BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 121–124 (1997) ARTICLE NO. RC976598 Sphingosine-1-phosphate Mobilizes Intracellular Calcium and Activates Transcription Factor NF- kB in U937 Cells Vladimir A. Shatrov,* , † Volker Lehmann,† and Salem Chouaib* *CJF 94-11 INSERM ‘‘Cytokines et Immunite ` Antitumorale,’’ Institut Gustave-Roussy, 94805 Villejuif, France; and †Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany Received March 24, 1997 lipid hydrolysis and arachidonic acid release (1,7,8). Sphingosine-1-phosphate (SPP), a metabolite of However, little is known about the effects of SPP on sphingolipids, has been implicated as a second mes- transcription factors. Recent studies have shown that senger in cell growth regulation and signal transduc- SPP markedly increased specific DNA binding activity tion via calcium mobilization from internal stores. of AP-1 in Swiss 3T3 fibroblasts (9). This study shows that SPP mobilizes intracellular cal- The inducible transcription factor NF-kB resides in cium in U937 cells and demonstrates for the first time the cytoplasm as an inactive form complexed to the the ability of SPP to activate the transcription factor inhibitory subunit IkB. Upon stimulation of cells with NF-kB in these cells. Furthermore, calcium release a large variety of agents including cytokines, mitogens, from the internal stores by thapsigargin (TG), an in- UV irradiation, LPS and viruses, NF-kB is rapidly acti- hibitor of the endoplasmic reticulum Ca 2/ pump, was vated by its release from IkB, which allows transloca- associated with activation of NF-kB. Moreover, we tion of the p50/p65 complex into the nucleus where it have shown that while an intracellular calcium chela- activates transcription of target genes such as interfer- tor BAPTA/AM was able to inhibit both SPP- and TG- ons, cytokines, cell adhesion molecules and hematopoe- induced NF-kB activation, it had no effect on TNF-in- tic growth factors (10). Here we demonstrate that SPP duced NF-kB activation. In addition, SPP-induced NF- kB activation was blocked both by cyclosporin A, mobilizes intracellular calcium and induces activation known to inhibit calcineurin phosphatase activity, of the transcription factor NF-kB in U937 cells. Our and by the antioxidant butylated hydroxyanisole. observations suggest that intracellular calcium re- These observations suggest that intracellular calcium lease, calcineurin- and redox-dependent mechanisms mobilization is required for SPP-induced NF-kB acti- may play a role in SPP-induced NF-kB activation. vation, which may involve calcineurin- and redox-de- pendent mechanisms.  1997 Academic Press MATERIALS AND METHODS Materials. SPP was purchased from BIOMOL (Plymouth, USA). Thapsigargin (TG), BAPTA/AM, and Indo-1 were obtained from CAL- BOICHEM (La Jolla, USA). Recombinant human TNF-a was pro- Sphingosine-1-phosphate (SPP), the initial product vided by BASF (Ludwigshafen, Germany). All other reagents were of sphingosine degradation by sphingosine kinase, is purchased from SIGMA (Munich, Germany). known to play an important role in cell growth regula- Cell culture. U937 monocytic cells were cultured in RPMI 1640 tion and signal transduction and is served as an intra- (GIBCO, Grand Island, NY), supplemented with 10% heat-inacti- cellular second messenger (1,2,3,4). Ghosh et al. (5,6) vated fetal calf serum (FCS), HEPES (0,02 M) and 50 mg/ml genta- demonstrated that sphingosine and SPP induced a mycin. rapid release of Ca 2/ from internal stores in smooth Ca 2/ fluorescence measurements. The assay of cytosolic Ca 2/ was muscle cells. They have shown that endoplasmic reticu- performed using Indo-1. Cells (1r10 6 /ml) were loaded with 5 mM Indo-1 at 37°C for 30 min and resuspended in calcium free medium lum membrane itself contains the sphingosine kinase containing 2 mM EGTA. After a base-line calcium level was mea- activity required to convert inactive sphingosine to SPP sured, SPP, TG or TNF were added, and changes in free calcium as well as the site at which Ca 2/ release was activated. levels were measured by flow cytometry at 37°C, as previously de- Studies by Spiegel and co-workers revealed that SPP scribed (11). acts as a potent mitogen on Swiss 3T3 fibroblasts by a Electrophoretic mobility shift assay (EMSA). Preparation of nu- pathway that is independent of protein kinase C and clear and cytosolic extracts and conditions for electrophoresis mobil- can cause mobilization of Ca 2/ from internal calcium ity shift (gel shift) assay have been previously described in detail (12,13). Equal amounts of extracts were incubated with an NF-kB- stores through a mechanism independent of inositol 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 121 AID BBRC 6598 / 692a$$$481 04-22-97 09:24:33 bbrcg AP: BBRC