Marianne Depreter

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARα-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARα independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

Biogenesis of peroxisomes in fetal liver

Peroxisomes are single membrane‐limited cell organelles that are involved in numerous metabolic functions. Peroxisomes do not contain DNA; the matrix and membrane proteins are encoded by the nuclear genome. It is assumed that new peroxisomes are formed by division of existing organelles. The present article gives an overview of microscopic studies and recent unpublished results dealing with peroxisome biogenesis in mammalian fetal liver and presents data on peroxisomes in oocytes.

Effects of extracellular matrix on the expression of peroxisomes in primary rat hepatocyte cultures.

BACKGROUND/AIMS Peroxisomes in wild-type cells vary between tissues and developmental stages. In the liver of some peroxisomal deficiency disorder patients, rare parenchymal cells express normal peroxisomes (mosaics); the mechanism is unknown. Our aim was to find factors regulating peroxisome expression. METHODS Liver-specific as well as peroxisome characteristics were studied in three types of primary rat hepatocyte cultures. RESULTS Total glutathione S-transferase activity and albumin secretion both increased in the collagen I sandwich and immobilization gel cultures. In contrast, in monolayers cultured on plastic, total glutathione S-transferase activity decreased and albumin secretion was only 30-40% compared to the collagen cultures. Glycogen rosettes typical of liver parenchymal cells were always abundant. Laminin and collagen IV-producing stellate cells were numerous in the monolayer but almost absent in the sandwich cultures. In 6-day-monolayer cultures, the number of liver-specific peroxisomes had decreased while atypical small or elongated peroxisomes appeared. Immunolabeling density for catalase and three beta-oxidation enzymes was decreased compared to adult rat liver; catalase specific activity in homogenates had dropped to 15% and 4% in the sandwich and monolayer cultures, respectively. In 17-day-sandwich cultures, some peroxisomes showed a very weak catalase reaction; total activity was 5%. Supplementation of the collagen type I cultures with several extracellular matrix factors could not prevent peroxisome dedifferentiation. CONCLUSION The presence of these extracellular matrix components is not sufficient for normal peroxisome expression. It is suggested that hepatocyte-specific and peroxisomal features are regulated differently. The sandwich preserves hepatocyte differentiation better than the monolayer.

Effect of epidermal growth factor in collagen gel cultures of rat hepatocytes.

Collagen gel cultures of hepatocytes represent a promising in vitro model in pharmaco-toxicology. Epidermal growth factor (EGF) is usually added to the culture medium, although one could question its value in a culture model aiming at maintaining a maximum of differentiated functional capacities. In this study, the effects of EGF (20 ng/ml) on albumin secretion, morphology and pentoxyresorufin O-depentylase (PROD), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities have been examined in both collagen gel sandwich and immobilization gel cultures of adult rat hepatocytes. Transmission electron microscopy did not show an obvious influence of epidermal growth factor (EGF) on the intracellular organization of organelles of the rat hepatocytes. It was found that EGF addition had no effect on albumin secretion in both culture models. On the contrary, the presence of EGF in the culture medium provoked in collagen gel sandwich cultures, after 7 days, significant decreases of 66% and 25% in EROD and PROD activities, respectively. On GST activities, no effect of EGF could be observed in both collagen gel cultures. Removal of EGF from the culture medium seemed to have a positive effect on the maintenance of the phase 1 biotransformation capacity of rat hepatocytes. Its addition should therefore be avoided in collagen gel cultures used in pharmaco-toxicology.

Maturation of the liver-specific peroxisome versus laminin, collagen IV and integrin expression

The interaction of cells with extracellular matrix components contributes to their specific differentiation. We studied hepatic peroxisomes and their changing features during embryonic development, and we immunolocalized in the same tissue the extracellular matrix components laminin and collagen IV as well as the integrin receptor subunits alpha 1, alpha 2, beta 1, and beta 4. Rat and human embryonic liver peroxisome expression were studied at the light- and electron-microscopic level by means of localizing catalase-, D-amino acid oxidase- and polyamine oxidase activities and by means of the immunocytochemistry of six peroxisomal proteins. The successive import of catalase and the peroxisomal beta-oxidation enzymes, the late appearance of the other enzymes, and the gradual increase of peroxisomal size and number to adult values occurred as asynchronous events. Although still immature, peroxisomes were recognized at every stage examined and coexisted with laminin and collagen IV in both species. The beta 1 integrin subunit was immunodetected as early as at 12.5 days in rat. It was concluded that these extracellular matrix factors may be important for the differentiation of liver parenchyma from the liverbud stage onwards. However, the stepwise maturation of the liver-specific peroxisome suggests the involvement of many other regulating factors.

Effect of extracellular matrix composition on the expression of glutathione S-transferase isoenzymes in organotypical hepatocyte cultures.

Collagen gel sandwich and immobilization cultures of hepatocytes, using hydrated collagen type I as extracellular matrix (ECM), have been proposed as long-term in vitro models in pharmaco-toxicology. The in vivo ECM composition in the space of Disse is, however, much more complex. As a differentiated hepatocyte phenotype is thought to be highly dependent on ECM composition and biophysical characteristics, we modulated the ECM to mimic the in vivo situation. Moreover, commercially available collagen type I (Boehringer-Ingelheim) was compared to the one prepared in the laboratory from rat tails. ECM composition had no effect on albumin secretion or hepatocyte morphology in both collagen gel sandwich and immobilization cultures. Total, Alpha and Mu class GST activities in organotypical cultures with a complex or a simple collagen type I ECM were similar. The Pi class GST activity increased as a function of culture time in all culture models. Thus, mimicking the in vivo composition of the ECM did not improve the changes in GST expression that were observed in simple collagen gel cultures. The collagen type I matrix is therefore assumed to confer sufficient protection to help the hepatocytes to maintain their differentiated phenotype to a certain extent. Moreover, we hypothesize that the collagen gel matrix may act as a scaffold to keep newly synthesized ECM components in the proximity of the basolateral surfaces of the hepatocytes.

Pharmacological Induction of Redundant Genes for a Therapy of X-ALD

X-linked adrenoleukodystrophy (X-ALD) is a recessive neurologic disease with an incidence among males of 1/17 000. Since the identification of the X-ALD gene (ABCD1) ten years ago (Mosser et al 1993), no satisfactory therapy has been available. A close homologue (ABCD2) was then cloned and presented as a putative modifier gene that could account for some of the extreme phenotypic variability of X-ALD (Lombard-Platet et al 1996). The inducibility of Abcd2 by the hypolipidemic drug fenofibrate in the liver of rodents (Albet et al 1997), correlated to a partial normalisation of the biochemical phenotype of X-ALD (Netik et al 1999), opened up the way of a pharmacological therapy of X-ALD. The basis of such a therapy and the results obtained chiefly in rodents will be presented in this chapter.

Modified Peroxisomes in Primary Hepatocyte Cultures

It is well known that when differentiated cells such as hepatocytes are cultured in vitro, they rapidly lose their specific features.

Regulation of Peroxisomal Genes by DHEA and Vitamin D

The aim of our study was to find factors that modify the phenotype of the normal peroxisomal compartment (number, size, shape, membrane proteins and enzyme content of the matrix) and/or the transcription and translation of genes involved in peroxisome biogenesis and function in normal wild-type cells.

Peroxisomes during Development and in Distinct Cell Types

In normal wild type cells of the same individual enzymatic content and activity of peroxisomes (Px) vary greatly with developmental stage and with cell-and tissue type; so do peroxisomal number, shape and volume. Wcell-and examples in humans are AGT and pipecolic acid oxidase, enzymes which are expressed only in liver parenchyma*, precluding their investigation in cultured fibroblasts and prenatal testing in chorionic villi. Since the genome is identical in all these circumstances, several delicate regulatory processes must be determining the final phenotype. Studying this variability could give insight into the control mechanisms, and possibly create opportunities to modify peroxisomal functions on purpose.