RESUMO
The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.
Assuntos
Coenzima A Ligases/biossíntese , Hepatócitos/enzimologia , Hiperlipidemias/enzimologia , PPAR gama/metabolismo , Animais , Colesterol na Dieta/metabolismo , Clonagem Molecular , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Indução Enzimática , Perfilação da Expressão Gênica/métodos , Células HEK293 , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Hiperlipidemias/genética , Masculino , Mesocricetus , Camundongos Endogâmicos C57BL , PPAR gama/agonistas , PPAR gama/genética , Fenoxiacetatos/farmacologia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/biossíntese , Testículo/metabolismo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
The transcription factors hepatic nuclear factor (HNF)1α and HNF1ß can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1ß) to examine the effects of knockdown of HNF1α or HNF1ß on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1ß, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (â¼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (â¼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1ß did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1ß infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1ß, is the primary positive regulator of PCSK9 transcription in mouse liver.
Assuntos
LDL-Colesterol/sangue , Técnicas de Silenciamento de Genes , Fator 1-alfa Nuclear de Hepatócito/deficiência , Fator 1-alfa Nuclear de Hepatócito/genética , Fígado/metabolismo , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Animais , Dieta , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células Hep G2 , Fator 1-beta Nuclear de Hepatócito/deficiência , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Fígado/citologia , Masculino , Camundongos , Especificidade de Órgãos , Pró-Proteína Convertase 9 , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) impedes lowdensity lipoprotein (LDL) receptor (LDLR)-mediated LDL-cholesterol uptake and has hence emerged as a critical regulator of serum cholesterol levels and a new therapeutic target for the treatment of hypercholesterolemia. Statins have been shown to elevate circulating PCSK9 levels by stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDLcholesterol reduction. The transcription of PCSK9 is partially controlled by the hepatocyte nuclear factor 1 (HNF1) binding site embedded in the proximal region of its promoter. In this study, we utilized adenoviral shRNA delivery vectors to generate liver-specific knockdown of HNF1α (AdshHNF1α) or HNF1ß (AdshHNF1ß) in hamsters to examine the impact of reduced hepatic expression of HNF1 transcription factors on statininduced elevation of PCSK9 expression and serum cholesterol levels. We showed that the administration of rosuvastatin (RSV) to normolipidemic hamsters significantly augmented hepatic PCSK9 expression and serum PCSK9 levels. In addition, RSV treatment increased hepatic HNF1α protein levels without a clear effect on HNF1α mRNA expression. Injection of Ad-shHNF1α or AdshHNF1ß into hamsters both blunted RSVinduced elevation of PCSK9 serum concentration and hepatic mRNA and protein levels, which led to significant increases in liver LDLR protein abundance. Furthermore, hepatic depletion of HNF1 factors lowered circulating total cholesterol and nonhigh density lipoprotein cholesterol levels in RSVtreated hamsters. Our study demonstrates that both HNF1α and HNF1ß are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver-specific knockdown of either HNF1α or HNF1ß could antagonize the RSVinduced elevation of serum PCSK9 and reduce circulating cholesterol levels.