RESUMEN
Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy.
Asunto(s)
Chaperonina 60/metabolismo , Regulación de la Expresión Génica/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Progesterona/biosíntesis , Línea Celular , Chaperonina 60/genética , Colesterol , Femenino , Humanos , Proteínas Mitocondriales/genética , Placenta/citología , Embarazo , Unión ProteicaRESUMEN
It is known the deleterious effects of diabetes on embryos, but the effects of diabetes on placenta and its mitochondria are still not well known. In this work we generated a mild hyperglycemia model in female wistar rats by intraperitoneal injection of streptozotocin in 48 hours-old rats. The sexual maturity onset of the female rats was delayed around 6-7 weeks and at 16 weeks-old they were mated, and sacrificed at day 19th of pregnancy. In placental total tissue and isolated mitochondria, the fatty acids composition was analyzed by gas chromatography, and lipoperoxidation was measured by thiobarbituric acid reactive substances. Membrane fluidity in mitochondria was measured with the excimer forming probe dipyrenylpropane and mitochondrial function was measured with a Clark-type electrode. The results show that even a chronic mild hyperglycemia increases lipoperoxidation and decreases mitochondrial function in placenta. Simultaneously, placental fatty acids metabolism in total tissue is modified but in a different way than in placental mitochondria. Whereas the chronic mild hyperglycemia induced a decrease in unsaturated to saturated fatty acids ratio (U/S) in placental total tissue, the ratio increased in placental mitochondria. The measurements of membrane fluidity showed that fluidity of placenta mitochondrial membranes increased with hyperglycemia, showing consistency with the fatty acids composition through the U/S index. The thermotropic characteristics of mitochondrial membranes were changed, showing lower transition temperature and activation energies. All of these data together demonstrate that even a chronic mild hyperglycemia during pregnancy of early reproductive Wistar rats, generates an increment of lipoperoxidation, an increase of placental mitochondrial membrane fluidity apparently derived from changes in fatty acids composition and consequently, mitochondrial malfunction.
Asunto(s)
Hiperglucemia/metabolismo , Hiperglucemia/patología , Peroxidación de Lípido , Peróxidos Lipídicos/metabolismo , Fluidez de la Membrana , Mitocondrias/metabolismo , Placenta/patología , Animales , Glucemia/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Femenino , Hiperglucemia/sangre , Masculino , Membranas Mitocondriales/metabolismo , Embarazo , Ratas , Ratas Wistar , Maduración SexualRESUMEN
BACKGROUND: STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS: Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS: STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION: Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE: Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Péptido Hidrolasas/metabolismo , Progesterona/biosíntesis , Trofoblastos/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Concentración de Iones de Hidrógeno , Mitocondrias/metabolismo , Consumo de Oxígeno , Fenantrolinas/farmacología , Placenta/citología , Placenta/metabolismo , Embarazo , Inhibidores de Proteasas/farmacología , Proteolisis/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: The aim of this study was to evaluate the actions of coenzyme Q10 (CoQ10) on rats with a cholesterol-rich diet (HD) and high doses of atorvastatin (ATV, 0.2, 0.56 or 1.42 mg/day). METHODS: Two experiments were done, the first one without coenzyme Q10 supplementation. On the second experiment all groups received coenzyme Q10 0.57 mg/day as supplement. After a 6-week treatment animals were sacrificed, blood and liver were analyzed and liver mitochondria were isolated and its oxygen consumption was evaluated in state 3 (phosphorylating state) and state 4 (resting state) in order to calculate the respiratory control (RC). RESULTS: HD increased serum and hepatic cholesterol levels in rats with or without CoQ10. ATV reduced these values but CoQ10 improved even more serum and liver cholesterol. Triacylglycerols (TAG) were also lower in blood and liver of rats with ATV + CoQ10. HDL-C decreased in HD rats. Treatment with ATV maintained HDL-C levels. However, these values were lower in HD + CoQ10 compared to control diet (CD) + CoQ10. RC was lessened in liver mitochondria of HD. The administration of ATV increased RC. All groups supplemented with CoQ10 showed an increment in RC. In conclusion, the combined administration of ATV and CoQ10 improved biochemical parameters, liver function and mitochondrial respiration in hypercholesterolemic rats. CONCLUSIONS: Our results suggest a potential beneficial effect of CoQ10 supplementation in hypercholesterolemic rats that also receive atorvastatin. This beneficial effect of CoQ10 must be combined with statin treatment in patient with high levels of cholesterol.
Asunto(s)
Ácidos Heptanoicos/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Hipercolesterolemia/tratamiento farmacológico , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Pirroles/administración & dosificación , Ubiquinona/análogos & derivados , Animales , Atorvastatina , Respiración de la Célula , Colesterol en la Dieta/efectos adversos , HDL-Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos , Evaluación Preclínica de Medicamentos , Hipercolesterolemia/sangre , Hipercolesterolemia/etiología , Hígado/fisiopatología , Masculino , Ratas , Ratas Wistar , Triglicéridos/sangre , Ubiquinona/administración & dosificaciónRESUMEN
The peripheral benzodiazepine receptor and protein kinase A have been proposed to modulate placental steroidogenesis. Binding of the radioactive benzodiazepine PK 11195 has been observed in membranes isolated from whole human placenta, but the presence of the peripheral benzodiazepine receptors, now called translocator protein, does not seem to be indispensable. We hypothesized that cAMP analogs could induce the translocator protein expression in BeWo cells increasing steroidogenesis in the presence of benzodiazepines. The effect of two benzodiazepines and of 8-Br-cAMP on steroidogenesis in BeWo cells or in isolated human placental mitochondria was studied. Benzodiazepines did not modify progesterone synthesis in either system. Progesterone increased three times in BeWo cells incubated in the presence of 8-Br-cAMP. The translocator protein was not identified by western blot in mitochondria isolated from either the human placenta or BeWo cells but it was present in isolated rat testicular mitochondria. Neither was it observed in isolated mitochondria from BeWo cells incubated with 8-Br-cAMP. An inhibitor of protein kinase A activity, H89, at 25 microM inhibited 90% the steroidogenesis in BeWo cells, even in the presence of 8-Br-cAMP, but protein phosphorylation in mitochondria increased in the presence of H89, suggesting that protein kinase A modulates the phosphorylation cycle of mitochondrial proteins. The results suggest that placental steroidogenesis is regulated via activation of protein kinase A modulated by cAMP.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Placenta/metabolismo , Progesterona/biosíntesis , Receptores de GABA-A/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Benzodiazepinonas/farmacología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Humanos , Isoquinolinas/farmacología , Placenta/efectos de los fármacos , Placenta/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacologíaRESUMEN
El colesterol es indispensable para el desarrollo y crecimiento celular. En varios organismos, como en los insectos, el colesterol se considera una vitamina, ya que debe ser ingerido en los alimentos. En Drosophila, el colesterol es esencial para la embriogénesis y en su ausencia estas moscas no desarrollan las alas. Esto se debe a la interacción entre el colesterol y las proteínas hedgehog. También es necesaria la presencia de colesterol en la regulación de varias enzimas, sobre todo en las que están en relación con su síntesis. Finalmente, se ha observado que algunos intermediarios de la síntesis del colesterol modulan la velocidad del ciclo celular al interactuar con el DNA nuclear y, al parecer, esta asociación también está relacionada con los procesos cancerígenos.