RESUMO
StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.
Assuntos
Fibroblastos/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células 3T3-L1 , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Immunoblotting , Técnicas In Vitro , Fígado/metabolismo , Lovastatina/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteróis/farmacologiaRESUMO
Cholesterol metabolized to 7alpha-hydroxylated bile acids is a principle pathway of cholesterol degradation. Cholesterol 7alpha-hydroxylase (CYP7A1) is the initial and rate-determining enzyme in the "classic pathway" of bile acid synthesis. An "alternative" pathway of bile acid synthesis begins with 27-hydroxylation of cholesterol by 27-hydroxylase (CYP27), followed by 7alpha-hydroxylation by oxysterol 7alpha-hydroxylase (CYP7B1). The aim of the current study was to investigate the regulation of CYP7B1 by bile acids, cholesterol, and thyroid hormone in a previously well-studied in vivo model of bile acid synthesis, and to compare its regulation to that of CYP7A1. Three study groups were examined. In the first, male Sprague-Dawley rats with intact enterohepatic circulations were fed normal chow (controls), cholestyramine (CT), cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), or cholesterol (Chol). In the second group, taurocholate (TCA) was continuously intraduodenally infused for 48 hours to chronic biliary diverted rats. In a third set of studies, squalestatin, an inhibitor of cholesterol synthesis, was intravenously infused for 48 hours. In a fourth set of studies, the diurnal variation in CYP7B1 was compared to that of CYP7A1. At the end of each study livers were harvested, and CYP7B1 and CYP7A1 activities and mRNA levels were determined. Complete biliary diversion significantly increased the specific activity (SA) of both CYP7B1 ( upward arrow 212%; P <.002) and CYP7A1 ( upward arrow 212%; P <.007). Intraduodenal infusion of TCA to rats with biliary diversion decreased SA of both CYP7B1 ( downward arrow 29%; P <.001) and CYP7A1 ( downward arrow 46%; P <.01). The addition of CA, CDCA, or DCA to rat chow led to downregulation of CYP7B1 SAs by 42% (P <.003), 51% (P <.009), and 47% (P <.003), and CYP7A1 SAs by 32% +/- 6% (P <.003), 73% +/- 9% (P <.002), and 60% +/- 13% (P <.004), respectively. CT feeding upregulated both CYP7B1 ( upward arrow 136%; P <.004) and CYP7A1 ( upward arrow 216%; P <.001) SAs. While Chol feeding significantly upregulated CYP7A1 SA, no significant increase in CYP7B1 SA was found. Conversely, as previously shown in vitro, inhibition of cholesterol synthesis significantly suppressed both CYP7A1 and CYP7B1 activity and mRNA levels. Both CYP7B1 and CYP7A1 underwent diurnal variation, with peak and trough values for CYP7B1 lagging approximately 6 hours behind CYP7A1. We conclude that, in the rat, like CYP7A1, CYP7B1 demonstrates diurnal rhythm and is regulated by bile acids and cholesterol.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Fígado/enzimologia , Fígado/fisiologia , Esteroide Hidroxilases/biossíntese , Animais , Ácidos e Sais Biliares/fisiologia , Northern Blotting , Colesterol/fisiologia , Ritmo Circadiano/fisiologia , Sistema Enzimático do Citocromo P-450/genética , Duodeno/fisiologia , Feminino , Hormônios/fisiologia , Infusões Intravenosas , Intubação Gastrointestinal , Masculino , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Ensaios de Proteção de Nucleases , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Esteroide Hidroxilases/genéticaRESUMO
StarD4 protein is a member of the StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins that includes StarD5 and StarD6, proteins whose functions remain poorly defined. The objective of this study was to isolate and characterize StarD4's sterol binding and to determine in a hepatocyte culture model its sterol transport capabilities. Utilizing purified full-length StarD4, in vitro binding assays demonstrated a concentration-dependent binding of [(14)C]cholesterol by StarD4 similar to that of the cholesterol binding START domain proteins StarD1 and StarD5. Other tested sterols showed no detectable binding to StarD4, except for 7alpha-hydroxycholesterol, for which StarD4 demonstrated weak binding on lipid protein overlay assays. Subsequently, an isolated mouse hepatocyte model was used to study the ability of StarD4 to bind/mobilize/distribute cellular cholesterol. Increased expression of StarD4 in primary mouse hepatocytes led to a marked increase in the intracellular cholesteryl ester concentration and in the rates of bile acid synthesis. The ability and specificity of StarD4 to bind cholesterol and, as a function of its level of expression, to direct endogenous cellular cholesterol suggest that StarD4 plays an important role as a directional cholesterol transporter in the maintenance of cellular cholesterol homeostasis.
Assuntos
Colesterol/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Ácidos e Sais Biliares/biossíntese , Células Cultivadas , Dicroísmo Circular , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Camundongos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.
Assuntos
Proteínas de Transporte/análise , Colesterol/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Western Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HL-60 , Células HT29 , Humanos , Imuno-Histoquímica , Células de Kupffer/metabolismo , Fígado/citologia , Macrófagos/metabolismo , Microscopia de Fluorescência , Monócitos/metabolismo , Ligação Proteica , Esteróis/metabolismoRESUMO
Recently identified StarD5 belongs to the StarD4 subfamily, a subfamily of steroidogenic acute regulatory related lipid transfer (START) domain proteins that includes StarD4 and StarD6, proteins whose functions remain unknown. The objective of this study was to confirm StarD5's protein localization and sterol binding capabilities as measures to pursue function. Using rabbit polyclonal antibody against newly purified human histidine-tagged/StarD5 protein, StarD5 was detected in human liver. In parallel studies, increased expression of StarD5 in primary hepatocytes led to a marked increase in microsomal free cholesterol. Cell fractionation studies demonstrated StarD5 protein in liver cytosolic fractions only, suggesting StarD5 as a directional cytosolic sterol carrier. Supportive in vitro binding assays demonstrated a concentration-dependent binding of cholesterol by StarD5 similar to that of the cholesterol binding START domain protein StarD1. In contrast to selective cholesterol binding by StarD1, StarD5 bound the potent regulatory oxysterol, 25-hydroxycholesterol, in a concentration-dependent manner. StarD5 binding appeared selective for cholesterol and 25-hydroxycholesterol, as no binding was observed for other tested sterols. The ability of StarD5 to bind not only cholesterol but also 25-hydroxycholesterol, a potent inflammatory mediator and regulatory oxysterol, raises basic fundamental questions about StarD5's role in the maintenance of cellular cholesterol homeostasis.
Assuntos
Colesterol/metabolismo , Citosol/química , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Colesterol/análise , Hepatócitos/química , Humanos , Hidroxicolesteróis/metabolismo , Fígado/química , Ligação Proteica , Esteróis/metabolismoRESUMO
There are two major pathways of bile acid synthesis: the "neutral" pathway, initiated by highly regulated microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" pathway, initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In hepatocyte cultures, overexpression of CYP7A1 increases bile acid synthesis by >8-fold. However, overexpression of CYP27A1 in hepatocytes only increases it by 1.5-fold, suggesting that additional rate-limiting steps must be involved in the regulation of this pathway. The effects of intracellular cholesterol transport proteins on bile acid synthesis have been investigated in the current study. Under culture conditions in which the neutral pathway was inactive, selective overexpression of the gene encoding steroidogenic acute regulatory protein (StAR), MLN64 (StAR homolog protein), and sterol carrier protein-2 (SCP-2) led to 5.7-, 1.2-, and 1.7-fold increases, respectively, in the rates of bile acid synthesis in primary rat hepatocytes. Surprisingly, co-overexpression of MLN64 with StAR, SCP-2, or CYP7A1 blunted the upregulated bile acid synthesis by 48, 47, and 45%, respectively. These results suggest that MLN64, in its full-length form, is not responsible for the transport of cholesterol to the mitochondria or the endoplasmic reticulum, where CYP27A1 or CYP7A1 is located, respectively.
Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/biossíntese , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Fosfoproteínas/biossíntese , Adenoviridae/genética , Animais , Transporte Biológico , Western Blotting , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilase/metabolismo , Humanos , Masculino , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.
Assuntos
Ácidos e Sais Biliares/biossíntese , Colesterol/metabolismo , Hepatócitos/metabolismo , Animais , Transporte Biológico , Colestanotriol 26-Mono-Oxigenase , Expressão Gênica , Hepatócitos/ultraestrutura , Masculino , Microssomos Hepáticos/enzimologia , Mitocôndrias/metabolismo , Fosfoproteínas/genética , Ratos , Ratos Sprague-Dawley , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismoRESUMO
Bile acid synthesis (BAS) occurs mainly via two pathways: the "neutral" pathway, which is initiated by highly regulated microsomal CYP7A1, and an "acidic" pathway, which is initiated by mitochondrial CYP27A1. Previously, we have shown that overexpression of the steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol transport protein, increases bile acid biosynthesis more than 5-fold via the acidic pathway in primary rat hepatocytes. This observation suggests that mitochondrial cholesterol transport is the rate-limiting step of BAS via this pathway. The objective of this study was to determine the effect of increased StAR on rates of BAS in vivo. Overexpression of StAR and CYP7A1 were mediated via infection with recombinant adenoviruses. BAS rates were determined in chronic biliary-diverted rats and mice, and in mice with an intact enterohepatic circulation. The protein/messenger RNA levels of StAR and CYP7A1 increased dramatically following overexpression. Overexpression of StAR or CYP7A1 led to a similar 2-fold (P <.01) increase in BAS over up-regulated (approximately 2-fold) 3-day chronic biliary-diverted control rats. Additionally, overexpression of StAR led to more than 3- and 6-fold increases over controls in the rates of BAS in biliary-diverted and intact mice, respectively (P <.01). In conclusion, in both rats and mice in vivo, overexpression of StAR led to a marked increase in the rates of BAS initiated by delivery of cholesterol to mitochondria containing CYP27A1.
Assuntos
Ácidos e Sais Biliares/biossíntese , Hepatócitos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Expressão Gênica , Vetores Genéticos , Hepatócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Esteroide Hidroxilases/metabolismoRESUMO
Conversion of cholesterol into 7alpha-hydroxylated bile acids is a principal pathway of cholesterol disposal. Cholesterol 7alpha-hydroxylase (CYP7A1) is the initial and rate-determining enzyme in the "classic" pathway of bile acid synthesis. An "alternative" pathway of bile acid synthesis is initiated by sterol 27-hydroxylase (CYP27) with subsequent 7alpha-hydroxylation of 27-hydroxycholesterol by oxysterol 7alpha-hydroxylase (CYP7B1). The regulation of CYP7B1, possibly a rate-determining enzyme in the alternative pathway, has not been thoroughly studied. The aims of this study were to (1) study the regulation of liver CYP7B1 by bile acids, cholesterol, adenosine 3', 5'-cyclic monophosphate (cAMP), and phorbol myristate acetate (PMA) in primary rat hepatocytes and (2) determine the effect of CYP7B1 overexpression on rates of bile acid synthesis. The effects of different bile acids (3-150 micromol/L), cAMP (50 micromol/L), PMA (100 nmol/L; protein kinase C stimulator), cholesterol (200 micromol/L), and squalestatin (1 micromol/L; cholesterol synthesis inhibitor) on CYP7B1 expression in primary rat hepatocytes were studied. Taurocholic acid and taurodeoxycholic acid decreased CYP7B1 activity by 45% +/- 10% and 36% +/- 7%, respectively. Tauroursodeoxycholic acid and taurochenodeoxycholic acid did not alter CYP7B1 activity. Inhibition of cholesterol synthesis with squalestatin decreased CYP7B1 activity by 35%, whereas addition of cholesterol increased activity by 39%. Both PMA and cAMP decreased CYP7B1 activity by 60% and 34%, respectively, in a time-dependent fashion. Changes in CYP7B1 messenger RNA (mRNA) levels correlated with changes in specific activities. Overexpression of CYP7B1 led to a marked increase in CYP7B1 mRNA levels and specific activity but no change in rates of bile acid synthesis. In conclusion, in the rat, CYP7B1 specific activity is highly regulated but does not seem to be rate limiting for bile acid synthesis.