Species differences in sequence and activity of the peroxisome proliferator response element (PPRE) within the acyl CoA oxidase gene promoter

In rats and mice, peroxisome proliferators (PP) cause liver enlargement, he patocarcinogenesis and peroxisome proliferation associated with induction o f enzymes such as acyl CoA oxidase (ACO). However, humans appear to be non- responsive to the adverse effects of PPs such as ACO induction. PPs activat e the peroxisome proliferator activated receptor alpha (PPAR alpha) that bi nds to DNA at peroxisome proliferator response elements (PPREs) within the promoters of PP-responsive genes. When the human ACO promoter was cloned pr eviously (Varanasi et al., 1996. Journal of Biological Chemistry, 271, 2147 -2155), it was reported to contain a PPRE (5' AGGTCA C TGGTCA 3') that boun d PPAR alpha and could be activated in vitro by Wyeth-14,643 (at >1 mM) or DEHP (at > 1.5 mM). In contrast, when we cloned the ACO gene promoter from a human liver biopsy, it was non-responsive to PPs and differed at three po sitions (5' AGGTCA G CTGTCA 3') from that reported previously (Woodyatt et al., 1999. Carcinogenesis, 20, 369-375). Subsequent to this, Varanasi et al . re-sequenced their constructs and obtained the same sequence as we have d escribed (Varanasi et al., 1998. Journal of Biological Chemistry, 273, 3083 2). However, the observation that the errant sequence (5' AGGTCA C TGGTCA 3 ') was able to bind PPAR alpha still remained since it appears that this se quence was used by Varanasi et al. (1996) to design oligonucleotides for th eir DNA binding analyses. Thus, if the 5' AGGTCA C TGGTCA 3' sequence did e xist in some individuals, it could be active. To address this, we used site -directed mutagenesis to create a promoter fragment that contained the erra nt sequence. This reporter gene was transfected into NIH3T3 cells together with a plasmid expressing mPPAR alpha, and assessed for its ability to driv e PP-mediated gene transcription using a non-toxic concentration of Wyeth-1 4,643 (100 mu M). This human ACO promoter was also inactive, unlike the equ ivalent rat ACO promoter fragment used as a positive control. Next, we used site directed mutagenesis to convert the PPRE found in the active rat ACO promoter (3' AGGACA A AGGTCA 5') to our inactive human sequence (AGGTCA G C TGTCA). This human PPRE was unable to drive PP-induced gene transcription e ven in the context of the rat ACO promoter suggesting that the activity of the rat promoter is conferred principally by the PPRE sequence, even though it may be enhanced by flanking sequences. These data confirm that neither the native nor the errant human ACO gene PPRE can respond to PPs. The absen ce of a responsive PPRE contributes to our understanding of the lack of res ponse of humans to some of the adverse effects of the PP class of non-genot oxic hepatocarcinogens. (C) 1999 Elsevier Science Ireland Ltd. All rights r eserved.