Leukemia (2000) 14, 1105–1111 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu The role of RAR and RXR activation in retinoid-induced tissue factor suppression T Tenno1, J Botling2,FO¨ berg2, S Jossan1, K Nilsson2 and A Siegbahn1 Departments of 1Medical Sciences and 2Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden Excessive expression of tissue factor (TF) is a common finding cells.9–11 RA therapy is also widely used in treatment of APL in leukaemic cells and may contribute to thrombotic compli- patients, giving a remission rate over 90%.12 Nevertheless, cations in patients. Retinoic acid has been shown to induce differentiation and reduce TF expression in acute promyelo- there is a limited knowledge about the mechanisms underly- cytic leukaemia (APL) cells in vitro, and to induce remission in ing the processes of RA-induced TF suppression in APL and APL patients. Treatment of the APL cell line NB4 with the spe- other myeloid leukaemic cells. It is known that RA acts cific retinoic acid receptor-α (RARα) agonists Ro40–6055 or through nuclear steroid receptors: retinoic acid receptors TTNPB resulted in down-regulation of TF expression and in (RARα, β, γ) and retinoid X receptors (RXRα, β, γ). RA treat- β γ induction of differentiation. The activation of RAR , RAR or ment of various cells induces formation of RAR-RXR transcrip- retinoid X receptor (RXR) did not suppress the constitutive TF expression in NB4 cells. Moreover, the RARα antagonist Ro41- tional complexes on the RA response element (RARE) in pro- 5253 blocked the retinoid-induced down-regulation of TF. In moters of RA-regulated genes. Although RXR has been contrast, in the monoblastic U-937 cell line only a partial sup- considered as a silent transcriptional partner,13 the activation pression of TF antigen expression and activity was observed of both RXR and RAR is essential for full transcriptional by treatment with the RAR agonist TTNPB or the RXR agonist activity and induction of differentiation in myeloid cells.14,15 SR11237 alone. However, the combination of TTNPB and The abnormal PML/RARα gene in APL cells results in defective SR11237 resulted in a pronounced down-regulation of TF RA signalling and is responsible for the leukaemic phenotype expression and induction of differentiation in U-937 cells. We 16 show for the first time that the activation of both subunits of of these cells. It was shown that in NB4 cells the activation the RARα-RXR transcriptional complex is needed for TF sup- of RARα or RARβ is sufficient to induce TF down-regu- pression in U-937 cells, whereas in NB4 cells RARα activation lation.17,18 However, the abnormal retinoic acid signalling by alone is sufficient. Thus, distinct molecular mechanisms for TF the PML/RARα nuclear oncoprotein in NB4 cells does not suppression seem to be operating in leukaemic cell lines of allow us to expand these data on TF regulation in other different origin. Leukemia (2000) 14, 1105–1111. Keywords: tissue factor; RAR; RXR; differentiation myeloid and leukaemic cells. In this report, we have used RA nuclear-receptor-specific ligands to reveal the mechanisms of retinoid-induced TF sup- pression in the APL cell line NB4 and in the monoblastic cell Introduction line U-937. Although RARα activation can be shown to be sufficient for TF suppression in NB4 cells, we show that in a Tissue factor is the main initiator of the blood coagulation cas- cell line with a normal RARα gene the full suppression of TF cades. Expression of this protein is restricted to subendothelial requires activation of both RARα and RXR. Moreover, TF sup- tissues, and the peripheral blood cells and endothelial cells of pression in both of the cell lines was found to be coherent healthy individuals are negative for TF.1 Monocytes, however, with induction of general myeloid differentiation markers, are the only blood cells that can be induced to express this which might imply that TF suppression and cell differentiation protein.2 In septic conditions TF expression in monocytes is are regulated concomitantly. known to play a major role in development of disseminated intravascular coagulation (DIC).3 Moreover, pathological expression of TF on leukaemic cells seems to be an important Materials and methods finding for thrombotic complications.4 Acute promyelocytic leukaemia (APL) is the most common myeloid leukaemia Cells associated with thrombotic complications, ultimately leading to DIC.5 APL is a subtype of acute myelogenous leukaemia The NB4 cells, established from a human acute promyelocytic with the characteristic chromosome translocation that disrupts leukaemia (M3)19 (kindly provided by Inge Olsson, University the retinoic acid receptor (RAR)-α gene on chromosome 17 of Lund, Lund, Sweden), and the human monoblastic cell line and fuses it to the promyelocytic leukaemia (PML) gene at U-937 (subline U-937-1), derived from a patient with histo- chromosome 15, thus resulting in the PML/RARα fusion pro- cytic lymphoma20,21 were maintained in RPMI 1640 medium tein. Beyond pathological expression, TF is also expressed on supplemented with 10% foetal bovine serum (Sigma, St Louis, the surface of bone marrow derived myeloid progenitor cells.6 MO, USA), 1% of L-glutamine, 100 U/ml of penicillin and The role of the protein during myeloid maturation is however 50 µg/ml of streptomycin (Biochrom, Berlin, Germany). The still poorly understood. Thus, understanding the mechanisms cells were routinely screened for mycoplasma infection at reg- of the specific regulation of TF in different cells of myeloid ular intervals. The cells at a concentration of 2 × 105/ml were origin is of great importance for interventions aiming at allevi- induced with different retinoids: all-trans retinoic acid (ATRA; ating the thrombotic complications. Sigma), TTNPB, SR11237, Ro40-6055, Ro48-2249, Ro44- Retinoic acid induces differentiation and growth arrest in 4753 and Ro41-5253 (Table 1). Retinoids were dissolved in myeloid precursor cells and in leukaemic cells.7,8 This differ- ethanol to the 1 mM concentration and further dissolved in entiation process has been shown to be accompanied by sup- the culture medium. The final concentration of ethanol in the pression of TF expression in various myeloid leukaemia culture media was less than 0.1%, which was found to have no effect on the TF expression in these cells. During the period Correspondence: A Siegbahn; Fax: 46 18 55 25 62 of incubation we did not observe any changes in the cell Received 16 November 1999; accepted 31 January 2000 viability (.90%). Cell culture medium was checked for endo- The role of RAR and RXR in tissue factor suppression T Tenno et al 1106 Table 1 The specificities of the retinoids used in the study primers and probe. The PCR reaction was carried out in ABI PRISM 7700 Sequence Detection System (Perkin Elmer Ligand Receptor specificities Ref. Applied Biosystems) in a two-step protocol: 10 min at 95°C (denaturation of DNA and activation of AmpliTaq Gold) and ATRA RARα/β/γ agonist 26 50 cycles amplification by 15 s at 95°C and 1 min at 60°C. α β γ TTNPB RAR / / agonist 39 The real-time detection of amplification signal information Ro40–6055 (Am580) RARα agonist 26 Ro48–2249 RARβ agonist 26 was captured by the ABI PRISM 7700 and the appearance Ro44–4753 RARγ agonist 26 of the amplification signal, threshold cycle number (CT), was Ro41–5253 RARα antagonist 40 registered for each sample. The differences in TF mRNA α β γ SR11237 (BMS649) RXR / / agonist 24, 37 expression were determined by the comparative CT method as described in ABI PRISM 7700 User Bulletin No. 2. Briefly, β ∆ the difference in TF and -actin CT values ( CT) was determ- ∆ ined and calibrated to the CT value of the uninduced sample ∆∆ = ∆ − ∆ toxin contamination with limulus amebocyte lysate assay ( CT CTX CT0). The relative TF mRNA expression (Chromogenix AB, Mo¨lndal, Sweden), and endotoxin concen- related to uninduced sample is determined by evaluating the tration was found less than 5 pg/ml at working concentrations expression: 2−∆∆CT. of the reagents. Analysis of TF mRNA Flow cytometry Total RNA was isolated from 2 × 106 cells with TRIZOL The surface antigen expression was analysed by immunofluo- reagent (Life Technologies, St Louis, MO, USA). RNA concen- rescence with a flow cytometer (EPICS XL, Coulter Electronics, tration was determined at 260 nm with a spectrophotometer Hialeah, FL, USA). For indirect immunofluorescence experi- (SPECTRAmax 250, Molecular Devices, Sunnyvale, CA, USA), ment the cells were washed twice with PBS + 0.1% BSA, incu- and the integrity of 28S and 18S ribosomal RNA was checked bated for 30 min on ice with the primary antibody, washed in ethidium bromide-stained agarose gel. Total RNA (1 µg) twice, incubated for 30 min on ice with fluorescein-isothiocy- was subjected to a reverse transcription (RT) reaction for anate (FITC)-labelled rabbit anti-mouse (Dakopatts, Glostrup, 90 min at 37°Cin20µl volume containing 200 U MMLV RT Denmark) and finally washed twice. The primary antibodies enzyme, 0.5 mM dNTP mixture, 10 mM DTT, 0.25 µg were monoclonal antibodies to TF (TF9-9C3; American Diag- × oligo(dT)12–18 primer and 1 first strand buffer (50 mM Tris- nostica, Greenwich, CT, USA) and Leu M5 (CD11c) HCI, 75 mM KCI, 3 mM MgCl2) (Life Technologies). (Camfolio, Becton Dickinson, Mountain View, CA, USA). The The relative amount of reverse transcribed TF mRNA was expression of CD11b and CD49f was determined by FITC- quantitated with the TaqMan real-time polymerase chain reac- labelled monoclonal antibodies BEAR1 and GoH3, respect- tion (PCR) assay. The method is based on 59–39 nuclease ively (Immunotech, Marseilles, France).