AGING INDUCED UP-REGULATION OF NUCLEAR-BINDING ACTIVITIES OF OXIDATIVE STRESS-RESPONSIVE NF-KB TRANSCRIPTION FACTOR IN MOUSE CARDIAC-MUSCLE

The accumulation of lipofuscin to cardiomyocytes is a classical parame ter of aging and is believed to reflect oxidative stress, NF-kB transc ription factor complex is one of the cellular sensors which responds t o oxidative stress and regulates gene expression. Our purpose was to s tudy whether aging affects the level and distribution of DNA binding a ctivities of NF-kB transcription factors both in cardiac sarcoplasm an d nuclear extracts. We used electrophoretic mobility shift assays (EMS A) to characterize the DNA binding activities of NF-kB and two other t ranscription factors, AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p 52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MA D-3 were assayed with Western blots. Surprisingly, aging upregulated b y 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not s how any change during aging. Aging-induced increase in nuclear NF-kB p rotein-DNA binding activity was observed both by gel retardation and U V-crosslinking assays. In immunoblotting, the level of p52 component b ut not those of p50 and p65 components of NF-kB-complex was slightly i ncreased in nuclear fractions. Aging did not affect the sarcoplasmic l evels of p50, p52, and p65 proteins. Supershift EMSA assays showed tha t the nuclear NF-kB complex contained p50, p52, and p65 components. Th e level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions, Aging down-regulated the n uclear Sp-1 binding activities but did not affect AP-1 binding activit ies. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicat e that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expre ssion. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies. (C) 1996 Academic Press Limited