Aging-dependent proteolysis of NF-κB in human fibroblasts

We investigated the NF-κB-like factor induced in the late-passage human oral mucosal fibroblasts stimulated with interleukin-1 (IL-1). Compared with the NFκBs of HeLa cells and early-passage fibroblasts, the NF-κB-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-κB could supershift the small NF-κB-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-κB of HeLa cells corresponded to that of the small NF-κB-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-κB band disappeared, leaving behind a small NF-κB-like band. This reduction of NF-κB was prevented by the addition of a cysteine protease inhibitor leupeptin. These results suggest that the small NF-κB-like factor of late-passage fibroblasts is a part of the NF-κB truncated by aging-induced protease(s).