RESUMO
UNLABELLED: Liver sinusoidal endothelial cells (LSECs) display a number of receptors for efficient uptake of potentially injurious molecules. The receptors for the Fc portion of immunoglobulin G (IgG) antibodies (FcgammaRs) regulate a number of physiological and pathophysiological events. We used reverse transcription polymerase chain reaction (RT-PCR) and Western blotting to determine the expression of different types of FcgammaRs in LSECs. Biochemical approaches and immunofluorescence microscopy were used to characterize the FcgammaR-mediated endocytosis of immune complexes (ICs). FcgammaRIIb2 was identified as the main receptor for the efficient uptake of ICs in LSECs. The receptor was shown to use the clathrin pathway for IC uptake; however, the association with lipid rafts may slow the rate of its internalization. Moreover, despite trafficking through lysosomal integral membrane protein-II (LIMP-II)-containing compartments, the receptor was not degraded. Finally, it was shown that the receptor recycles to the cell surface both with and without IC. CONCLUSION: FcgammaRIIb2 is the main receptor for endocytosis of ICs in rat LSECs. Internalized ICs are degraded with slow kinetics, and IC internalization is not linked to receptor downregulation. After internalization, the receptor recycles to the cell surface both with and without ICs. Thus, FcgammaRIIb2 in rat LSECs is used as both a recycling receptor and a receptor for efficient IC clearance.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD/metabolismo , Endocitose/imunologia , Fígado/imunologia , Receptores de IgG/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/análise , Antígenos CD/genética , Western Blotting , Membrana Celular/química , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/genética , Células Endoteliais/imunologia , Cinética , Fosforilação , Transporte Proteico , Ratos , Ratos Wistar , Receptores de IgG/análise , Receptores de IgG/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina/metabolismoRESUMO
BACKGROUND: Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. RESULTS: By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. CONCLUSION: Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dexametasona/metabolismo , Hepatócitos/metabolismo , Insulina/metabolismo , Receptores Depuradores Classe B/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Colesterol/metabolismo , Humanos , Macrófagos/metabolismo , Ratos , Ratos Wistar , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.
Assuntos
Ácido Quenodesoxicólico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hepatócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor X Retinoide alfa/metabolismo , Receptores Depuradores Classe B/biossíntese , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , HDL-Colesterol/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Receptor X Retinoide alfa/agonistas , Transdução de SinaisRESUMO
Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Imunológicos/metabolismo , Receptores de Esteroides/agonistas , Animais , Sequência de Bases , Antígenos CD36 , Células Cultivadas , Primers do DNA , Fígado/metabolismo , Receptor de Pregnano X , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Receptores Imunológicos/genética , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.
Assuntos
Colágeno/metabolismo , Hepatócitos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Mitogênicos/metabolismo , Animais , Anticorpos/farmacologia , Cálcio/metabolismo , Bovinos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Eptifibatida , Concentração de Íons de Hidrogênio , Radioisótopos do Iodo/metabolismo , Ligantes , Fígado/citologia , Fígado/metabolismo , Ovalbumina/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Desnaturação Proteica , Inibidores da Bomba de Prótons , Ratos , Ratos Wistar , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Excessive cellular cholesterol is transported to the liver by a pathway called 'reverse cholesterol transport.' Scavenger receptor class B, type I (SR-BI) mediates cholesterol uptake in the liver. Polyunsaturated fatty acids, known to activate peroxisome proliferator-activated receptor (PPAR), have been reported to increase hepatic cholesterol uptake. We found in the present study that PPARgamma induces expression of SR-BI in rat hepatocytes, liver endothelial cells, and Kupffer cells. In contrast, PPARalpha increased SR-BI levels only in hepatocytes and liver endothelial cells. PPARgamma/RXR binds to a response element between -459 and -472 bp in the human SR-BI promoter. Furthermore, hepatocyte nuclear factor 4alpha (HNF4alpha) was found to enhance PPARgamma-mediated SR-BI transcription. Thiazolidinedione (TZD)-activated PPARgamma/RXR increased hepatic SR-BI levels, which may lead to increased hepatic cholesterol uptake and less accumulation of lipids in peripheral tissues. The present results are in agreement with previous reports, indicating that specific PPARgamma-agonists (such as TZDs) protect against atherosclerosis.