Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: novel link to expression of major histocompatibility complex class II genes

Abstract

NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.

FIGURE 1.

Accumulation of 24-DHL in the enterocytes of IE-Cpr-null mice. Enterocytes from two 4-month-old male WT or IE-Cpr-null mice were pooled. Sample extraction, preparation of trimethylsilyl derivatives, and GC/MS analysis were as described under “Experimental Procedures.” The data shown represent typical results from one of three independent experiments. Shown are extracted ion chromatograms (XIC) of m/z 395 from the analysis of 5 μg of 24-DHL standard, derivatized to form 24-DHL-TMS (A); a derivatized lipid extract from WT mice (B); and a derivatized lipid extract from IE-Cpr-null mice (C). The mass spectrum for the 24-DHL-TMS peak at 40.7 min (D) from the analysis of the lipid extract from IE-Cpr-null enterocytes is identical to the mass spectrum for the peak at 40.6 min (E) for the 24-DHL-TMS standard. A peak corresponding to 24-DHL-TMS was not detected in the derivatized extract from WT mice (B).

FIGURE 2.

Differential expression of genes related to antigen presentation and processing in the enterocytes of WT and IE-Cpr-null mice. RNA samples prepared from enterocytes of 2.5–3-month-old male mice (n = 3) were used for quantitative RNA-PCR analysis. The levels of various target transcripts were normalized to the level of GAPDH mRNA in the same RNA sample. Relative levels of each transcript in the two mouse strains were determined, and the results are shown in arbitrary units obtained by setting the GAPDH-normalized values for the WT samples to 1. The values represent mean ± S.D. (error bars). *, p ≤ 0.05; **, p ≤ 0.01. Data represent typical results from two experiments.

FIGURE 3.

Immunoblot analysis of MHC II protein expression in enterocytes of WT and IE-Cpr-null mice. A, mice were fasted overnight, and whole-cell lysates (30 μg/lane) were prepared from enterocytes of three individual, 3-month-old male WT or IE-Cpr-null mice and analyzed on immunoblot with an anti-mouse MHC II antibody. As a loading control, the same samples were also analyzed with an anti-GAPDH antibody. B, results from densitometry analysis. The two bands were combined for determination. Data represent typical results (normalized to GAPDH) from three experiments. *, p < 0.05. Error bars, S.D.

FIGURE 4.

Immunoblot analysis of STAT1 protein expression and phosphorylation in enterocytes of WT and IE-Cpr-null mice. A, whole cell lysates (40 μg/lane), prepared from enterocytes of individual 3-month-old male WT or IE-Cpr-null mice, were subjected to immunoblot analysis using either anti-STAT1 or anti-phosphorylated STAT 1. As a loading control, the same samples were also analyzed using an anti-β-actin antibody. B, results from densitometry analysis. For STAT1, the two bands were combined for determination. Data represent typical results (normalized to β-actin) from three experiments.

FIGURE 5.

Proposed mechanistic link between Por deletion and up-regulation of MHC II expression in enterocytes. Metabolic pathways are indicated by black arrows, whereas signaling pathways are represented by blue dashed arrows. Genes found to have increased expression, at either mRNA or protein level, and metabolites found to have increased levels in the IE-Cpr-null enterocytes are shown in red. The GTPase is expected to have increased activity, due to increased prenylation (as indicated by square brackets). The criteria for inclusion of genes in the cholesterol biosynthesis pathway were p < 0.05 and fold change > 25%. The metabolic step catalyzed by CYP51 is completely blocked (solid ×), whereas the step catalyzed by SQLE is partially inhibited (dashed ×), by the loss of POR, leading to accumulation of 24-dihydrolanosterol as well as the isoprenoids. Abbreviations and gene symbols not already listed in Table 1 or mentioned elsewhere include the following: Hmgcs1, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1; Hmgcr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Mvk, mevalonate kinase; FDPS, farnesyl diphosphate synthetase.