Pregnane X Receptor and Yin Yang 1 Contribute to the Differential Tissue Expression and Induction of CYP3A5 and CYP3A4

The hepato-intestinal induction of the detoxifying enzymes CYP3A4 and CYP3A5 by the xenosensing pregnane X receptor (PXR) constitutes a key adaptive response to oral drugs and dietary xenobiotics. In contrast to CYP3A4, CYP3A5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. Using cell lines and mice transgenic for a CYP3A5 promoter we demonstrate that the CYP3A5 expression in these organs is non-inducible and independent from PXR. Instead, it is enabled by the loss of a suppressing yin yang 1 (YY1)-binding site from the CYP3A5 promoter which occurred in haplorrhine primates. This YY1 site is conserved in CYP3A4, but its inhibitory effect can be offset by PXR acting on response elements such as XREM. Taken together, the loss of YY1 binding site from promoters of the CYP3A5 gene lineage during primate evolution may have enabled the utilization of CYP3A5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. Our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. They also suggest an explanation for the considerable tissue expression differences between CYP3A5 and CYP3A4

The effect of PCN on the expression of firefly luciferase driven by 6.2 kb of a human <i>CYP3A5</i> promoter in the duodenum, kidney, adrenal gland, and lung of transgenic mice.

<p>Mice (3 males and 3 females per treatment group) were injected i.p. with PCN (50 mg/kg) or the DMSO solvent. Organ homogenates were assayed with luciferase reporter gene assay (Promega) using a luminometer. Data are represented as ratio of RLU per µg protein of PCN over DMSO, shown as mean values (± SEM). Statistically significant differences are indicated by asterisks (*** p<0.001).</p

Sequence comparison and distribution of regulatory elements in the human <i>CYP3A4</i> and <i>CYP3A5</i> proximal promoters.

<p>Identical nucleotides are denoted by asterisks. The 57 bp region absent from the <i>CYP3A5</i> promoter is represented as a stretch of hyphens. The transcription start sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Iwano1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Hashimoto1" target="_blank">[38]</a> are indicated by arrows. The sequence is numbered relative to the transcription start site taken as +1. The binding sites for previously characterized transcriptional regulators CCAAT-box, ER6, BTE, TATA-box, and NF1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Iwano1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Hashimoto1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Saito1" target="_blank">[39]</a> are underlined. The portion of the NF1 binding site described to constitute a CCAAT box, the YY1 site, and the two E-box motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Biggs1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#pone.0030895-Saito1" target="_blank">[39]</a>, all contained in the 57 bp region, are boxed. The positions of binding sites are shown separately for <i>CYP3A4</i> and, if applicable, in brackets for <i>CYP3A5</i>.</p

Mutational analysis of the <i>CYP3A4</i>-derived YY1 binding site expressed (A) in MDCK.2 cells in the <i>CYP3A5-57ins</i> promoter construct and (B) in LS174T cells in the native <i>CYP3A4</i> promoter.

<p>The uppercase and lowercase letters represent preferred and tolerated nucleotides, respectively. The bolded and underlined letters indicate the mutated nucleotides. The mutations either restore the consensus core motif (M1), or progresively disrupt the YY1 binding site (M2 to M7). The construction of <i>CYP3A4</i> and <i>CYP3A5</i> mutants is described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030895#s4" target="_blank">Materials and Methods</a>.” Promoter-driven firefly luciferase activities were normalized using activities of the co-transfected renilla luciferase driven by a constitutive promoter and compared in (A) to that of the <i>CYP3A5</i>-ins57 construct and in (B) to that of the wild-type <i>CYP3A4</i>. Data are expressed as mean values (±SEM) of four to eight independent experiments, conducted as triplicates. Statistically significant differences are indicated by asterisks (** p<0.01,*** p<0.001).</p

Genomic and functional conservation of the 57 bp <i>CYP3A</i> promoter region in primates.

<p>(A) Representation of the evolution of the 57 bp region. Deletions are shown as stretch of hyphens, with the widest one corresponding to the deletion of the entire 57 bp region. 1a–b and 2 indicate the two alternative scenarios of the 57 bp deletion. “CYP” has been removed from gene names to improve legibility. A 7 bp fragment present only in all <i>CYP3A43</i> genes has been removed for clarity and it position in the human CYP3A43 gene is indicated by an arrow. (B) The effect of galago CYP3A91- and CYP3A92-derived 57 bp regions on the human CYP3A5 promoter activity in MDCK.2 cells. Data are expressed as mean values (±SEM) of five independent experiments conducted as triplicates. Promoter-driven firefly luciferase activities in the individual wells were normalized using activities of the co-transfected renilla luciferase driven by a constitutive promoter. Statistically significant differences are indicated by asterisks *p<0.05, ***p<0.001.</p

The activities of proximal <i>CYP3A4</i> (374 bp) and <i>CYP3A5</i> (370 bp) promoters in kidney-derived MDCK.2 cells (A) and in small intestine-derived LS174T cells (B).

<p>Data are expressed as mean values (±SEM) of six independent experiments conducted as triplicates. Promoter-driven firefly luciferase activities in the individual wells were normalized using activities of the co-transfected renilla luciferase driven by a constitutive promoter. Statistically significant differences are indicated by asterisks (*** <i>p</i><0.001).</p

The effect of the <i>CYP3A4</i>-derived 57 bp region on the activities of the proximal <i>CYP3A4</i> and <i>CYP3A5</i> promoters in MDCK.2 cells.

<p>(<b>A</b>) The effect of a deletion of the 57 bp region from the proximal <i>CYP3A4</i> promoter, or of its replacement with an unrelated “spacer” (SP) sequence of identical length. (<b>B</b>) The effect of the insertion of the 57 bp region, or of the “spacer” into the <i>CYP3A5</i> promoter. Data are expressed as mean values (± SEM) of three to six independent experiments conducted as triplicates. Promoter-driven firefly luciferase activities in the individual wells were normalized using activities of the co-transfected renilla luciferase driven by a constitutive promoter. Statistically significant differences are indicated by asterisks (** <i>p</i><0.01,*** <i>p</i><0.001).</p

The effect of PXR overexpression on the <i>XREM-CYP3A4</i>-driven luciferase activity in MDCK.2 cells.

<p>The wild-type 374 bp <i>CYP3A4</i> and the chimeric <i>XREM-CYP3A4</i> constructs were transiently transfected in MDCK.2 cells. (+) and (−) indicate transfection with a PXR-expressing plasmid and with the same empty plasmid, respectively. Data are expressed as mean values (± SEM) of three to five independent experiments conducted as triplicates. Promoter-driven firefly luciferase activities in the individual wells were normalized using activities of the co-transfected renilla luciferase driven by a constitutive promoter. Statistically significant differences are indicated by asterisks (*** <i>p</i><0.001).</p