Aging-dependent proteolysis of NF-κB in human fibroblasts

We investigated the NF-kB-like factor induced in the late-passage human oral mucosal fibroblasts stimulated with interleukin-l (IL-l). Compared with the NF-kBs Of HeLa cells and early-passage fibroblasts, the NF-kB-like factor of late-passage (passage 15) fibroblasts migrated faster in the electrophoretic mobility shift assay (EMSA) and behaved like a 70-80 kDa protein in the gel filtration chromatography. Both antibodies against p50 and p65 subunits of NF-kB could supershift the small NF-kB-like factor of late-passage cells in the EMSAs. A 47-kDa band was detected in late-passage fibroblasts by immunoblotting against p50. The mobility of the trypsin-degraded NF-kB of HeLa cells corresponded to that of the small NF-kB-like factor of late-passage fibroblasts in the EMSAs. Furthermore, when the nuclear extracts of the IL-1-stimulated HeLa cells were incubated with those of the IL-1-stimulated old fibroblasts, the p65-p50 NF-kB band disappeared, leaving behind a small NF-kB-like band. This reduction of NF-kB was prevented by the addition of a cysteine protease inhibitor leupeptin. These results suggest that the small NF-kB-like factor of late-passage fibroblasts is a part of the NF-kB truncated by aging-induced protease(s). (C) 2000 Wiley-Liss, Inc.