Aging-dependent proteolysis of NF-kappaB in human fibroblasts.
We investigated the NF-kappaB-like factor induced in the late-passage human oral mucosal fibroblasts stimulated with interleukin-1 (IL-1). Compared with the NF-kappaBs of HeLa cells and early-passage fibroblasts, the NF-kappaB-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-kappaB could supershift the small NF-kappaB-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-kappaB of HeLa cells corresponded to that of the small NF-kappaB-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-kappaB band disappeared, leaving behind a small NF-kappaB-like band. This reduction of NF-kappaB was prevented by the addition of a cysteine protease inhibitor leupeptin. These results suggest that the small NF-kappaB-like factor of late-passage fibroblasts is a part of the NF-kappaB truncated by aging-induced protease(s).
Ikebe
T
T
Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, Higashi-ku, Fukuoka, Japan. ikb@dent.kyushu-u.ac.jp
Jimi
E
E
Beppu
M
M
Takeuchi
H
H
Nakayama
H
H
Shirasuna
K
K
D000293:Adolescent; D000328:Adult; D000375:Aging / Q000502:physiology*; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004586:Electrophoresis; D005347:Fibroblasts / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D015151:Immunoblotting; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D020033:Protein Isoforms / Q000032:analysis
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009841:Oligonucleotides; D020033:Protein Isoforms; D000072000:NF-KappaB Inhibitor alpha; D010447:Peptide Hydrolases