Leukemia (1997) 11, 1290–1297  1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Upregulation of CD9 expression during TPA treatment of K562 cells F Le Naour, C Francastel, M Prenant, O Lantz, C Boucheix and E Rubinstein INSERM U268, Ho ˆ pital Paul Brousse, 94807 Villejuif Cedex, France The CD9 antigen, a major platelet glycoprotein, is a member of expressed by megakaryocytes and platelets, on which it is a the tetraspan superfamily. We show that treatment of K562 major surface protein with 35 000–60 000 molecules per cell, cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which a level of expression comparable to that of glycoprotein induces megakaryocytic differentiation, leads to a seven-fold IIb/IIIa. 29–31 Because it is recognized by platelet-activating increase in CD9 expression, which becomes associated with mAbs, the CD9 antigen has been suggested to play a role in the integrin 1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of platelet activation. However, platelet activation is triggered the megakaryocytic-specific marker GPIIb (CD41) as well as after recruitment of the platelet Fc receptor, a mechanism also integrins 3 (GPIIIa/CD61), v (CD51) and VLA-2 (CD49b). Both found with activating mAbs directed against other target anti- GPIIb/IIIa expression and CD9 upregulation are dependent on gens. 32,33 Another possibility is that CD9 antigen could play protein kinase C (PKC) activation since they are blocked by the a role in the differentiation of megakaryocytes. In this paper, specific inhibitor GF109203X. Steady-state levels of CD9 and we have employed the K562 model for megakaryocytic differ- GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to entiation and have shown that treatment of these cells with accumulate during TPA treatment. However, the increase of the 12-O-tetradecanoylphorbol-13-acetate (TPA) results in an CD9 mRNA was detected much earlier than the increase of early increase of CD9 expression preceding the acquisition of GPIIb mRNA (1–2 h vs 24–48 h). Using different constructs of other megakaryocytic markers. the 5′-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregul- Materials and methods ation in the megakaryocytic lineage could occur at early stages of differentiation. Cell lines and induction of differentiation Keywords: CD9; tetraspan; K562; megakaryocytic differentiation; TPA K562 is a pluripotent hematopoietic cell line initially derived from a patient with a Philadelphia chromosome-positive chronic myelogenous leukemia. 34 HEL is derived from an Introduction erythroleukemia. 35 Nalm6 and Reh6 cell lines, respectively CD9 positive and CD9 negative or weakly positive, are The CD9 antigen is a 24 kDa glycoprotein member of the derived from B lineage acute lymphoblastic leukemias. CEM, tetraspan superfamily. 1,2 These proteins are characterized by Raji and Daudi are CD9-negative cell lines, respectively four transmembrane domains with intracytoplasmic extremi- derived from a human T cell leukemia and from Burkitt lym- ties and sequence homologies that distinguish them from the phomas. The cells were cultured in RPMI 1640 medium sup- other proteins with four transmembrane domains. Several dif- plemented with 10% FCS, 2 mML-glutamine and antibiotics ferentiation and tumoral antigens are members of the tetras- (all from Gibco, Gaithersburg, MD, USA). 12-O-tetra- pan family, including CD37, CD53, CD63, CD81/TAPA-1, decanoylphorbol-13-acetate (TPA) and hemin were added to CD82, CD151/PETA-3, L6, Co029 and TALLA-1. 3–5 The differ- give a final concentration of 16 nM (10 ng/ml) and 100 M, ent tetraspans may derive from the duplication of an ancestral respectively. Erythroid differentiation was evaluated by detec- gene because they are encoded by genes with similar genomic tion of benzidine-stainable hemoglobin after addition of structure. 6–11 Furthermore, two proteins of the tetraspan fam- 100 l of benzidine solution to 100 l of cells suspended in ily were found in the parasite worm Schistosoma 12,13 and one culture medium. 36 Cells were incubated on ice for 5 min and is expressed by motoneurons in Drosophila 14 which strongly examined for benzidine positivity (blue staining). Cyclohexi- suggests an early appearance in evolution. mide (2 and 10 g/ml) and actinomycin D (1 g/ml) (both The exact function of these molecules is still unknown. from Boehringer, Meylan, France), were added 30 min before Recent studies have suggested that CD9 antigen may be TPA treatment. The PKC inhibitor GF109203X (Calbiochem, involved in migration and adhesion processes 15–18 functions Meudon, France) was added 45 min before TPA treatment at that may be related to its association with VLA integrins. 17,19,20 the concentration of 5 M. The CD9 antigen is also associated with the transmembrane precursor of heparin-binding EGF (HB-EGF) and upregulates the juxtacrine activity of this receptor as well as its activity as Monoclonal antibodies and flow cytometry analysis the receptor for diphtheria toxin. 21,22 Certain members of this family have been reported to trigger an activation signal The CD9 mAb SYB-1 has been previously described. 17,32 Anti- (CD81, CD82 and CD53). 23–26 body Gi9 (VLA-2) was purchased from Immunotech (Luminy, The CD9 antigen is expressed in various tissues. In the France). The anti-VLA1 mAb C9 was provided by Dr Zardi hematopoietic tissue it is found at the surface of basophils, (Genoa, Italy). The GPIIb mAb D33C and the GPIIIa mAb B2A eosinophils, PHA-activated T lymphocytes and early B were obtained from Dr Gulino. 37 The glycophorin A mAb cells. 27,28 It is not found on erythrocytes but is strongly MAS 517P was purchased from Valbiotech (Paris, France) and the anti-integrin v mAb LM609 was purchased from Chemicon (Temecula, CA, USA). Correspondence: C Boucheix Received 19 July 1996; accepted 3 April 1997 Immunofluorescence analysis was carried out on a Profile