Leukemia (2005) 19, 1439–1445 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu SET-induced calcium signaling and MAPK/ERK pathway activation mediate dendritic cell-like differentiation of U937 cells A Kandilci1 and GC Grosveld1 1Department of Genetics and Tumor Cell Biology, Mail Stop 331, St Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA Human SET, a target of chromosomal translocation in human G1/S transition by allowing cyclin E–CDK2 activity in the leukemia encodes a highly conserved, ubiquitously expressed, presence of p21.11 Second, SET interacts with cyclin B–CDK1.19 nuclear phosphoprotein. SET mediates many functions includ- ing chromatin remodeling, transcription, apoptosis and cell Overexpression of SET inhibits cyclin B-CDK1 activity, which in cycle control. We report that overexpression of SET directs turn, blocks the G2/M transition; this finding suggests a negative 13 differentiation of the human promonocytic cell line U937 along regulatory role for SET in G2/M transition. Overexpression of the dendritic cell (DC) pathway, as cells display typical cell division autoantigen-1 (CDA1), another member of the morphologic changes associated with DC fate and express NAP/SET family, inhibits proliferation and decreases bromo- the DC surface markers CD11b and CD86. Differentiation occurs deoxyuridine uptake in HeLa cells.20 Acidic and basic domains via a calcium-dependent mechanism involving the CaMKII and 20 MAPK/ERK pathways. Similar responses are elicited by inter- of CDA1 show 40% identity and 68% similarity to SET. feron-c (IFN-c) treatment with the distinction that IFN-c signaling We have recently shown that overexpression of SET in the activates the DNA-binding activity of STAT1 whereas SET human promonocytic cell line U937 causes G0/G1 arrest and overexpression does not. In addition, unlike IFN-c signaling, stimulates differentiation, an effect dependent on the acidic SET generated stress-induced p38/MAPK activity. Interestingly, domain of SET.21 Therefore, the level of expression of SET may IFN-c treatment transiently upregulated endogenous SET in a affect cellular processes, as determined by cell type and context. dose-dependent manner. These results suggest that SET is part of both IFN-c-mediated and stress-mediated cellular responses Herein, we further analyzed the mechanisms of SET-induced and that SET induces cell differentiation via calcium and MAPK/ differentiation of U937 cells and showed that it occurs along a ERK pathways. dendritic pathway as a result of calcium signaling and MAPK/ Leukemia (2005) 19, 1439–1445. doi:10.1038/sj.leu.2403826; ERK activation. published online 2 June 2005 Keywords: SET; dendritic cell differentiation; Ca2 þ signaling; IFN-g signaling Materials and methods Cell culture and FACS analysis Introduction Stable U937 cell clones expressing tetracycline-regulatable Flag-epitope tagged SET (FS) were generated and maintained The human SET gene, a member of the NAP/SET family,1 is as described previously.21 Cells that were either treated or non- located on chromosome 9q34. SET was initially identified as À7 treated with vitamin D (10 M) were cultured in the presence one partner in the SET-CAN fusion gene, which results from the 3 (FS-uninduced) or absence of tetracycline (FS-induced) after t(9;9)(q34;q34).2 SET encodes a 39-kDa ubiquitously expressed, initial washing (tet[ þ ] cells with tet[ þ ] medium and tet[À] predominantly nuclear phosphoprotein. One-third of its C-ter- cells with tet[À] medium) and the expression of CD11b minal acidic amino acids form an acidic tail.2–4 SET (also known (Beckman Coulter Inc., Fullerton, CA, USA), CD14, CD86, as template-activating factor I beta (TAF-Ib)) physically interacts CD83, CD80 and HLA-DR (all from BD Biosciences, Franklin with several protein complexes, which suggests that it has Lakes, NJ, USA) were analyzed by FACS at day 4 of culture. For diverse functions including Granzyme A–induced apoptosis,5,6 inhibition assays, cells were treated with specific inhibitors (CsA chromosome remodeling,7 transcriptional regulation,8 mRNA (2 mg/ml; Sigma, St Louis, MO, USA), W-7 (15 mM; Calbiochem, stabilization9 and cell cycle regulation.10–13 San Diego, CA, USA), KN-93 (3 mM; Calbiochem) SB202190 SET also forms a complex with the MLL leukemic fusion (5 mM; Upstate, Charlottesville, VA, USA), PD98059 (50 mM; Cell protein and type-2A protein phosphatase (PP2A).14 Among other Signaling Technology, Beverly, MA, USA) or DMSO vehicle as a functions, PP2A regulates cell cycle progression,15,16 and one control. At day 4 of culture, each sample was divided into three target of PP2A is the mitogen-activated protein kinase (MAPK)/ aliquots, which were used for light microscopy, FACS and extracellular signal-regulated protein kinase (ERK) pathway.17 immunohistochemical analysis. For light microscopy, 1x106 SET inhibits PP2A activity,10,12 and overexpression of SET cells were resuspended in 2 ml serum-free RPMI, transferred activates MAPK and prevents Fas-mediated apoptosis.17 onto polylysine-coated six well plates (Becton Dickinson It has been proposed that SET has opposite functions during Labware, MA, USA) and cultured for 1 additional hour. Cells cell cycle progression:13,18 First, SET binds p21. This interaction were photographed with an Olympus IX-70 inverted micro- reverses the inhibitory effect of p21 on cyclin E–CDK2 scope. All the inhibitors were first tested for toxicity and complexes and suggests a positive regulatory role for SET on optimized nontoxic doses were used in the further experiments. Correspondence: Dr GC Grosveld, Department of Genetics, St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA; Fax: þ 1 901 526 2907; Microarray and data analysis E-mail: [email protected] Received 18 February 2005; accepted 29 April 2005; published Total RNA was isolated from parental U937 and FS-induced online 2 June 2005 cells at 8, 24 or 48 h of culture using TRIzol (Life Technologies, SET induces dendritic cell-like differentiation of U937 cells A Kandilci and GC Grosveld 1440 Gaithersburg, MD, USA). The RNA was processed and colorimetric readout was quantified by spectrophotometry. Each hybridized to human genome U95Av2 gene chips, following sample and positive control was analyzed in triplicate. the manufacturer’s protocols (Affymetrix, Santa Clara, CA, USA). Chips were scanned and analyzed using Microarray software 22 Results (Affymetrix) as described previously. Upregulation of a gene in the FS-induced cells was determined by comparing its level of expression with that in the control U937 cells. SET and vitamin D3 stimulate the expression of different markers on U937 cells Real-time RT-PCR We recently reported that tetracycline (tet)-regulatable over- expression of flag-tagged SET (FS) in U937 cells blocked G1/S Quantitative real-time RT-PCR (TaqMan) analysis was per- transition and stimulated differentiation as detected by upregu- 21 formed with an ABI Prism 7900HT sequence detection system lation of the cell surface marker CD11b. Vitamin D3 stimulates (Applied Biosystems, Foster City, CA, USA). The amplification monocytic differentiation in U937 cells via vitamin D3 receptor- 23 mix included 100 ng RNA, 0.2 mM of each primer, 0.2 mM of dependent expression of surface markers CD11b and CD14. each probe and TaqMan one-step RT-PCR master mix reagent in To determine whether overexpression of SET mimics other a total reaction volume of 40 ml. As an internal control, cellular responses to vitamin D3, we assessed CD14 expression amplification of GAPDH was performed in the same reaction in FS-induced (tet [À]) cells. A 4-day induction of FS stimulated mix and detected with an alternatively labeled probe. Triplicates the expression of CD11b in 76% of the cells but CD14 of each standard and sample were assessed. Primer sequences expression remained undetectable (Figure 1a), whereas addition are available upon request. of vitamin D3 activated the expression of CD14 and CD11b in over 75% of the cells, regardless of whether the cells were maintained in the presence (FS-uninduced) or absence of tet (FS- Immunocytochemistry induced) (Figure 1b). These results suggest that overexpression of SET induces differentiation of U937 cells along a pathway Cells were fixed with 4% paraformaldehyde, permeabilized distinct from that induced by vitamin D3. with 0.2% PBS–TritonX-100 and incubated with mouse anti-flag antibody (Sigma, St Louis, MO, USA) and Alexa-488 conjugated Ectopic SET stimulates the expression of calcium- secondary antibody (Molecular Probes, Eugene, OR, USA) for responsive genes and dendritic cell (DC) markers 1 h at room temperature. Cells were covered with mounting medium containing 4,6-diamidino-2-phenylindole (DAPI, Vec- To determine the comprehensive profile of genes whose tor Laboratories, Burlingame, CA, USA) and then analyzed with expression is induced by SET overexpression, we performed an Olympus BX-51 fluorescence microscope. microarray analyses. We identified 18 genes that were significantly upregulated (two-fold or greater increase in Western blot analysis expression) and eight genes that were significantly down- regulated (two-fold or greater decrease in expression). Interest- Total cell lysates were separated on 10% SDS-PAGE gels and ingly, six out of 18 upregulated genes (GTP-cyclohydroxylase I, transferred to polyvinylidene diflouride membranes (Millipore, protein kinase JNK2, myocyte specific enhancer factor 2A Bedford, MA, USA). Membranes were blocked with a solution of (MEF2A), Ins(1,3,4,5)P4-binding protein (IP4BP), lymphocyte- 5% low-fat milk in tris-buffered saline and 0.05% Tween 20 for specific protein 1 (LSP1) and type 3 inositol 1,4,5-trisphosphate 1 h. Samples were incubated with primary antibodies overnight receptor (IP3R3) (Table 1)) are directly or indirectly regulated by 24–29 at 41C and then with horseradish peroxidase (HRP)-conjugated calcium. secondary antibodies for 1 h at room temperature.