Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated L...Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-media展开更多
Protein phosphatase 2 A（PP2 A） was reported to play an important role in cancer development;however,the relationship between PP2 A and cervical cancer development has yet to be fully understood.The present study aim...Protein phosphatase 2 A（PP2 A） was reported to play an important role in cancer development;however,the relationship between PP2 A and cervical cancer development has yet to be fully understood.The present study aimed to explore the role of PP2 A in the development of cervical cancer.Serum levels of PP2 A were detected by ELISA in 23 patients with cervical cancer and 30 patients with benign cervical lesions.Furthermore,the PP2 A activities and the m RNA and protein levels of PP2 A were measured in cervical cancer（n=8） and chronic cervicitis（n=10） tissues.The results showed that the serum levels of PP2 A were significantly reduced in patients with cervical cancer.Further studies showed that not only the activities of PP2 A but also the m RNA and protein levels of PP2 A were significantly decreased in cervical cancer tissues.Wound healing and Transwell assays demonstrated that pharmacological and genetic upregulation of PP2 A could inhibit the migration of He La cells,but the downregulation of PP2 A promoted cellular migration.The activation of PP2 A also inhibited the remodeling of actin and the activity of mitogen-activated protein kinases（MAPKs） including p-JNK,p-p38 and p-ERK.Meanwhile,the activation of PP2 A was found to downregulate MMP-9 levels,which further inhibited the migration and invasion of He La cells.In conclusion,our data suggest that the activity and expression of PP2 A are significantly reduced in cervical cancer tissues,and the activation of PP2 A may inhibit the migration of cervical cancer cells by inhibiting the phosphorylation of p-JNK,p-p38 and the p-ERK/MAPK signaling pathway as well as by downregulating MMP-9,implying that PP2 A plays an important role in cervical cancer development.展开更多
BACKGROUND It is well known that nonalcoholic fatty liver disease(NAFLD)is associated with insulin resistance(IR).LB100,a serine/threonine protein phosphatase 2A(PP2A)inhibitor,is closely related to IR.However,there i...BACKGROUND It is well known that nonalcoholic fatty liver disease(NAFLD)is associated with insulin resistance(IR).LB100,a serine/threonine protein phosphatase 2A(PP2A)inhibitor,is closely related to IR.However,there is little data regarding its direct influence on NAFLD.AIM To elucidate the effect and underlying mechanism of LB100 in NAFLD.METHODS After 10 wk of high fat diet(HFD)feeding,male C57BL/6 mice were injected intraperitoneally with vehicle or LB100 for an additional 6 wk(three times a week).The L02 cell line was treated with LB100 and free fatty acids(FFAs)for 24 h.Hematoxylin and eosin and oil red O staining were performed for histological examination.Western blot analysis was used to detect the protein expression of Sirtuin 1(Sirt1),total and phosphorylated AMP-activated protein kinaseα(AMPKα),and the proteins involved in lipogenesis and fatty acid oxidation.The mRNA levels were determined by qPCR.Pharmacological inhibition of AMPK was performed to further examine the exact mechanism of LB100 in NAFLD.RESULTS LB100 significantly ameliorated HFD-induced obesity,hepatic lipid accumulation and hepatic injury in mice.In addition,LB100 significantly downregulated the protein levels of acetyl-CoA carboxylase,sterol regulatory element-binding protein 1 and its lipogenesis target genes,including stearoyl-CoA desaturase-1 and fatty acid synthase,and upregulated the levels of proteins involved in fatty acidβ-oxidation,such as peroxisome proliferator-activated receptorα(PPARα),peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α),carnitine palmitoyltransferase 1α,acyl-CoA oxidase 1 and uncoupling protein 2,as well as the upstream mediators Sirt1 and AMPKαin the livers of HFD-fed mice.In vitro,LB100 alleviated FFA-induced lipid accumulation in L02 cells through the AMPK/Sirt1 signaling pathway.Further studies showed that the curative effect of LB100 on lipid accumulation was abolished by inhibiting AMPKαin L02 cells.CONCLUSION PP2A inhibition by LB100 significantly ameliorates hepatic 展开更多
基金This study was supported by grants from the National Natural Science Foundation of China(Key Program,No.30530360)the National Basic Research Program of China(No.2006CB503907)Royal Free Hospital Special Trustees grant
文摘Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-media
基金supported by the National Natural Science Foundation of China(No.81100959)Natural Science Foundation of Hubei Province(Nos.2015CFB185,2015CFB723)National Clinical Key Specialty Construction Projects of Ministry of Health of China(No.2010-305)
文摘Protein phosphatase 2 A（PP2 A） was reported to play an important role in cancer development;however,the relationship between PP2 A and cervical cancer development has yet to be fully understood.The present study aimed to explore the role of PP2 A in the development of cervical cancer.Serum levels of PP2 A were detected by ELISA in 23 patients with cervical cancer and 30 patients with benign cervical lesions.Furthermore,the PP2 A activities and the m RNA and protein levels of PP2 A were measured in cervical cancer（n=8） and chronic cervicitis（n=10） tissues.The results showed that the serum levels of PP2 A were significantly reduced in patients with cervical cancer.Further studies showed that not only the activities of PP2 A but also the m RNA and protein levels of PP2 A were significantly decreased in cervical cancer tissues.Wound healing and Transwell assays demonstrated that pharmacological and genetic upregulation of PP2 A could inhibit the migration of He La cells,but the downregulation of PP2 A promoted cellular migration.The activation of PP2 A also inhibited the remodeling of actin and the activity of mitogen-activated protein kinases（MAPKs） including p-JNK,p-p38 and p-ERK.Meanwhile,the activation of PP2 A was found to downregulate MMP-9 levels,which further inhibited the migration and invasion of He La cells.In conclusion,our data suggest that the activity and expression of PP2 A are significantly reduced in cervical cancer tissues,and the activation of PP2 A may inhibit the migration of cervical cancer cells by inhibiting the phosphorylation of p-JNK,p-p38 and the p-ERK/MAPK signaling pathway as well as by downregulating MMP-9,implying that PP2 A plays an important role in cervical cancer development.
文摘BACKGROUND It is well known that nonalcoholic fatty liver disease(NAFLD)is associated with insulin resistance(IR).LB100,a serine/threonine protein phosphatase 2A(PP2A)inhibitor,is closely related to IR.However,there is little data regarding its direct influence on NAFLD.AIM To elucidate the effect and underlying mechanism of LB100 in NAFLD.METHODS After 10 wk of high fat diet(HFD)feeding,male C57BL/6 mice were injected intraperitoneally with vehicle or LB100 for an additional 6 wk(three times a week).The L02 cell line was treated with LB100 and free fatty acids(FFAs)for 24 h.Hematoxylin and eosin and oil red O staining were performed for histological examination.Western blot analysis was used to detect the protein expression of Sirtuin 1(Sirt1),total and phosphorylated AMP-activated protein kinaseα(AMPKα),and the proteins involved in lipogenesis and fatty acid oxidation.The mRNA levels were determined by qPCR.Pharmacological inhibition of AMPK was performed to further examine the exact mechanism of LB100 in NAFLD.RESULTS LB100 significantly ameliorated HFD-induced obesity,hepatic lipid accumulation and hepatic injury in mice.In addition,LB100 significantly downregulated the protein levels of acetyl-CoA carboxylase,sterol regulatory element-binding protein 1 and its lipogenesis target genes,including stearoyl-CoA desaturase-1 and fatty acid synthase,and upregulated the levels of proteins involved in fatty acidβ-oxidation,such as peroxisome proliferator-activated receptorα(PPARα),peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α),carnitine palmitoyltransferase 1α,acyl-CoA oxidase 1 and uncoupling protein 2,as well as the upstream mediators Sirt1 and AMPKαin the livers of HFD-fed mice.In vitro,LB100 alleviated FFA-induced lipid accumulation in L02 cells through the AMPK/Sirt1 signaling pathway.Further studies showed that the curative effect of LB100 on lipid accumulation was abolished by inhibiting AMPKαin L02 cells.CONCLUSION PP2A inhibition by LB100 significantly ameliorates hepatic
