INCREASE OF CALCIUM LEVELS IN EPITHELIAL-CELLS INDUCES TRANSLOCATION OF CALCIUM-BINDING PROTEINS MIGRATION-INHIBITORY FACTOR-RELATED PROTEIN-8 (MRP8) AND MRP14 TO KERATIN INTERMEDIATE FILAMENTS

Migration inhibitory factor-related protein 8 (MRP8) and MRP14, two S-100-like Ca2+-binding proteins, have been described in cells of the epithelial lineage where they are either expressed constitutively (e.g. by mucosal squamous epithelium) or induced during disease (e.g. in keratinocytes during the course of psoriasis). Their biological function, however, is not yet clear. Recent studies have provided evidence that S-100-like proteins may interact with cytoskeletal components; we have therefore studied the biochemical properties and subcellular distribution of MRP8 and MRP14 in epithelial cells. TR146 human squamous carcinoma cells, which were found to express MRP8 and MRP14 in Northern and Western blot studies, were chosen for analysis. Cross-linking experiments using bis(sulpho succinimidyl)suberate followed by SDS/PAGE and Western blot analysis revealed formation of heteromeric MRP8-MRP14 complexes. On subjecting TR146 cell lysates to two-dimensional gel electrophoresis and Western blotting, four distinct MRP14 isoforms could be identified resembling those described earlier in macrophages. A differential centrifugation technique revealed a Ca2+-dependent translocation of MRP8-MRP14 from the cytoplasm to the membrane and the Nonidet P40-insoluble cytoskeletal fraction. Double-label immunofluorescence microscopy of Ca2+ ionophore A23187-stimulated TR146 cells and cytochalasin B and demecolcine cytoskeleton disruption studies identified these structures as keratin intermediate filaments. Ca2+-dependent binding of MRP8-MRP14 to keratin filaments was additionally confirmed by an in vitro binding assay. In conclusion, our data suggest that MRP8 and MRP14 may be involved in Ca2+-dependent reorganization of cytoskeletal filaments in epithelial cells, which could be of importance for events associated with differentiation and inflammatory activation.