NUCLEAR FACTOR KAPPA-B FUNCTIONS AS A NEGATIVE REGULATOR FOR THE RAT ANDROGEN RECEPTOR GENE AND NF-KAPPA-B ACTIVITY INCREASES DURING THE AGE-DEPENDENT DESENSITIZATION OF THE LIVER

Transcriptional regulation of the steroid hormone receptor genes plays a central role in temporal changes of target cell sensitivity during development, maturation, and aging, Sequence-specific DNA-protein interactions mediate these regulatory functions, Progressive 5' deletion of the rat androgen receptor (rAR) gene immediately beyond the -572 base pair (bp) region causes a marked increase in its promoter activity. DNase I footprinting with nuclear proteins revealed a protected area encompassing -574- to -554-bp positions that begins with a perfectly palindromic nuclear factor kappa B (NF-kappa B) motif. Electrophoretic mobility shift analyses (EMSA) showed that the decameric rAR NF-kappa B site at positions -574 to -565 cross-competes with the authentic kappa immunoglobulin light chain enhancer for specific protein binding, Supershift with specific antibodies to NP-kappa B subunits confirmed that the two retarded bands observed in the EMSA with the labeled rAR probe are due to p50/p65 and p50/p50 dimers of the NF-kappa B/Rel proteins, Fragments of rAR promoter with either deletion or point mutation of the NF-kappa B site are found to be about 2- to S-fold more effective as compared to the wild type control in driving a heterologous reporter gene in cellulo. Thus, unlike most other known cases, NF-kappa B acts as a negative regulator for the rAR gene, The physiological relevance of this repressor function is evident from a 10-fold increase in the p50/p50 form of the NF-kappa B activity in the liver of aged rats exhibiting hepatic androgen desensitization. The newly identified repressor element is a rare example of a naturally occurring perfect palindromic binding motif for the NF-kappa B/Rel family of transcription factors, This repressor factor and the positively acting age-dependent factor, ADF, described earlier (Supakar, P. C., Song C. S., Jung, M., H., Slomczynska, M. A., Kim, J.-M., Vellanoweth, R. L., Chatterjee, B. & Poy, A. K. (1993) J. Biol. Chem. 268, 26400-26408) function to coordinate the tissue-specific down-regulation of the rAR gene during aging.