Lessons from the CYP3A4 promoter.

There is considerable interest in determining the molecular basis for human variation in drug response. Investigations over the past 20 years have largely focused on identifying polymorphisms in genes that encode drug metabolism enzymes. Significant progress has been made for many of the cytochromes P450, including CYP2D6, CYP2C19, and CYP2C9 (http://www.imm.ki.se/CYPalleles/). However, the molecular basis for individual variation in CYP3A4 has remained elusive. This is unfortunate, because CYP3A4 is the most abundant hepatic and intestinal cytochrome P450, catalyzes the metabolism of more than half of all drugs, and represents the primary route of elimination for many drugs. Some studies have reported a 10-fold variation in clearance of CYP3A probe drugs (Floyd et al., 2003; Rogers et al., 2003), although 90-fold variability in CYP3A4 protein expression has been reported in liver (Lamba et al., 2002a). Some of the variation occurs because in the general population, CYP3A activity can be influenced by concurrent administration of CYP3A inhibitors and inducers. Some variation in CYP3A activity stems from the fact that there are four human CYP3A genes (CYP3A4, CYP3A5, CYP3A7, and CYP3A43) in the 231-kb CYP3A locus located on chromosome 7q21–22.1 (Finta and Zaphiropoulos, 2000). The molecular basis for polymorphic expression of CYP3A5 and CYP3A7 in adults has been described (Kuehl et al., 2001), and CYP3A43 is expressed at too low a level to contribute significantly to hepatic CYP3A activity (Koch et al., 2002). Thus, the continuing quest is to determine the genetic basis for variable expression of CYP3A4, the isoform believed by many to be the major source of CYP3A-mediated drug metabolism. Ozdemir et al. (2000) reported a significant genetic component to variable CYP3A expression. Several groups resequenced the CYP3A4 coding region in attempts to identify single nucleotide polymorphisms (SNPs) or insertions/deletions (Sata et al., 2000; Eiselt et al., 2001; Lamba et al., 2002b). However, none of the SNPs are polymorphic, with frequencies of less than 1% in the populations studied (http:// www.imm.ki.se/CYPalleles/), and thus cannot be the major factors influencing CYP3A expression. Resequencing of the CYP3A4 promoter has identified multiple variants (Kuehl et al., 2001; Lamba et al., 2002b), including one common variant allele, a 392A3G transition that has a higher allelic frequency in African Americans than in white persons (Felix et al., 1998; Rebbeck et al., 1998; Wandel et al., 2000); however, studies of the relationship of this SNP to CYP3A4 expression have been inconclusive (Lamba et al., 2002a). Because the pregnane X receptor (PXR)/steroid and xenobiotic receptor is a major regulator of CYP3A-inducible expression (Blumberg et al., 1998; Lehmann et al., 1998), the proximal and distal PXR binding elements in CYP3A4 have also been resequenced but have so far shown no sequence variation (Kuehl et al., 2001; Lamba et al., 2002b). A consensus is building that human variation in CYP3A4 activity is caused by regulatory polymorphisms rather than structural polymorphisms in the CYP3A4 gene. The supportive evidence came first from a report that there is significant correlation in human livers between expression of CYP3A4 and CYP3A5 (in polymorphic expressors) (Lin et al., 2002). Further analysis revealed strong correlation between expression of each of the CYP3A family members (CYP3A4, CYP3A5, CYP3A7, and CYP3A43) and between CYP3A4 and PXR (Chang et al., 2003; Westlind-Johnsson et al., 2003). Although at first glance one could conclude that PXR is a likely transcription factor regulating constitutive CYP3A4 expression, mice nullizygous for PXR show either no change, or even a small decrease, in constitutive expression of hepatic CYP3A (Xie et al., 2000; Staudinger et al., 2001). Furthermore, human PXR sequence variants are so rare (Zhang et al., 2001) that they cannot explain variation in CYP3A expression. These findings, coupled with reports of significant correlation between the constitutive androstane receptor (CAR) and CYP3A4, CAR, and PXR (Pascussi et al., 2001) and between CYP2B6, CAR, CYP3A4, and PXR (Chang et al., 2003) in human liver strongly supports the notion that genetic variation in a common hepatic regulatory factor(s) underlies human variability in hepatic CYP3A expression. Thus, the article by Matsumara and colleagues (2004) in this issue of Molecular Pharmacology is particularly valuable in their further characterization of transcription factors important for hepatic regulation of CYP3A4. Matsumara et al. (2004) identify a region between 10.5 and 11.4 kb that functions as a constitutive liver enhancer module (CLEM4). Significantly, this region binds and is reg-