts, and antibodies against TNF-R2 had no effects.

Our data on normal CD34-derived myeloid precursors confirm and expand previous data of Richter et al. [18 ], who have demonstrated that TNF-{alpha} plays a crucial role for the up-regulation of the newly described, costimulatory molecule ICOS ligand during differentiation of primary CD34+/CD38+ hemopoietic progenitor cells, in a NF-{kappa}B-dependent manner. In support of our findings claiming that NF-{kappa}B is important for monocytic differentiation, a key role for this transcription factor in monocytopoiesis has been strongly suggested by gene knockout experiments for p65/RelA and double-knockout for p65/RelA and p75/c-Rel [34 , 35 ]. In those studies, because of the death of mutant fetuses at early days of gestation, day 12 fetal liver hemopoietic progenitors were used for in vitro cultures. The frequency of macrophage and granulocyte-macrophage colonies was reduced, and the majority of mature macrophages in these colonies had undergone apoptosis [34 , 35 ].

NF-{kappa}B activation depends on the formation of a multiprotein complex, comprising TNF receptor-associated factors IKK{alpha}, IKKß, NF-{kappa}B essential modulator, I{kappa}Bs, and receptor interacting protein (RIP), resulting in phosphorylation and degradation of I{kappa}Bs and the release of NF-{kappa}B for nuclear translocation [21 ]. Although the role of each of these proteins starts to be delineated in details in mediating the TNF-{alpha}/TNF-R interactions [21 ], the mechanisms by which TRAIL signaling affects the different components of this multiprotein complex in myeloid progenitors and leukemic blasts remain to be elucidated. In this respect, it has been shown that the death domain kinase RIP plays an essential role in TRAIL-mediated activation of I{kappa}B kinase [36 ]. Moreover, although some studies have shown that the addition of z-VAD-fmk potentiated the ability of TRAIL to activate NF-{kappa}B, implying a negative role for activated caspases in TRAIL-mediated NF-{kappa}B activation [26 , 27 ], other studies [23 , 25 ] have shown that activation of NF-{kappa}B required functional Fas-associated death domain (FADD), casper (cellular FADD-like interleukin-1ß-converting enzyme-like inhibitory protein), and pro-caspase-8 and was inhibited by z-VAD-fmk. The exact role of caspase-8 and/or its homologs in the TRAIL-induced NF-{kappa}B activation is not clear at present. However, although these proteins may represent one of the several possible pathways for the induction of NF-{kappa}B by the interaction of TNF-{alpha} with TNF-Rs [23 ], our data suggest that their role is prominent in the case of the TRAIL/TRAIL-R system. Consistent with our findings, other studies have shown that the caspase activation in intact cells does not necessarily lead to cell death and argue for a checkpoint in the apoptotic pathway downstream of caspases. In particular, knockouts of caspases in mice by homologous recombination revealed that caspase-8-/- mice have defective heart muscle development and die as embryos [37 , 38 ]. Surprisingly, the number of hemopoietic progenitors is dramatically reduced in caspase-8-/- mice, suggesting that caspase-8 is essential for the growth and differentiation of heart muscle and hemopoietic progenitors. Thus, TRAIL might play a significant role in promoting monocytic differentiation in normal and malignant hemopoietic cells through a caspase-8/NF-{kappa}B pathway. This possibility might have an important, therapeutic perspective, also taking into account the low in vivo toxicity of recombinant TRAIL, at least in animal models [39 , 40 ].


   ACKNOWLEDGEMENTS
 
This research was supported by A.I.R.C., C.I.B., and FIRB project grants.

Received January 7, 2003; revised March 28, 2003; accepted April 3, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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