RESUMO
Lipoprotein disorder is a common feature of chronic pancreatitis (CP); however, the relationship between lipoprotein disorder and pancreatic fibrotic environment is unclear. Here, we investigated the occurrence and mechanism of pancreatic stellate cell (PSC) activation by lipoprotein metabolites and the subsequent regulation of type 2 immune responses, as well as the driving force of fibrotic aggressiveness in CP. Single-cell RNA sequencing revealed the heterogeneity of PSCs and identified very-low-density lipoprotein receptor (VLDLR)+ PSCs that were characterized by a higher lipid metabolism. VLDLR promoted intracellular lipid accumulation, followed by interleukin-33 (IL-33) expression and release in PSCs. PSC-derived IL-33 strongly induced pancreatic group 2 innate lymphoid cells (ILC2s) to trigger a type 2 immune response accompanied by the activation of PSCs, eventually leading to fibrosis during pancreatitis. Our findings indicate that VLDLR-enhanced lipoprotein metabolism in PSCs promotes pancreatic fibrosis and highlight a dominant role of IL-33 in this pro-fibrotic cascade.
Assuntos
Células Estreladas do Pâncreas , Pancreatite Crônica , Receptores de LDL/metabolismo , Células Cultivadas , Fibrose , Humanos , Imunidade Inata , Interleucina-33/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas VLDL/metabolismo , Linfócitos/metabolismo , Pâncreas/patologia , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologiaRESUMO
Obesity is associated with excess lipid deposition in non-adipose tissues, leading to increased oxidative stress and insulin resistance. Very-low-density lipoprotein receptor (VLDLR), a member of the LDL receptor family, binds and increases the catabolism of triglyceride-rich lipoproteins. Although VLDLR is highly expressed in the heart, its role in obesity-associated oxidative stress and insulin resistance is unclear. Here, we used lean (WT), genetically obese leptin-deficient (ob/ob), and leptin-VLDLR double-null (ob/ob-VLDLR-/-) mice to determine the impact of VLDLR deficiency on obesity-induced oxidative stress and insulin resistance in the heart. While insulin sensitivity and glucose uptake were reduced in the hearts of ob/ob mice, VLDLR expression was upregulated and was associated with increased VLDL uptake and excess lipid deposition. This was accompanied by an upregulation of cardiac NADPH oxidase (Nox) expression and increased production of Nox-dependent superoxides. Silencing the VLDLR in ob/ob mice had reduced VLDL uptake and prevented excess lipid deposition in the heart, in addition to a reduction of superoxide overproduction and the normalization of insulin sensitivity and glucose uptake. In isolated cardiomyocytes, VLDLR deficiency had prevented VLDL-mediated induction of Nox activity and superoxide overproduction while improving insulin sensitivity and glucose uptake. Our findings indicate that VLDLR deficiency prevents excess lipid accumulation and moderates oxidative stress and insulin resistance in the heart of obese mice. This effect is linked to the active role of VLDLR in VLDL uptake, which triggers a cascade of events leading to increased NOX activity, overproduction superoxide and insulin resistance.
RESUMO
Subretinal fibrosis is a key pathological feature in neovascular age-related macular degeneration (nAMD). Previously, we identified soluble very low-density lipoprotein receptor (sVLDLR) as an endogenous Wnt signaling inhibitor. This study investigates whether sVLDLR plays an anti-fibrogenic role in nAMD models, including Vldlr-/- mice and laser-induced choroidal neovascularization (CNV). We found that fibrosis factors including P-Smad2/3, α-SMA, and CTGF were upregulated in the subretinal area of Vldlr-/- mice and the laser-induced CNV model. The antibody blocking Wnt co-receptor LRP6 significantly attenuated the overexpression of fibrotic factors in these two models. Moreover, there was a significant reduction of sVLDLR in the interphotoreceptor matrix (IPM) in the laser-induced CNV model. A transgenic strain (sVLDLR-Tg) with sVLDLR overexpression in the IPM was generated. Overexpression of sVLDLR ameliorated the profibrotic changes in the subretinal area of the laser-induced CNV model. In addition, Wnt and TGF-ß signaling synergistically promoted fibrogenesis in human primary retinal pigment epithelium (RPE) cells. CRISPR/Cas9-mediated LRP6 gene knockout (KO) attenuated this synergistic effect. The disruption of VLDLR expression promoted, while the overexpression of sVLDLR inhibited TGF-ß-induced fibrosis. These findings suggest that overactivated Wnt signaling enhances the TGF-ß pathway in subretinal fibrosis. sVLDLR confers an antifibrotic effect, at least partially, through the inhibition of Wnt signaling and thus, has therapeutic potential for fibrosis.
Assuntos
Neovascularização de Coroide/complicações , Modelos Animais de Doenças , Fibrose/prevenção & controle , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Degeneração Macular/complicações , Receptores de LDL/fisiologia , Epitélio Pigmentado da Retina/patologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Epitélio Pigmentado da Retina/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização WntRESUMO
We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.
Assuntos
Metabolismo dos Lipídeos , Proteínas WT1/metabolismo , Adulto , Células Cultivadas , AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Endométrio/citologia , Feminino , Regulação da Expressão Gênica , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/genética , Receptores de LDL/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Proteínas WT1/antagonistas & inibidores , Proteínas WT1/genéticaRESUMO
OBJECTIVE: To investigate the effect of miR-17-5p on vascular lesion and expression of very low density lipoprotein receptor (VLDLR) in atherosclerotic (AS) mice. METHODS: ApoE-/- mice were fed with high fat diet for 15 weeks to establish atherosclerotic mice models, and these mice were injected with miR-17-5p inhibitor antagomiR-17-5p 20 mg/kg from week 13 to week 15 to interfere the expression of miR-17-5p. AS model group (injection of normal saline) and NC miRNA group (injection of negative control inhibitors) were set and C57BL/6 mice were fed with normal diet for 15 weeks as normal control group (NC group, injection of normal saline during week 13-15). HE staining was used to detect the pathological changes of arterial vessels in each group and the vascular morphological changes were measured as well, so as to investigate the therapeutic effect of interfering miR-17-5p on AS vascular lesions. According to the prediction of Targetscan target gene prediction database, VLDLR as the target gene of miR-17-5p, the distribution of VLDLR in vascular tissues of mice in each group was observed by immunofluorescence. The effect of miR-17-5p on the expression of VLDLR mRNA in the arterial tissues of each group was detected by real-time PCR, and the changes of VLDLR protein expression caused by miR-17-5p in the arterial tissues in each group was detected by Western blot. RESULTS: The results of HE staining showed thatcompared with the NC group, the AS model group had obvious plaques in vascular endothelium, smooth muscle cell disorder and intimal hyperplasia, while the antagomiR-17-5p treated mice had significantly less lesions compared with the NC miRNA group. The intimal area of mice in the AS model group was bigger compared with NC group, but decreased after the inhibition of miR-17-5p. There was no statistically significant difference in the area of the media in each group. Vascular lumen area was smaller and intima/media ratio (I/M) values were lower in the AS model group and the NC miRNA group compared with the NC group, while the antagomiR-17-5p group alleviated this effect (P<0.05). Immunofluorescence showed that the expression of VLDLR in the AS model group was decreased, and that in the antagomiR-17-p group was higher than that in the NC miRNA group. The expression of VLDLR gene in the AS model group was lower than that in the NC group (P<0.01), while the VLDLR gene expression was higher in the antagomiR17-p group than that in the NC miRNA group (P<0.05). The results of VLDLR expression detected by Western blot were similar. CONCLUSION: miR-17-5p inhibitors may effectively alleviate the pathological changes of arterial vessels in AS mice by up-regulating the expression of VLDLR in arterial tissues, and may become a new therapeutic target for AS disease.
Assuntos
Aterosclerose/terapia , MicroRNAs/antagonistas & inibidores , Receptores de LDL/metabolismo , Animais , Aterosclerose/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoERESUMO
The LDL receptor (LDLR) family has long been studied for its role in cholesterol transport and metabolism; however, the identification of ApoE4, an LDLR ligand, as a genetic risk factor for late-onset Alzheimer's disease has focused attention on the role this receptor family plays in the CNS. Surprisingly, it was discovered that two LDLR family members, ApoE receptor 2 (Apoer2) and VLDL receptor (Vldlr), play key roles in brain development and adult synaptic plasticity, primarily by mediating Reelin signaling. This review focuses on Apoer2 and Vldlr signaling in the CNS and its role in human disease.
Assuntos
Doenças do Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/metabolismo , Processamento Alternativo , Animais , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiologia , Sistema Nervoso Central/fisiopatologia , Doenças do Sistema Nervoso Central/patologia , Doenças do Sistema Nervoso Central/fisiopatologia , Doenças do Sistema Nervoso Central/terapia , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Receptores de LDL/química , Proteína Reelina , Transdução de SinaisRESUMO
OBJECTIVE: To propose and verify a hypothesis that miR-17-5p knockdown may mitigate atherosclerotic lesions using atherosclerotic ApoE-/- mice as serum microRNA-17-5p (miR-17-5p) is elevated in patients with atherosclerosis. RESULTS: The level of miR-17-5p was higher while the level of very low density lipoprotein receptor (VLDLR), a predicted target of miR-17-5p, was lower in the peripheral blood lymphocytes (PBLs) of atherosclerosis patients as compared with control PBLs. ApoE-/- mice fed with a high-cholesterol diet displayed marked atherosclerotic vascular lesions, which were ameliorated after treatment with antagomiR-17-5p. Moreover, the decreased VLDLR in atherosclerotic mice was partly restored when miR-17-5p was antagonized. Further, luciferase assay confirmed VLDLR as a direct target of miR-17-5p in vascular smooth muscle cells (VSMCs). In addition, the elevated expression of proprotein convertase subtilisin kexin 9 (PCSK9), a secreted protease that binds to and promotes VLDLR degradation, in the atherosclerotic mice was suppressed by antagomiR-17-5p. CONCLUSIONS: A novel interaction between miR-17-5p and VLDLR is revealed and suggests that miR-17-5p may be a potential therapeutic target for AS.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/patologia , MicroRNAs/biossíntese , Receptores de LDL/biossíntese , Animais , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Leucócitos Mononucleares/química , Luciferases/análise , Camundongos Knockout para ApoERESUMO
OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and αVß3. APPROACH AND RESULTS: We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to ß3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. CONCLUSIONS: PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving αVß3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.
Assuntos
Células Endoteliais/metabolismo , Integrina alfaVbeta3/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptor Cross-Talk , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Endocitose , Células Endoteliais/efeitos dos fármacos , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ácidos Indolacéticos/administração & dosagem , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Interferência de RNA , Receptor Cross-Talk/efeitos dos fármacos , Receptores de LDL/metabolismo , Proteínas Recombinantes/metabolismo , Inibidores de Serina Proteinase/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Vitronectina/deficiência , Vitronectina/genéticaRESUMO
Clusterin, also known as apolipoprotein J, is a multifunctional glycoprotein with the capacity to interact with a wide range of molecules. Although clusterin has been implicated in a broad spectrum of physiological and pathological processes, such as Alzheimer disease or cancer, its precise functions remain elusive. Here we report, that clusterin binds to apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR) and is internalized by cells expressing either one of these receptors. Binding of clusterin to these receptors triggers a Reelin-like signal in cells expressing disabled-1 (Dab1). It induces phosphorylation of Dab1, which leads to activation of PI3K/Akt and n-cofilin. Cell proliferation and neuroblast chain formation in subventricular zone (SVZ) explants are compromised when clusterin, which is present in the subventricular zone, is blocked in vitro. These data suggest that in the subventricular zone where Reelin is not present but ApoER2, VLDLR, and Dab1, clusterin might be involved in maintaining neurogenesis in vivo.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Clusterina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Núcleos da Linha Média do Tálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Proliferação de Células , Células Cultivadas , Clusterina/genética , Proteínas da Matriz Extracelular/genética , Células HEK293 , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Núcleos da Linha Média do Tálamo/citologia , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Neurogênese/fisiologia , Receptores de LDL/genética , Proteína Reelina , Serina Endopeptidases/genéticaRESUMO
Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.
Assuntos
Alisma/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Extratos Vegetais/farmacologia , Animais , Apolipoproteínas B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dieta Hiperlipídica , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Camundongos , Camundongos Obesos , Substâncias Protetoras/farmacologia , Receptores de LDL/genética , Receptores de LDL/metabolismo , Triglicerídeos/metabolismo , Tunicamicina/efeitos adversosRESUMO
The liver and the VLDL receptor (VLDLR) play major roles in TG and VLDL metabolism. However, the exact role of liver VLDLR is not well known because of the absence of or difficulty in detecting VLDLR in the liver. In this study, we demonstrate that fenofibrate, a PPARα agonist and widely used TG-lowering drug, markedly upregulated hepatic VLDLR, which is essential for lowering TG. This study also shows that the distinct regulatory roles of PPARα agonists on VLDLR in the liver and peripheral tissues including adipose tissues, heart, and skeletal muscles are due to the pattern of expression of PPARα. The in vivo portion of our study demonstrated that oral fenofibrate robustly increased liver VLDLR expression levels in hyperlipidemic and diabetic mice and significantly reduced the increase in serum TG observed in wt mice after feeding with high-fat diet (HFD) but not in Vldlr(-/-) mice or Pparα(-/-) mice. However, overexpression of mouse VLDLR in livers of Vldlr(-/-) mice significantly prevented the increase in serum TG induced by HFD. The in vitro portion of our study showed that fenofibrate upregulated VLDLR transcriptional activity through PPAR response element binding to the VLDLR promoter. The conclusions of our study provide a novel mechanism for the TG-lowering effects of fenofibrate in the treatment of dyslipidemia.
Assuntos
Fenofibrato/farmacologia , Fígado/metabolismo , PPAR alfa/metabolismo , Receptores de LDL/biossíntese , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacos , Células 3T3-L1 , Animais , Camundongos , Camundongos Knockout , PPAR alfa/genética , Receptores de LDL/genética , Elementos de Resposta , Transcrição Gênica/efeitos dos fármacos , Triglicerídeos/genéticaRESUMO
Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism.
Assuntos
Bass , Proteínas de Peixes/metabolismo , Receptores de Lipoproteínas/metabolismo , Vitelogeninas/metabolismo , Animais , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Ligação Proteica , Transporte Proteico , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genéticaRESUMO
The precise functions and mechanisms of microRNAs (miR) in gallbladder cancer (GBC) remain elusive. In this study, we found that miR-135a-5p expression is often dampened and correlated with neoplasm histologic grade in GBC. MicroRNA-135a-5p introduction clearly inhibited GBC cell proliferation in vitro and in vivo. Moreover, very low density lipoprotein receptor (VLDLR), which is often upregulated in GBC tissues, was identified as a direct functional target of miR-135a-5p. Furthermore, the p38 MAPK pathway was proven to be involved in miR-135a-VLDLR downstream signaling. Together, these results suggested that the miR-135a-VLDLR-p38 axis may contribute to GBC cell proliferation.
Assuntos
Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Receptores de LDL/biossíntese , Idoso , Western Blotting , Feminino , Genes Supressores de Tumor , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor's glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway.
Assuntos
Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/fisiologia , Receptores de LDL/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Células Hep G2 , Humanos , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/genética , Transporte Proteico/genética , Proteínas Qa-SNARE/genética , Receptores de LDL/genética , Via Secretória/genética , Rede trans-Golgi/metabolismoRESUMO
Here we describe methods for investigating alpha-1 antitrypsin (AAT) and very-low-density lipoprotein receptor (VLDLR) interactions with infectious and non-infectious HIV-1 virions. Using silencing RNA to transiently block expression of VLDLR and the receptor-associated protein (RAP) to continuously block VLDLR activity, AAT is demonstrated to participate with VLDLR during internalization and infectivity of HIV-1 virions.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/genética , Mecanismos de Defesa , HIV-1/genética , Vírion/genética , Lipoproteínas VLDLRESUMO
The very low density lipoprotein receptor (VLDLr) is highly upregulated during hypoxia in mouse cardiomyocytes and in human and mouse ischemic hearts causing a detrimental lipid accumulation. To know how the gene is regulated is important for future studies. In this study, we have thoroughly mapped the 5'-flanking region of the mouse VLDLr promoter and show that the hypoxia-mediated increase in VLDLr expression is dependent on Hif-1α binding to a hypoxia responsive element (HRE) located at -162 to -158bp 5'of translation start. We show that classical HRE sites and the previously described PPARγ and Sp1 binding are not involved in the hypoxia-induced regulation of the VLDLr promoter. Using a chromatin immunoprecipitation (ChIP) assay, we show that Hif-1α specifically binds and activates the mouse VLDLr promoter at the previously described non-classical HRE in HL-1 cells. We also show that the same HRE is present and active in response to hypoxia in human cardiomyocytes, however at a different location (-812bp from translation start). These results conclude that in the hypoxic hearts of mice and men, the VLDLr gene is regulated by a direct binding of Hif-1α to the VLDLr promoter.
Assuntos
Hipóxia/fisiopatologia , Receptores de LDL/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Primers do DNA , Camundongos , PPAR gama/metabolismo , Regiões Promotoras Genéticas , Receptores de LDL/genética , Fator de Transcrição Sp1/metabolismoRESUMO
Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species.
Assuntos
Proteínas de Peixes/genética , Folículo Ovariano/metabolismo , Receptores de LDL/genética , Truta/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Feminino , Proteínas de Peixes/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Oócitos/metabolismo , Especificidade de Órgãos , Folículo Ovariano/citologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Truta/metabolismoRESUMO
Reproductive modes of vertebrates are classified into two major embryonic nutritional types: yolk deposits (i.e., lecithotrophy) and maternal investment (i.e., matrotrophy). Vitellogenin (VTG), a major egg yolk protein synthesized in the female liver, is one of the molecules relevant to the lecithotrophy-to-matrotrophy shift in bony vertebrates. In mammals, all VTG genes are lost following the lecithotrophy-to-matrotrophy shift, and it remains to be elucidated whether the lecithotrophy-to-matrotrophy shift in nonmammalians is also associated with VTG repertoire modification. In this study, we focused on chondrichthyans (cartilaginous fishes)-a vertebrate clade that underwent multiple lecithotrophy-to-matrotrophy shifts. For an exhaustive search of homologs, we performed tissue-by-tissue transcriptome sequencing for two viviparous chondrichthyans, the frilled shark Chlamydoselachus anguineus and the spotless smooth-hound Mustelus griseus, and inferred the molecular phylogeny of VTG and its receptor very low-density lipoprotein receptor (VLDLR), across diverse vertebrates. As a result, we identified either three or four VTG orthologs in chondrichthyans including viviparous species. We also showed that chondrichthyans had two additional VLDLR orthologs previously unrecognized in their unique lineage (designated as VLDLRc2 and VLDLRc3). Notably, VTG gene expression patterns differed in the species studied depending on their reproductive mode; VTGs are broadly expressed in multiple tissues, including the uterus, in the two viviparous sharks, and in addition to the liver. This finding suggests that the chondrichthyans VTGs do not only function as the yolk nutrient but also as the matrotrophic factor. Altogether, our study indicates that the lecithotrophy-to-matrotrophy shift in chondrichthyans was achieved through a distinct evolutionary process from mammals.
Assuntos
Tubarões , Animais , Feminino , Tubarões/genética , Tubarões/metabolismo , Vertebrados , Evolução Biológica , Mamíferos/metabolismo , Vitelogeninas/genéticaRESUMO
To investigate the regulation of soluble very low-density lipoprotein receptor (sVLDL-R), which is cleaved mostly from the extracellular domain of VLDL-R II, we generated two rat monoclonal antibodies (mAbs) against human sVLDL-R, and used them to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to measure sVLDL-R levels in human serum or plasma. The ELISA had a linear range from 0.20 ng/mL to 13.02 ng/mL and allowed for the quantification of sVLDL-R in serum and culture cell medium. The coefficient of variation (CV) was less than 10% for both the intra- and inter-assays. The bililubin F, and C, triglyceride (TG), and hemoglobin levels did not affect assay precision. The sVLDL-R concentration was negatively associated with body fat percentage, TG, and HbA1c, suggesting the possibility of obesity and diabetes in middle-aged Japanese women.
RESUMO
Introduction: Mesial temporal lobe epilepsy (MTLE) is the most frequent form of partial epilepsy. Granule cell dispersion, resulting from aberrant neuronal migration in the hippocampus, is pathognomonic of MTLE. Reelin, a secreted neurodevelopmental glycoprotein has a crucial role in controlling the radial migration of neurons. Several animal studies have implicated Reelin in the MTLE pathogenesis Mesial temporal lobe epilepsy (MTLE) is the most frequent form of partial epilepsy. Granule cell dispersion, resulting from aberrant neuronal migration in the hippocampus, is pathognomonic of MTLE. Reelin, a secreted neurodevelopmental glycoprotein has a crucial role in controlling the radial migration of neurons. Several animal studies have implicated Reelin in the MTLE pathogenesis. Methods: The aim of this study was to investigate the Reelin signalling pathway in the MTLE patients. Therefore, we studied each step in the Reelin signalling pathway for the gene and protein expressions, in the hippocampal tissue obtained from patients undergoing surgery for MTLE and compared it with age matched normal autopsy cases. Results: We found statistically significant decrease (P<0.001) in the Reelin mRNA expression in MTLE patients. Among the two reelin receptors, apolipoprotein E receptor 2 (ApoER2) was significantly increased whereas very low density lipoprotein receptor (VLDLR) was decreased among the patients. Disabled 1 (Dab1), the downstream target of reelin, was found to be decreased. Dab1 in turn inhibits Cofilin, which is responsible for cytoskeletal reorganization, thus limiting aberrant neuronal migration. Statistically significant over expression of Cofilin protein was found in the patient group. Matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteases-1 (TIMP-1), both of which are involved in processing of Reelin, were down regulated in 70-85% of cases. Conclusion: The whole pathway was found to be deranged in MTLE. These results indicate that Reelin signalling pathway is disturbed at various points in the MTLE patients and might be involved in the pathogenesis & progression of MTLE. Our results extend the existing information regarding the components of the Reelin pathway and further, establish a link between pathway disturbance and MTLE.