Relationship between the activation of heat shock factor and the suppression of nuclear factor-kappaB activity in rat hepatocyte cultures treated with cyclosporine A.
We investigated on primary cultures of rat hepatocytes the effect of cyclosporine A (CsA) on the activation of nuclear factor-kappaB (NF-kappaB), activator protein 1 (AP-1), and heat shock factor 1 (HSF1), three transcription factors involved in cellular response pathways. Hepatocytes were subjected to a time-course (1, 3, 6, and 22 hr) incubation and CsA treatment in the range 1-50 microM. NF-kappaB, AP-1, and HSF1 binding activities were established through electrophoretic mobility shift assay. Levels of HSP70 mRNA and protein were measured by Northern and Western blot analysis respectively. In cells incubated for 1 and 3 hr, electrophoretic mobility shift assay experiments showed a dose-dependent increase of the NF-kappaB binding activity; while following 22hr of incubation, a suppression of the positive effect of CsA at shorter times was detected. At all periods of incubation assayed, CsA induced the activation of AP-1 which was detected by DNA-binding activity of this transcription factor. A dose-dependent activation of HSF1 was observed at 22 hr of incubation. We conclude that in rat hepatocyte cultures, CsA induces the transcriptional activation of NF-kappaB, AP-1, and HSF1. However, the time point at which activation of each transcription factor occurs is different. Thus, at 22 hr of incubation, the CsA-induced activation of HSF1 is accompanied by the reduction of the positive effect of CsA on NF-kappaB activation at earlier time points.
Andrés
David
D
Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal sn, 28040 Madrid, Spain.
Díez-Fernández
Carmen
C
Castrillo
Antonio
A
Cascales
María
M
D000818:Animals; D016572:Cyclosporine / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; C000619709:Hsf1 protein, rat; D007166:Immunosuppressive Agents; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016572:Cyclosporine