Identification of a peroxisome-proliferator-activated-receptor response element in the apolipoprotein E gene control region.

Apolipoprotein E (apoE) is a protein involved in reverse cholesterol transport. Among other tissues, apoE is expressed in macrophages where its expression increases when macrophages develop into foam cells. It has been recently shown that peroxisome-proliferator-activated receptor gamma (PPARgamma) is involved in this conversion. Northern-blot analysis was carried out in the macrophage cell line THP1 to determine whether apoE mRNA levels were regulated by ciglitazone, a PPARgamma inducer. The results indicated that treatment with ciglitazone doubled the levels of apoE mRNA. To identify a possible PPARgamma response element (PPRE), several portions of apoE gene control region were used to construct luciferase reporter plasmids. In U-87 MG cells, a 185 bp fragment located in the apoE/apoCI intergenic region was sufficient to induce a 10-fold increase in the luciferase activity of the extract of cells co-transfected with a PPARgamma expression plasmid. Subsequent analysis revealed the presence of a sequence with a high level of sequence similarity to the consensus PPRE. Mutations in this sequence resulted in a lack of functionality both in transient transfection and in electrophoretic-mobility-shift assays. These results demonstrated the presence of a functional PPRE in the apoE/apoCI intergenic region. These results have implications for the regulation of apoE gene expression and could be relevant for understanding the anti-atherogenic effect of thiazolidinediones.

Insulin-like growth factor I receptor gene expression during postnatal development of rabbit kidney.

BACKGROUND: Insulin-like growth factor-I (IGF-I) is a peptide growth factor whose biological effects are mediated through a specific receptor (IGF-IR). IGF-I and IGF-IR are detected in both fetal and adult kidneys and have both metabolic and growth effects. IGF-IR expression during postnatal kidney development is not well defined and the biological role of this receptor during the postnatal stage is not clearly established. The purpose of the present study was to analyze IGF-IR gene expression during the postnatal development of rabbit kidney to achieve a better understanding of the correlation between growth and differentiation of kidney tissues and IGF-IR expression. METHODS: Using in situ hybridization, we studied changes in IGF-IR expression in the kidneys of newborn rabbits and those up to 35 days old. Evaluation of the stage of kidney development and morphological maturation was made on histological sections stained with hematoxylin-eosin. RESULTS: High levels of IGF-IR gene expression in the rabbit kidney occurred in the last stages of postnatal development and in the adult stages; during the development of the subcapsular metanephrogenic zone, IGF-IR gene expression was not observed. IGF-IR mRNA was expressed by proximal and distal tubules and by collecting ducts after these tissues attained morphological maturation. The appearance of IGF-IR mRNA in these kidney structures followed a precise temporo-spatial sequence. IGF-IR was not expressed by renal corpuscles, Henle's loops, inner medullary collecting ducts, vessels, or interstitial cells at any study stage. CONCLUSIONS: The temporal and spatial patterns of IGF-IR gene expression during postnatal development of the rabbit kidney suggest that IGF-IR and its ligands are relevant for the acquisition of the function, and not for development events, by proximal and distal tubules and collecting ducts. This study also suggests that IGF-IR mRNA localization constitutes a useful marker to determine the functional maturation of these renal structures.

Apoptosis induced by methylazoxymethanol in developing rat cerebellum: organization of the cell nucleus and its relationship to DNA and rRNA degradation.

We present a cytological and biochemical study of the cell death of granule cell precursors in developing rat cerebellum following treatment with the cytotoxic agent methylazoxymethanol (MAM) during the first postnatal week. The density of apoptotic figures per square millimeter progressively increases after 6, 12, 24 and 44 h of treatment, whereas cells immunoreactive for proliferating cell nuclear antigen tend to disappear in the external granular layer (EGL). DNA migration on gel electrophoresis reveals a typical ladder pattern of internucleosomal cleavage following MAM treatment, whereas gel electrophoresis of rRNA shows a conspicuous degradation of both 28S and 18S rRNAs. Ultrastructural analysis has revealed the alterations of structures containing chromatin and ribonucleoprotein (RNP) in dying cells of the EGL. The typical granular beaded configuration of the condensed chromatin changes to a denser, more homogeneous texture suggesting nucleosomal disruption. The reorganization of RNP nuclear domains is reflected by the appearance of dispersed nucleoplasmic RNP particles and the formation of a coiled-body-like structure. However, typical nuclear domains involved in the splicing of RNAs, namely interchromatin granule clusters and typical "coiled bodies", are not found in apoptotic cells. Intranuclear bundles of filaments have also been detected. In the cytoplasm, the presence of dispersed single ribosomes is an initial sign of apoptosis. The massive dispersion and disruption of ribosomes detected after 24 h and 44 h of MAM treatment is reflected by the degradation of both 28S and 18s rRNAs. These results show that MAM treatment provides a useful experimental model for the study of apoptosis in the developing central nervous system. The organization of the cell nucleus in cells undergoing apoptosis clearly reflects a disruption of the nuclear compartments involved in transcription and the processing and transport of RNA and is related to the patterns of DNA and rRNA degradation.

Induction of insulin-like growth factor receptor (IGF IR) mRNA levels by low density lipoproteins.

Smooth-muscle cell (SMC) proliferation is a major step in the development of atherosclerotic lesions. Insulin-like growth factor (IGF I) is a progression factor for smooth muscle cells. We have previously reported an increased level of expression of insulin-like growth factor I receptor (IGF IR) gene in SMC of atherosclerotic plaques of rabbit aortas. Here we report that low density lipoproteins (LDL) produced an almost twofold increase in the steady state levels of IGF I R mRNAs in cultured smooth muscle cells of the rat aortic cell line A10. The increase was specific for LDL and was not detected in the presence of high density lipoproteins (HDL). These results indicated that increased IGF IR mRNA levels may be related to the genesis of the atherosclerotic lesion and suggested a new explanation for the atherogenic effect of LDL.

Expression of insulin-like growth factor receptor mRNA in rabbit atherosclerotic lesions.

We have found by 'in situ' hybridization a high level of expression of insulin- like growth factor I receptor (IGF-I R) gene in foam cells of atherosclerotic rabbit aortas. By reaction with either anti- rabbit macrophage (RAM11) or anti smooth muscle cell antibodies we also found that most cells expressing increased amounts of IGF-I R mRNA were of smooth muscle cell origin. Thus, increased IGF-I R mRNA levels might be related to the genesis of the atheroma plaque.

Expression of apolipoprotein E in cholesterol-loaded macrophages of extrahepatic tissues during experimental hypercholesterolemia.

To study the expression of extrahepatic apolipoprotein E (apoE) under hypercholesterolemic conditions, apoE mRNA levels were evaluated in 14 tissues of rabbits fed on a cholesterol rich diet and compared to age-matched control animals. In hypercholesterolemic rabbits apoE expression was significantly induced in adipose tissue, adrenals, aorta, lung and spleen. The increase in apoE mRNA levels in lung and spleen was associated with the presence of cholesterol-loaded macrophages. These cells were found to express high levels of apoE mRNA as demonstrated by in situ mRNA hybridization. Our results suggest that extrahepatic tissues would be partially responsible for the rise in serum apoE levels detected under hypercholesterolemic conditions.