S100A12 Is Expressed Exclusively by Granulocytes and Acts Independently from MRP8 and MRP14

Abstract

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/ MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.Granulocytes and monocytes are major effector cells during inflammatory processes. Undue activation of these cells is a pathophysiological factor in many diseases, e.g. rheumatoid arthritis and chronic inflammatory bowel disease (1-4). In monocytes and granulocytes, intracellular Ca 2ϩ regulates various acute response activities, such as respiratory burst, phagocytosis, degranulation, and release of degrading enzymes (5-8). One molecular pathway of calcium signal transduction is calcium-binding proteins of the S100 multigene family, which comprises a group of small, acidic proteins with a tissue-and cell cycle-specific expression (9 -11). S100 proteins contain two calcium-binding sites per molecule (12). Two members of this family have been found in human granulocytes and monocytes, called macrophage migration inhibitory factor-related protein 8 (MRP8) 1 (S100A8) and MRP14 (S100A9), which represent up to 40% of the calcium binding capacity in monocytes (13,14). Both proteins form noncovalently associated complexes in a calciumdependent manner (15, 16). These complexes translocate from cytoplasm to membranes, as well as to intermediate filaments, after elevation of intracellular Ca 2ϩ levels, and this correlates with the induction of inflammatory actions of granulocytes and monocytes. Thus, MRP8 and MRP14 seem to be important regulators of cytoskeletal/membrane interactions during phagocyte activation (17-19).The calcium-induced change in the complex pattern of these two proteins has been considered to be important for their biological function (15,19). For example, only the ...

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“…4 and 6). Most of the S100 proteins formed homodimers, but when both MRP8 and MRP14 were expressed, the heterodimer MRP8/MRP14 was preferred (17,52). More sensitive immunochemical labeling will be necessary to determine which MRP oligomeric structure is involved, but we can reasonably suggest that the heterodimer MRP8/MRP14 is indeed implicated.…”

Monitoring neutrophil activation in juvenile rheumatoid arthritis by S100A12 serum concentrations

“…Concentrations of S100A12 were determined by a double sandwich enzymelinked immunosorbent assay (ELISA) system established in our laboratory, as described previously (27). Antibodies and protein standards were generated as reported previously (16). The ELISA was calibrated with recombinant S100A12 in concentrations ranging from 0.25 ng/ml to 250 ng/ml.…”

“…The importance of the innate immune system in chronic arthritis is emphasized by the fact that the most effective available therapies, such as anti-tumor necrosis factor ␣ (anti-TNF␣) agents, are specifically targeting phagocytes (13)(14)(15). S100A12 (EN-RAGE; calgranulin C) is expressed and secreted by granulocytes (16,17). Secreted S100A12 exhibits strong chemotactic activity (18).…”

The three-dimensional structure of human S100A12

“…S100B has also been shown to form heterodimers with S100A6 (Yang et al, 1999) and S100A1 with S100A4 (Wang et al, 2000;Tarabykina et al, 2000), while S100A8 forms heterodimers with S100A9 (Edgeworth et al, 1991). In contrast, S100A12 does not form heterocomplexes with the two other calgranulins S100A8 and S100A9 (Vogl et al, 1999).…”