tretinoin and 25-hydroxycholesterol

We studied the effects of cholesterol, its oxidized derivatives mevalonate, and nuclear receptor agonists LXR, RXR, and FXR on the production of transforming growth factor-beta1 (TGF- beta1) by macrophages. After recruiting of macrophage monocytes into the focus of inflammation, the production of TGF-beta1 increased by 3.5 times in comparison with control macrophages. Cholesterol diet stimulated the production of TGF-beta1 by 2.5 times. Cholesterol directly stimulated macrophage production of TGF-beta1 in vitro, while addition of mevalonate to the incubation medium effectively reduced this induced production. Agonists of nuclear receptor sharply reduced the production of TGF-beta1 in recruited macrophages. Under conditions of inflammation, hypercholesterolemia can be a factor of fibrogenesis due to TGF-beta1 induction in macrophages, which depends on the products of mevalonate biochemical chain.

Topics: Alitretinoin; Animals; Cells, Cultured; Cholesterol; Farnesol; Hydroxycholesterols; Hydroxysteroids; Ketocholesterols; Lipopolysaccharides; Liver X Receptors; Macrophages; Male; Mevalonic Acid; Mice; Mice, Inbred C57BL; Orphan Nuclear Receptors; Receptors, Cytoplasmic and Nuclear; Retinoid X Receptors; Transforming Growth Factor beta; Tretinoin

Changes in the expression level of the skeletal muscle LIM protein 1 (SLIM1) in cultured A10 cells were monitored in response to 25-hydroxycholesterol (25-HC), an oxidized form of cholesterol present in the oxidized low-density lipoproteins. The level of SLIM1 mRNA was elevated in a time- and concentration-dependent manner by treatment of 25-HC. Expressions of smooth muscle (SM) alpha-actin and calponin-1 (CNN-1), early markers for SMC differentiation, were also increased by the 25-HC treatments. Expressions of all three genes (SLIM1, SM alpha-actin and CNN-1) were simultaneously elevated in the cells treated with 9-cis retinoic acid (RA). On the other hand, the SLIM1 expression induced by the 25-HC or 9-cis RA (as well as SM alpha-actin and CNN-1) was decreased by the treatment of 15d-PGJ2. Since the 25-HC, 9-cis RA and 15d-PGJ2 were ligands for the LXR, RXRalpha and PPARgamma respectively, there might be a functional positive cross-talk between LXR and RXRalpha pathways and a negative cross-talk between PPARgamma and LXR and/or RXRalpha pathways in the regulation of SLIM1 expression. The cells stably transfected with the expressional vector for SLIM1 also showed an elevation in the levels of SM alpha-actin and CNN-1. In addition, an over-production of SLIM1 in the cells resulted in a change in the cell-shape into a spindle-like form, which is identical to that observed after a prolonged treatment of the cells with cholesterol.

Topics: Actins; Alitretinoin; Animals; Aorta; Calcium-Binding Proteins; Calponins; Cell Differentiation; Cell Line; Cell Shape; Dose-Response Relationship, Drug; Gene Expression Regulation; Hydroxycholesterols; LIM Domain Proteins; Microfilament Proteins; Muscle Proteins; Muscle, Skeletal; Muscle, Smooth, Vascular; Prostaglandin D2; Rats; Time Factors; Tretinoin; Up-Regulation

The adiponectin receptors AdipoR1 and AdipoR2 have been identified to mediate the insulin-sensitizing effects of adiponectin. Although AdipoR2 was suggested to be the main receptor for this adipokine in hepatocytes, AdipoR1 protein is highly abundant in primary human hepatocytes and hepatocytic cell lines. Nuclear receptors are main regulators of lipid metabolism and activation of peroxisome proliferator-activated receptor alpha and gamma, retinoid X receptor (RXR), and liver X receptor (LXR) by specific ligands may influence AdipoR1 abundance. AdipoR1 protein is neither altered by RXR or LXR agonists nor by pioglitazone. In contrast, fenofibric acid reduces AdipoR1 whereas hepatotoxic troglitazone upregulates AdipoR1 protein in HepG2 cells. Taken together this work shows for the first time that AdipoR1 protein is expressed in human hepatocytes but that it is not a direct target gene of nuclear receptors. Elevated AdipoR1 induced by hepatotoxic troglitazone may indicate a role of this receptor in adiponectin-mediated beneficial effects in liver damage.

Topics: Adiponectin; Alitretinoin; Animals; Caco-2 Cells; Cell Line; CHO Cells; Chromans; Cricetinae; DNA-Binding Proteins; Fenofibrate; HeLa Cells; Hepatocytes; Humans; Hydroxycholesterols; Liver X Receptors; Orphan Nuclear Receptors; Peroxisome Proliferator-Activated Receptors; Pioglitazone; PPAR alpha; Receptors, Adiponectin; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Retinoid X Receptors; Thiazolidinediones; Tretinoin; Troglitazone; Up-Regulation

The infection and inflammation process is associated with disturbances in lipid and lipoprotein metabolism. The apolipoprotein E (apo E) plays an important role in the lipoprotein metabolism and has been linked to inflammatory disease such as atherosclerosis and Alzheimer disease. An anti-inflammatory effect has also been suggested. The heterodimer nuclear receptor Liver-X-Receptor(alpha)/Retinoid-X-Receptor (LXR(alpha)/RXR) is considered to be a transcription factor for apo E. The aim of this study was to determine whether lipopolysaccharide (LPS) (principal component of the outer membrane Gram-negative bacteria) has an effect on apo E secretion by intestinal mucosa cells, using the Caco-2 cell line. Differentiated Caco-2 cells grown on filter inserts were incubated apically with LPS and/or 25-hydroxycholesterol (25-OH chol) and 9 cis retinoic acid (9cRA), ligands of LXR and RXR, respectively. The apical and basolateral media were separately collected. Apo E was detected by specific antibodies after protein separation by Two-dimensional nondenaturing gradient gel electrophoresis and apo E secreted in the cell culture media was measured by enzyme linked immunosorbent assay (ELISA). Apo E mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). LXR(alpha) and RXR mass was analyzed by Western Blot. We demonstrate here that CaCo-2 cells secrete apo E, by either apical or basolateral sides, associated with a high-density like lipoprotein, with a stoke's diameter comprised between 7.10 and 8.16 nm. We show that only apical secretion is decreased by LPS in a dose and time dependent manner. This is associated with a decrease in apo E gene expression contrasting with an increase of Il-8, a chemokine factor. Moreover, we demonstrate that only basolateral apo E secretion by CaCo-2 is significantly increased by 25-OH chol and 9cRA while apical secretion remains unchanged. LPS does not decrease the 25-OH chol and 9cRA mediated apo E secretion in basolateral compartment, while apical secretion is diminished under these circumstances. Our results provide evidence for the polarized secretion of apo E by intestinal epithelium. They also demonstrate that apo E secretion by CaCo-2 cell line is decreased by LPS through an LXR(alpha)/RXR independent signaling pathway.

Topics: Alitretinoin; Apolipoproteins E; Caco-2 Cells; Cell Differentiation; Cell Polarity; Gene Expression Regulation; Humans; Hydroxycholesterols; Interleukin-8; Lipopolysaccharides; RNA, Messenger; Tretinoin

Neural apoptosis-regulated convertase (NARC)-1 is the newest member of the proprotein convertase family implicated in the cleavage of a variety of protein precursors. The NARC-1 gene, PCSK9, has been identified recently as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The 2 other known genes implicated in ADH encode the low-density lipoprotein receptor and apolipoprotein B. As an approach toward the elucidation of the physiological role(s) of NARC-1, we studied its transcriptional regulation.. Using quantitative RT-PCR, we assessed NARC-1 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that NARC-1 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. NARC-1 mRNA level was increased by cholesterol depletion but insensitive to liver X receptor activation. Human, mouse, and rat PCSK9 promoters contain 2 typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site.. PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of SRE-binding protein (SREBP)-2.

Topics: Alitretinoin; Animals; Atorvastatin; Base Sequence; Cell Line; Cholesterol; Consensus Sequence; DNA-Binding Proteins; Gene Expression Regulation; Hepatocytes; Heptanoic Acids; Homeostasis; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Liver X Receptors; Lovastatin; Mevalonic Acid; Mice; Orphan Nuclear Receptors; Promoter Regions, Genetic; Proprotein Convertase 9; Proprotein Convertases; Pyridines; Pyrroles; Quinolines; Rats; Receptors, Cytoplasmic and Nuclear; Regulatory Sequences, Nucleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Simvastatin; Sp1 Transcription Factor; Species Specificity; Sterol Regulatory Element Binding Protein 2; Transcription Factors; Tretinoin

The inflammation that occurs during rheumatoid arthritis or atherosclerosis is characterized by the release of large amounts of sPLA(2) (group IIA secretory phospholipase A(2)). We have shown previously that the sPLA(2) promoter in SMC (smooth-muscle cells) is activated by interleukin-1beta and cAMP-signalling pathways, through the interplay of multiple transcription factors [Antonio, Brouillet, Janvier, Monne, Bereziat, Andreani, and Raymondjean (2002) Biochem. J. 368, 415-424]. In the present study, we have investigated the regulation of sPLA(2) gene expression in rat aortic SMCs by oxysterols. We found that oxysterol ligands that bind to the LXR (liver X receptor), including 25-HC (25-hydroxycholesterol) and 22( R )-HC, cause the accumulation of sPLA(2) mRNA and an increased enzyme activity. Transient transfection experiments demonstrated that the sPLA(2) promoter is synergistically activated by 22( R )-HC in combination with 9- cis -retinoic acid, a ligand for the LXR heterodimeric partner RXR (retinoid X receptor). Promoter activity was also increased in a sterol-responsive fashion when cells were co-transfected with LXRalpha/RXRalpha or LXRbeta/RXRalpha. Mutagenesis studies and gel mobility-shift assays revealed that LXR/RXR heterodimers regulate sPLA(2) transcription directly, by interacting with a degenerated LXRE (LXR response element) at position [-421/-406] of the sPLA(2) promoter. Chromatin immunoprecipitation revealed the in vivo occupancy of LXR on the sPLA(2) promoter. In addition, the orphan nuclear receptor LRH-1 (liver receptor homologue-1) potentiated the sterol-dependent regulation of the sPLA(2) promoter by binding to an identified promoter element (TCAAGGCTG). Finally, we have demonstrated that oxysterols act independent of interleukin-1beta and cAMP pathways to activate the sPLA(2) promoter. In the present study, we have identified a new pathway activating sPLA(2) gene expression in SMCs.

Topics: Alitretinoin; Animals; Cells, Cultured; Cyclic AMP; DNA-Binding Proteins; Enzyme Induction; Group II Phospholipases A2; Hydroxycholesterols; Interleukin-1; Liver X Receptors; Muscle, Smooth, Vascular; Orphan Nuclear Receptors; Phospholipases A; Phospholipases A2; Promoter Regions, Genetic; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Response Elements; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Transcriptional Activation; Tretinoin

In an attempt to identify transcription factors which activate sterol-regulatory element-binding protein 1c (SREBP-1c) transcription, we screened an expression cDNA library from adipose tissue of SREBP-1 knockout mice using a reporter gene containing the 2.6-kb mouse SREBP-1 gene promoter. We cloned and identified the oxysterol receptors liver X receptor (LXRalpha) and LXRbeta as strong activators of the mouse SREBP-1c promoter. In the transfection studies, expression of either LXRalpha or -beta activated the SREBP-1c promoter-luciferase gene in a dose-dependent manner. Deletion and mutation studies, as well as gel mobility shift assays, located an LXR response element complex consisting of two new LXR-binding motifs which showed high similarity to an LXR response element recently found in the ABC1 gene promoter, a reverse cholesterol transporter. Addition of an LXR ligand, 22(R)-hydroxycholesterol, increased the promoter activity. Coexpression of retinoid X receptor (RXR), a heterodimeric partner, and its ligand 9-cis-retinoic acid also synergistically activated the SREBP-1c promoter. In HepG2 cells, SREBP-1c mRNA and precursor protein levels were induced by treatment with 22(R)-hydroxycholesterol and 9-cis-retinoic acid, confirming that endogenous LXR-RXR activation can induce endogenous SREBP-1c expression. The activation of SREBP-1c by LXR is associated with a slight increase in nuclear SREBP-1c, resulting in activation of the gene for fatty acid synthase, one of its downstream genes, as measured by the luciferase assay. These data demonstrate that LXR-RXR can modify the expression of genes for lipogenic enzymes by regulating SREBP-1c expression, providing a novel link between fatty acid and cholesterol metabolism.

Topics: Alitretinoin; Base Sequence; CCAAT-Enhancer-Binding Proteins; Cell Line; Cholesterol; DNA-Binding Proteins; Humans; Hydroxycholesterols; Liver; Molecular Sequence Data; Promoter Regions, Genetic; Receptors, Retinoic Acid; Retinoid X Receptors; Sterol Regulatory Element Binding Protein 1; Trans-Activators; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

To determine whether differentiation therapy is useful in treating patients with hepatoma, we assayed the effects of 25-hydroxycholesterol (25-OH) and/or all-trans retinoic acid (ATRA) on rat AH136B ascites hepatoma cells. Flow cytometric DNA analysis showed that treatment of cells with 25-OH alone induced entry into the sub-G1 phase in a dose-dependent manner. While ATRA alone was ineffective, it enhanced the activity of 25-OH. Condensed and fragmented nuclei occurred mainly in cells treated with 25-OH (4 microg/ml). When cells treated with 25-OH (4 microg/ml), or 25-OH (4 microg/ml) + ATRA (1 microM) were transplanted into Donryu rats, we found that tumor development was completely inhibited; in contrast, rats administered the methanol-treated AH136B cells developed tumors. These findings suggest that AH136B cells in the sub-G1 phase can not recover tumorigenicity, and that the administration of a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, such as 25-OH, and ATRA may be effective in treating patients with hepatoma.

Topics: Animals; Carcinoma, Hepatocellular; Cell Transplantation; Diagnosis, Differential; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Liver Neoplasms, Experimental; Male; Rats; Tretinoin; Tumor Cells, Cultured

Receptors for retinoids in the immature rat ovary and the effects of retinol and retinoic acid on luteinizing granulosa cells were studied. Radioreceptor assay demonstrated the presence of specific cellular retinol-binding protein and cellular retinoic acid-binding protein in the ovaries of rats injected with PMSG alone or PMSG and hCG. In addition, when luteinizing granulosa cell from PMSG/hCG-injected immature rats were cultured with or without retinoic acid, the morphology, viability, number of cells in culture, and progesterone (P) accumulation were not affected by up to 10 microM retinoic acid. Beyond 10 microM, the cells began to round up, which was associated with a decrease in cell viability. Surprisingly, the deleterious concentrations of retinoic acid increased progesterone accumulation significantly higher than the medium control value. This increase in progesterone, however, was not accompanied by an increase in cAMP. When cells preincubated for 2 days with 1 microM of either retinoic acid or retinol were subsequently incubated in retinoid-free medium containing various substrates for steroidogenesis, the following results were obtained. Basal progesterone and its accumulation in response to human low density lipoprotein were significantly higher in cells preincubated with retinoids than in the control cells. However, no difference was seen in the degree of stimulation between retinol and retinoic acid pretreatments. Both 25-hydroxycholesterol, a substrate for side-chain cleavage enzyme, and pregnenolone, a substrate for 3 beta-hydroxysteroid dehydrogenase, significantly stimulated the accumulation of progesterone in cells preincubated with retinoids over the control value. Again, no appreciable difference was observed between retinol and retinoic acid pretreatments. Our results suggest that receptors for retinoids are present in gonadotropin-primed immature rat ovaries, retinoids increase luteal cell progesterone accumulation, and no difference exists between retinol and retinoic acid in their ability to increase the accumulation of progesterone by these cells.

Topics: 3-Hydroxysteroid Dehydrogenases; Animals; Carrier Proteins; Cells, Cultured; Corpus Luteum; Cyclic AMP; Female; Granulosa Cells; Hydroxycholesterols; Lipoproteins, LDL; Luteal Cells; Progesterone; Rats; Rats, Inbred Strains; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Vitamin A