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First published online 13 May 2003
doi: 10.1242/jcs.00471

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S100A13 mediates the copper-dependent stress-induced release of IL-1 from both human U937 and murine NIH 3T3 cells

¹ Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME 04074, USA
² Department of Oncology, Catholic University of Rome, School of Medicine, Rome 00168, Italy
³ Department of Geriatric Medicine, University of Florence, School of Medicine, Florence 50139, Italy

View larger version (18K): [in a new window]   Fig. 1. The stress-induced release of IL-1, FGF1 and S100A13 is Cu2+-dependant. (A) Recombinant human IL-1 (1 µg in 100 mM Tris HCl, pH 7.2) was adsorbed to a Cu2+-chelating column (Hi-Trap, Amersham), the column was washed with five column volumes of 0.2 M sodium phosphate buffer, pH 7.2 and eluted with an imidazole gradient in the wash buffer. The column was stripped with 50 mM EDTA and all fractions including the flow-through fraction were resolved by 15% acrylamide SDS-PAGE and evaluated using an IL-1 antibody. (B) Conditioned medium obtained from heat-shocked PMA-stimulated U937 and stable FGF1 U937 cell transfectants was processed by Cu2+ chelator affinity chromatography. Eluted proteins were resolved by 15% acrylamide SDS-PAGE, and evaluated by IL-1 (top-left panel), FGF1 (top-right panel) and S100A13 (bottom panel) immunoblot analysis. (C) NIH 3T3 cells stably transfected with IL-1 were either incubated for 18 hours at 37°C in the absence and presence of the Cu2+ chelator, TTM or for 2 hours in the absence or presence of the specific calpain inhibitor, ZLL, as indicated and the untreated and treated cells either maintained at 37°C or subjected to heat shock. The conditioned medium was processed and evaluated for IL-1 release as described in A. Cell lysates from TTM- and ZLL-treated cells were used to monitor the intracellular level of IL-1 expression. The TTM- and ZLL-negative control cell lysates exhibited a similar level of IL-1 expression (data not shown).

View larger version (16K): [in a new window]   Fig. 2. S100A13 is involved in the release of IL-1. (A) (left panel) The interaction of recombinant human IL-1 with S100A13 was assessed by the incubation of these proteins in 100 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl, followed by ultracentrifugation and S100A13 immunoblot analysis of the pellet fractions; (right panel) the Cu2+-dependent interaction of S100A13 with itself was assessed using the ultracentrifugation method described in the left panel. (B) Myc-S100A13 and IL-1-ßGal, insert-less vector and IL-1-ßGal NIH 3T3 cell cotransfectants were subjected to heat shock. Conditioned media were collected and processed as described (LaVallee et al., 1998). IL-1 was mmunoprecipitated with an IL-1 antibody, the immunoprecipitants were resolved by 8% and 12% acrylamide SDS-PAGE, respectively, and evaluated by IL-1 (top panel) and Myc (bottom panel) immunoblot analysis. (C) IL-1 NIH 3T3 cell transfectants were transiently transduced with a Myc-S100A13 adenoviral construct. 48 hours following transduction the cells were subjected to heat shock (42°C, 2 hours), cell lysates were obtained, immunoprecipitated with an anti-Myc antibody and resolved by IL-1 immunoblot analysis. (D) Myc-S100A13 and IL-1-ßGal NIH 3T3 cell cotransfectants and insert-less vector and IL-1-ßGal NIH 3T3 cell transfectants were evaluated for the release of IL-1-ßGal in the presence and absence of actinomycin D (10 µg/ml), as indicated, in response to heat shock. Conditioned media were processed and evaluated for IL-1-ßGal immunoblot analysis as described (Tarantini et al., 2001).

View larger version (52K): [in a new window]   Fig. 3. Heat shock induces the cytosolic redistribution of IL-1 and S100A13. NIH 3T3 cells were transiently cotransfected with IL-1-DsRed1 and EGFP-S100A13 constructs. 24 hours following transfection, the cells were fixed after a 2 hour incubation at either 37°C (a-c) or 42°C (d-f). Confocal images of median horizontal cell sections were obtained and the intracellular distribution of IL-1-DsRed1 (a,d), EGFP-S100A13 (b,e) and their respective overlays (c,f) recorded.

View larger version (24K): [in a new window]   Fig. 4. Deletion of the basic residue-rich C-terminus functions as a dominant negative effector of IL-1 Release. (A) The recombinant form of S100A13 lacking the BR domain (S100A13BR) was incubated with recombinant IL-1 as described in Fig. 2A at the molar ratios indicated. (B) S100A13BR mutant and IL-1-ßGal NIH 3T3 cell cotransfectants were subjected to heat shock and, following DTT treatment, conditioned media were concentrated and immunoprecipitated with anti-IL-1 antibody for the evaluation of IL-1 release. Immunoprecipitated proteins were resolved by 12% (w/v) SDS-PAGE, and evaluated by IL-1 immunoblot analysis. (C) S100A13BR mutant and IL-1 NIH 3T3 cell cotransfectants as well as insert-less vector and IL-1 NIH 3T3 cell cotransfectants were transiently transduced with a wild-type S100A13 adenoviral construct; 48 hours following transduction the cells were subjected to heat shock and, following DTT treatment, media conditioned at either 37°C or 42°C for 2 hours was resolved by IL-1 immunoprecipitation followed by Myc immunoblot analysis (12% acrylamide SDS-PAGE).

View larger version (22K): [in a new window]   Fig. 5. IL-1 exhibits the ability to displace carboxyfluorescein from the dioleylphosphoglycerol micelles. DOPG liposomes containing CF were prepared as described in Materials and Methods; the micelle was introduced into the cuvette at a 1:60 dilution in 10 mM HEPES, pH 7.0 containing 150 mM NaCl and allowed to equilibrate. At the 120 second time point, the experiment was initiated by introducing either 0.1% Triton X-100 (positive control, ), 0.5 µM of -Chymotrypsin (negative control, —), and different concentrations of IL-1, 0.5 µM (), 0.25 µM (), 0.125 µM () 0.062 µM (x) and 0.031 µM ().