RESUMO
Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.
Assuntos
Epiderme/metabolismo , Regulação Enzimológica da Expressão Gênica , Glicerol-3-Fosfato O-Aciltransferase/genética , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Queratinócitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Epiderme/efeitos dos fármacos , Epiderme/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos , Tiazolidinedionas/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
The acute-phase response (APR) induces alterations in lipid metabolism, and our data suggest that this is associated with suppression of type II nuclear hormone receptors that are key regulators of fatty acid, cholesterol, and bile acid metabolism. Recently, the farnesoid X receptor (FXR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) were found to regulate DHEA sulfotransferase (Sult2A1), which plays an important role in DHEA sulfation and detoxification of bile acids. Because FXR, PXR, and CAR are suppressed during the APR, we hypothesized that Sult2A1 is downregulated during the APR. To induce the APR, mice were treated with LPS, which will then trigger the release of various cytokines, and the mRNA levels of Sult2A1 and the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2), as well as the enzyme activity of Sult2A1, were determined in the liver. We found that mRNA levels of Sult2A1 decrease in a time- and dose-dependent manner during the LPS-induced APR. Similar changes were observed in the mRNA levels of PAPSS2, the major synthase of PAPS in the liver. Moreover, hepatic Sult2A1 activity and serum levels of DHEA-sulfate (DHEA-S) were significantly decreased in LPS-treated animals. These results suggest that decreased levels or activities of FXR, PXR, and CAR during the APR could contribute to decreases in Sult2A1, resulting in decreased sulfation of DHEA and lower circulating level of DHEA-S. Finally, we found that both TNF and IL-1 caused a significant decrease in the mRNA level of Sult2A1 in Hep3B human hepatoma cells, suggesting that the proinflammatory cytokines TNF and IL-1 mediate the inhibitory effect of LPS on Sult2A1 mRNA level. Our study provides a possible mechanism by which infection and inflammation are associated with altered steroid metabolism and cholestasis.
Assuntos
Reação de Fase Aguda/enzimologia , Desidroepiandrosterona/metabolismo , Sulfotransferases/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Receptor Constitutivo de Androstano , Citocinas/farmacologia , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desidroepiandrosterona/sangue , Sulfato de Desidroepiandrosterona/metabolismo , Regulação para Baixo/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Hidroxiesteroides/metabolismo , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/biossíntese , Receptor de Pregnano X , Carbonitrila de Pregnenolona/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Sulfato Adenililtransferase/biossíntese , Fatores de Transcrição/metabolismoRESUMO
Severe sepsis results in the decreased uptake and oxidation of fatty acids in the heart and cardiac failure. Some of the key proteins required for fatty acid uptake and oxidation in the heart have been shown to be downregulated after endotoxin (LPS) administration. The nuclear hormone receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid receptor (TR), which heterodimerize with the retinoid X receptor (RXR), are important regulators of fatty acid metabolism and decrease in the liver after LPS administration. In the present study, we demonstrate that LPS treatment produces a rapid and marked decrease in the mRNA levels of all three RXR isoforms, PPARalpha and PPARdelta, and TRalpha and TRbeta in the heart. Moreover, LPS administration also decreased the expression of the coactivators CREB-binding protein (CBP)/p300, steroid receptor coactivator (SRC)-1, SRC-3, TR-associated protein (TRAP)220, and PPARgamma coactivator (PGC)-1, all of which are required for the transcriptional activity of RXR-PPAR and RXR-TR. In addition, the mRNA levels of the target genes malic enzyme, Spot 14, sarcoplasmic reticulum Ca2+-ATPase, or SERCA2, the VLDL receptor, fatty acyl-CoA synthetase, fatty acid transporter/CD36, carnitine palmitoyltransferase Ibeta, and lipoprotein lipase decrease in the heart after LPS treatment. The decrease in expression of RXRalpha, -beta, and -gamma, PPARalpha and -delta, and TRalpha and -beta, and of the coactivators CBP/p300, SRC-1, SRC-3, TRAP220, and PGC-1 and the genes they regulate, induced by LPS in the heart, could account for the decreased expression of key proteins required for fatty acid oxidation and thereby play an important role in cardiac contractility. These alterations could contribute to the myocardial dysfunction that occurs during sepsis.
Assuntos
Reação de Fase Aguda/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Feminino , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Hexoquinase/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Transporte de Monossacarídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Receptores X de Retinoides , Fatores de Transcrição/genéticaRESUMO
The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 microg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response.