Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model.

Cholesterol is an essential structural constituent of cellular membranes and a precursor molecule for the synthesis of steroid hormones, bile acids, and regulatory oxysterols. Cholesterol homeostasis is achieved by a series of regulatory steps that control cholesterol biosynthesis, its uptake from plasma lipoproteins, and the conversion of cholesterol to bile acids (27, 45). The prevailing view has been that cholesterol biosynthetic reactions occur in the cytoplasm and endoplasmic reticulum. However, recent studies suggest that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes, since all of the enzymes required for the conversion of acetyl coenzyme A (acetyl-CoA) to farnesyl diphosphate (FPP) are localized in peroxisomes and the enzymes catalyzing the conversion of mevalonate to FPP appear to be exclusively peroxisomal (30). Proteins imported into the peroxisome matrix use either C-terminal tripeptide (PTS 1) or N-terminal nonapeptide (PTS 2) peroxisome targeting signals that are recognized by the cytosolic receptors Pex5p and Pex7p, respectively (40). Both peroxisomal import pathways are utilized by peroxisomal cholesterol biosynthetic enzymes, and PTS1 and PTS2 are found in an alternating order (30). 3-Hydroxy-3-methylglutaryl (HMG) CoA reductase, localized in the endoplasmic reticulum and peroxisomes, is the only enzyme in the cholesterol biosynthetic pathway required for the conversion of HMG-CoA to mevalonate for which we, as yet, have no peroxisomal targeting information. The importance of peroxisomes for normal cellular functioning is illustrated by the disorders associated with the Zellweger spectrum (Zellweger syndrome [ZS], neonatal adrenoleukodystrophy and infantile Refsum's disease). All of these diseases are characterized by a loss of functional peroxisomes resulting in the presence of matrix enzymes in the cytosol (21). In Zellweger syndrome (ZS), the most severe of these disorders, infants have central nervous system neuronal migration defects associated with neonatal hypotonia, seizures, and hepatic dysfunction. The effect of peroxisomal dysfunction on cholesterol biosynthesis in cells and tissues from patients with the Zellweger spectrum disorders has been examined, with conflicting results (30). Briefly, reduced plasma cholesterol levels have been found in Zellweger spectrum patients (30). Normal enzyme activities for mevalonate kinase (MvK), mevalonate diphosphate decarboxylase (MPD), and isopentenyl diphosphate isomerase (IPPI) have been measured in fibroblasts from ZS patients in one study (54), but decreased MvK activity was found in fibroblasts from ZS and neonatal adrenoleukodystrophy patients in another study (6). While three groups reported significantly decreased cholesterol biosynthesis in fibroblasts from Zellweger spectrum patients (25, 34; J. C. Collins, F. Keyserman, S. C. Rumsey, and R. J. Deckelbaum, Am. Heart Assoc. abstr. 1219, PG I-228, 1993), two other studies showed that cholesterol biosynthesis rates were equivalent to or slightly higher than those in control fibroblasts (33, 37). A study by Krisans et al. (31) analyzing liver samples from Zellweger spectrum patients has shown that enzymatic activities of HMG-CoA reductase, MvK, phosphomevalonate kinase, MPD, IPPI, and FPP synthase were reduced, while another group reported decreased or normal MvK activities in livers of ZS patients (54). In addition, one group reported a decrease in cholesterol biosynthesis in peroxisome-deficient PEX2−/− hamster cells (1), while another group reported increased cholesterol biosynthesis in the same cell lines (52). Recently, peroxisome-deficient mouse models for human ZS have become available for study. In one such model, the PEX5 knockout mouse, the cholesterol biosynthesis rate was normal in immortalized as well as primary PEX5−/− mouse fibroblast cultures (53). Cholesterol levels were also normal in the plasma, brain, and liver of newborn PEX5−/− mice, while hepatic enzyme activities for HMG-CoA reductase and IPP isomerase were slightly elevated (26). These PEX5-deficient mice die within 24 h after birth and thus cannot be used to study postnatal effects (2). The second mouse model for human ZS has a targeted deletion of the PEX2 gene (13), and homozygous mutants survive in the early postnatal period up to 13 days (12, 14). Thus, the PEX2 mouse provides an excellent model to assess the effect of peroxisomal deficiency on cholesterol homeostasis in the early postnatal period. The focus of our study was to characterize the effects of peroxisome deficiency on an array of cholesterol biosynthetic enzymes, bile acid proteins, ATP-binding cassette (ABC) transporters, transcription factors, and nuclear receptors that are involved in maintaining cholesterol homeostasis. Our data illustrate that the loss of peroxisomes leads to an upregulation of the sterol regulatory element-binding protein 2 (SREBP-2) pathway in the liver and a corresponding increase in the activities and the protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, due to the loss of compartmentalization, the cholesterol biosynthetic enzymes function with altered catalytic efficiency. The rate of cholesterol biosynthesis is increased in most tissues, except in the brain and kidneys, where it is reduced. The cholesterol contents of all tissues examined were found to be similar in wild-type and PEX2 knockout mice, with the exception of the liver, where the cholesterol content is significantly reduced in the PEX2 knockout mice. In addition, compared with wild-type mice, PEX2−/− mice have decreased plasma total and high-density lipoprotein (HDL) cholesterol levels. The classic pathway of bile acid biosynthesis is downregulated, while the alternate pathway is unexpectedly increased. Our studies demonstrate that there is a loss of coordinate regulation of cholesterol homeostasis in the PEX2 knockout mice and emphasize the importance of the peroxisome in this process.