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1.
Nat Commun ; 14(1): 6493, 2023 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-37838725

RESUMO

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), a leading hereditary kidney disease. There are no targeted therapies. In our generated mouse model recapitulating human ADTKD-UMOD carrying a leading UMOD mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are impaired, leading to cGAS-STING activation and tubular injury. Moreover, we demonstrate that inducible tubular overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF), a secreted endoplasmic reticulum protein, after the onset of disease stimulates autophagy/mitophagy, clears mutant UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, thus protecting kidney function in our ADTKD mouse model. Conversely, genetic ablation of MANF in the mutant thick ascending limb tubular cells worsens autophagy suppression and kidney fibrosis. Together, we have discovered MANF as a biotherapeutic protein and elucidated previously unknown mechanisms of MANF in the regulation of organelle homeostasis, which may have broad therapeutic applications to treat various proteinopathies.


Assuntos
Doenças Renais Policísticas , Humanos , Camundongos , Animais , Autofagia/genética , Homeostase , Fibrose , Fatores de Crescimento Neural/genética
2.
Exp Mol Med ; 55(7): 1348-1356, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37394581

RESUMO

Thioredoxin-interacting protein (TXNIP), which is also known as thioredoxin-binding protein 2 (TBP2), directly interacts with the major antioxidant protein thioredoxin (TRX) and inhibits its antioxidant function and expression. However, recent studies have demonstrated that TXNIP is a multifunctional protein with functions beyond increasing intracellular oxidative stress. TXNIP activates endoplasmic reticulum (ER) stress-mediated nucleotide-binding oligomerization domain (NOD)-like receptor protein-3 (NLRP3) inflammasome complex formation, triggers mitochondrial stress-induced apoptosis, and stimulates inflammatory cell death (pyroptosis). These newly discovered functions of TXNIP highlight its role in disease development, especially in response to several cellular stress factors. In this review, we provide an overview of the multiple functions of TXNIP in pathological conditions and summarize its involvement in various diseases, such as diabetes, chronic kidney disease, and neurodegenerative diseases. We also discuss the potential of TXNIP as a therapeutic target and TXNIP inhibitors as novel therapeutic drugs for treating these diseases.


Assuntos
Antioxidantes , Proteína 3 que Contém Domínio de Pirina da Família NLR , Antioxidantes/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Estresse Oxidativo , Tiorredoxinas/genética , Inflamassomos/metabolismo
3.
bioRxiv ; 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36711449

RESUMO

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- UMOD ), one of the leading hereditary kidney diseases, and Alzheimer’s disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide. For the first time, in our newly generated mouse model recapitulating human ADTKD- UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

4.
Proc Natl Acad Sci U S A ; 119(35): e2116505119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994650

RESUMO

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop-/- and Txnip-/- mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1-dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.


Assuntos
Albuminúria , Proteínas de Transporte , Rim , Mitocôndrias , Síndrome Nefrótica , Tiorredoxinas , Fator de Transcrição CHOP , Albuminúria/complicações , Albuminúria/genética , Albuminúria/prevenção & controle , Animais , Apoptose , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Deleção de Genes , Inflamassomos/metabolismo , Rim/metabolismo , Rim/patologia , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Síndrome Nefrótica/complicações , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Síndrome Nefrótica/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/metabolismo , Fator de Transcrição CHOP/deficiência , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
5.
Am J Pathol ; 191(2): 256-265, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33245915

RESUMO

Calcium (Ca2+) homeostasis is a crucial determinant of cellular function and survival. Endoplasmic reticulum (ER) acts as the largest intracellular Ca2+ store that maintains Ca2+ homeostasis through the ER Ca2+ uptake pump, sarco/ER Ca2+ ATPase, ER Ca2+ release channels, inositol 1,4,5-trisphosphate receptor channel, ryanodine receptor, and Ca2+-binding proteins inside of the ER lumen. Alterations in ER homeostasis trigger ER Ca2+ depletion and ER stress, which have been associated with the development of a variety of diseases. In addition, recent studies have highlighted the role of ER Ca2+ imbalance caused by dysfunction of sarco/ER Ca2+ ATPase, ryanodine receptor, and inositol 1,4,5-trisphosphate receptor channel in various kidney diseases. Despite progress in the understanding of the importance of these ER Ca2+ channels, pumps, and binding proteins in the pathogenesis of kidney disease, treatment is still lacking. This mini-review is focused on: i) Ca2+ homeostasis in the ER, ii) ER Ca2+ dyshomeostasis and apoptosis, and iii) altered ER Ca2+ homeostasis in kidney disease, including podocytopathy, diabetic nephropathy, albuminuria, autosomal dominant polycystic kidney disease, and ischemia/reperfusion-induced acute kidney injury.


Assuntos
Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Homeostase/fisiologia , Nefropatias/metabolismo , Animais , Humanos
6.
Proc Natl Acad Sci U S A ; 116(28): 14154-14163, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31235574

RESUMO

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.


Assuntos
Cálcio/metabolismo , Síndrome Nefrótica/genética , Fatores de Crescimento Neural/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Albuminúria/tratamento farmacológico , Albuminúria/genética , Albuminúria/patologia , Animais , Sinalização do Cálcio/genética , Calpaína/genética , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/genética , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Camundongos , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/patologia , Podócitos/metabolismo , Podócitos/patologia , Talina/genética , Tiazepinas/farmacologia
7.
Pediatr Nephrol ; 34(9): 1493-1500, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30099615

RESUMO

The advent of next-generation sequencing (NGS) in recent years has led to a rapid discovery of novel or rare genetic variants in human kidney cell genes, which is transforming the risk assessment, diagnosis, and treatment of kidney disease. Mutations may lead to protein misfolding, disruption of protein trafficking, and endoplasmic reticulum (ER) retention. An imbalance between the load of misfolded proteins and the folding capacity of the ER causes ER stress and unfolded protein response. Mutations in nephrin (NPHS1), podocin (NPHS2), laminin ß2 (LAMB2), and α-actinin-4 (ACTN4) have been shown to induce ER stress in HEK293 cells and podocytes in hereditary nephrotic syndromes; various founder mutations in collagen IV α chains (COL4A) have been demonstrated to activate podocyte ER stress in collagen IV nephropathies; and mutations in uromodulin (UMOD) have been reported to trigger tubular ER stress in autosomal dominant tubulointerstitial kidney disease. Meanwhile, ER resident protein SEC63 may modify disease severity in autosomal dominant polycystic kidney disease. These findings underscore the importance of ER stress in the pathogenesis of monogenic kidney disease. Recently, we have identified mesencephalic astrocyte-derived neurotrophic factor (MANF) and cysteine-rich with EGF-like domains 2 (CRELD2) as urinary ER stress biomarkers in ER stress-mediated kidney diseases.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Nefrologia/métodos , Medicina de Precisão/métodos , Biomarcadores/análise , Análise Mutacional de DNA , Estresse do Retículo Endoplasmático/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nefropatias/diagnóstico , Nefropatias/genética , Nefropatias/patologia , Mutação , Podócitos/efeitos dos fármacos , Podócitos/patologia
8.
Free Radic Biol Med ; 123: 27-38, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29777756

RESUMO

Elevated levels of reactive oxygen species (ROS) are a hallmark of obesity. Peroxiredoxin 5 (Prx5), which is a cysteine-dependent peroxidase enzyme, has an intensive ROS scavenging activity because it is located in the cytosol and mitochondria. Therefore, we focused on the role of Prx5 in regulating mitochondrial ROS and adipogenesis. We demonstrated that Prx5 expression was upregulated during adipogenesis and Prx5 overexpression suppressed adipogenesis by regulating cytosolic and mitochondrial ROS generation. Silencing Prx5 promoted preadipocytes to differentiate into adipocytes accumulating lipids by activating adipogenic protein expression. Prx5-deletion mice fed on a high-fat diet (HFD) exhibited significant increase in body weight, enormous fat pads, and adipocyte hypertrophy in comparison to wild type mice. Prx5 deletion also remarkably induced adipogenesis-related gene expression in white adipose tissue. These phenotypic changes in Prx5-deletion mice were accompanied with lipid metabolic disorders, such as excessive lipid accumulation in the liver, severe hepatic steatosis, and high levels of triglyceride in the serum. These results demonstrated that Prx5 deletion increased the susceptibility to HFD-induced obesity and several of its associated metabolic disorders. In conclusion, we suggest that Prx5 inhibits adipogenesis by modulating ROS generation and adipogenic gene expression, implying that Prx5 may serve as a potential strategy to prevent and treat obesity.


Assuntos
Adipogenia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Obesidade/etiologia , Estresse Oxidativo , Peroxirredoxinas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Obesidade/patologia
9.
Free Radic Biol Med ; 123: 96-106, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29778464

RESUMO

Reactive oxygen species (ROS) produced in biological reactions have been shown to contribute to ovarian aging. Peroxiredoxin 2 (Prx2) is an antioxidant enzyme that protects cells by scavenging ROS; however, its effect on age-related, oxidative stress-associated ovarian failure has not been reported. Here, we investigated its role in age-related ovarian dysfunction and 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure using Prx2-deficient mice. Compared to those in wildtype (WT) mice, serum levels of anti-Müllerian hormone, 17ß-estradiol, and progesterone and numbers of follicles and corpora lutea were significantly lower in 18-month-old Prx2-/- mice. Moreover, levels of Bax, cytochrome c, cleaved caspase-3, and phosphorylated JNK proteins were higher and numbers of apoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cells were considerably greater in 18-month-old Prx2-/- ovaries than WT ovaries. Furthermore, the effects of the ovarian toxicant VCD in significantly enhancing ROS levels and apoptosis through activation of JNK-mediated apoptotic signaling were more pronounced in Prx2-/- than WT mouse embryonic fibroblasts. Expression of the steroidogenic proteins StAR, CYP11A1, and 3ß-HSD and serum levels of 17ß-estradiol and progesterone were also reduced to a greater extent in Prx2-/- mice than WT mice after VCD injection. This reduced steroidogenesis was rescued by addition of the Prx mimic ebselen or JNK inhibitor SP600125. This constitutes the first report that Prx2 deficiency leads to acceleration of age-related or VCD-induced ovarian failure by activation of the ROS-induced JNK pathway. These findings suggest that Prx2 plays an important role in preventing accelerated ovarian failure by inhibiting ROS-induced JNK activation.


Assuntos
Envelhecimento , Sistema de Sinalização das MAP Quinases , Doenças Ovarianas/patologia , Folículo Ovariano/patologia , Estresse Oxidativo , Peroxirredoxinas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose , Carcinógenos/toxicidade , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Corpo Lúteo/patologia , Cicloexenos/toxicidade , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Ovarianas/induzido quimicamente , Doenças Ovarianas/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Transdução de Sinais , Compostos de Vinila/toxicidade
10.
Int J Biochem Cell Biol ; 99: 80-90, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29605633

RESUMO

Insulin signaling is essential for regulating glucose homeostasis. Numerous studies have demonstrated that reactive oxygen species (ROS) affect insulin signaling, and low ROS levels can act as a signal to regulate cellular function. Peroxiredoxins (Prxs) are highly abundant and widely expressed antioxidant enzymes. However, it is unclear whether antioxidant enzymes, such as Prx2, mediate insulin signaling. The aim of our study was to investigate the influence of Prx2 deficiency on insulin signaling. Our western blot results showed that Prx2 deficiency enhanced insulin signaling and increased oxidation of protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homologue (PTEN) in mouse embryonic fibroblasts (MEFs) treated with insulin. In addition, we assessed ROS levels with a Cytosol-HyPer H2O2 sensor. As a result, increased ROS levels and Akt activation were decreased by N-acetyl-cysteine (Nac), which acted as an antioxidant in Prx2-deficient MEFs. Body weight measurements and glucose tolerance test (GTT) revealed significant body weight reduction and increase in glucose clearance in Prx2-/- mice fed a high-fat diet. Interestingly, glucose transporter type 4 (GLUT4) was significantly higher in Prx2-/- mice than in wild-type mice according to western blotting results. Western blotting also revealed that Akt phosphorylation was higher in Prx2-/- MEFs and muscle tissue than in wild-type. Together, our findings indicate that increased ROS due to Prx2 deficiency promotes insulin sensitivity and glucose clearance in skeletal muscles by increasing protein tyrosine phosphatase (PTPs) oxidation. These results provide novel insights into the fundamental mechanisms of insulin signaling induced by Prx2 deficiency and suggest that ROS-based therapeutic strategies can be used to suppress insulin resistance.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/fisiologia , Resistência à Insulina , Insulina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Proteínas Tirosina Fosfatases/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucose/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxirredução , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais
11.
JCI Insight ; 2(23)2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212948

RESUMO

ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress-mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress-induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion-induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.


Assuntos
Injúria Renal Aguda/urina , Moléculas de Adesão Celular/urina , Estresse do Retículo Endoplasmático/fisiologia , Proteínas da Matriz Extracelular/urina , Síndrome Nefrótica/urina , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Animais , Biomarcadores/urina , Procedimentos Cirúrgicos Cardíacos , Moléculas de Adesão Celular/fisiologia , Criança , Proteínas da Matriz Extracelular/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mutação , Nefrite Intersticial/genética , Nefrite Intersticial/fisiopatologia , Nefrite Intersticial/urina , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/fisiopatologia , Podócitos/metabolismo , Complicações Pós-Operatórias/urina , Uromodulina/genética
12.
PLoS One ; 12(10): e0185764, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28968439

RESUMO

Adipocyte differentiation is regulated by intracellular reactive oxygen species (ROS) generation and mitochondrial fission and fusion processes. However, the correlation between intracellular ROS generation and mitochondrial remodeling during adipocyte differentiation is still unknown. Here, we investigated the effect on adipocyte differentiation of 3T3-L1 cells of intracellular ROS inhibition using N-acetyl cysteine (Nac) and Mito-TEMPO and of mitochondrial fission inhibition using Mdivi-1. Differentiated 3T3-L1 adipocytes displayed an increase in mitochondrial fission, ROS generation, and the expression of adipogenic and mitochondrial dynamics-related proteins. ROS scavenger (Nac or Mito-TEMPO) treatment inhibited ROS production, lipid accumulation, the expression of adipogenic and mitochondrial dynamics-related proteins, and mitochondrial fission during adipogenesis of 3T3-L1 cells. On the other hand, treatment with the mitochondrial fission inhibitor Mdivi-1 inhibited mitochondrial fission but did not inhibit ROS production, lipid accumulation, or the expression of adipogenic and mitochondrial dynamics-related proteins, with the exception of phosphorylated Drp1 (Ser616), in differentiated 3T3-L1 adipocytes. The inhibition of mitochondrial fission did not affect adipocyte differentiation, while intracellular ROS production decreased in parallel with inhibition of adipocyte differentiation. Therefore, our results indicated that ROS are an essential regulator of adipocyte differentiation in 3T3-L1 cells.


Assuntos
Dinaminas/antagonistas & inibidores , Radicais Livres/metabolismo , Insulina/farmacologia , Metabolismo dos Lipídeos , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antioxidantes/metabolismo , Camundongos
13.
Transl Res ; 188: 1-9, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28719799

RESUMO

Mesencephalic astrocyte-derived neurotrophic factor (MANF), a newly identified 18-kDa soluble protein, localizes to the luminal endoplasmic reticulum (ER), whose stress can stimulate MANF expression and secretion. In Drosophila and zebrafish, MANF regulates dopaminergic neuron development. In contrast, in mice, MANF deficiency leads to diabetes and activation of the unfolded protein response. Recent studies in rodent models have demonstrated that MANF mitigates diabetes, exerts neurotrophic function in neurodegenerative disease, protects cardiomyocytes and neurons in myocardial infarction and cerebral ischemia, respectively, and promotes immune cell phenotype switch from proinflammatory macrophages to prorepair anti-inflammatory macrophages. The cytoprotective mechanisms of MANF on ER stress are currently under active investigation. In addition, for the first time, we have discovered that MANF can potentially serve as a urinary ER stress biomarker in ER stress-mediated kidney disease. These studies have underscored the diagnostic and therapeutic importance of MANF in ER diseases.


Assuntos
Retículo Endoplasmático/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Biomarcadores , Humanos , Fatores de Crescimento Neural/genética , Conformação Proteica , Especificidade da Espécie
14.
Free Radic Biol Med ; 108: 44-55, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28323129

RESUMO

Luteal regression is a natural and necessary event to regulate the reproductive process in all mammals. Prostaglandin F2α (PGF2α) is the main factor that causes functional and structural regression of the corpus luteum (CL). It is well known that PGF2α-mediated ROS generation is closely involved in luteal regression. Peroxiredoxin 2 (Prx2) as an antioxidant enzyme plays a protective role against oxidative stress-induced cell death. However, the effect of Prx2 on PGF2α-induced luteal regression has not been reported. Here, we investigated the role of Prx2 in functional and structural CL regression induced by PGF2α-mediated ROS using Prx2-deficient (-/-) mice. We found that PGF2α-induced ROS generation was significantly higher in Prx2-/- MEF cells compared with that in wild-type (WT) cells, which induced apoptosis by activating JNK-mediated apoptotic signaling pathway. Also, PGF2α treatment in the CL derived from Prx2-/- mice promoted the reduction of steroidogenic enzyme expression and the activation of JNK and caspase3. Compared to WT mice, serum progesterone levels and luteal expression of steroidogenic enzymes decreased more rapidly whereas JNK and caspase3 activations were significantly increased in Prx2-/- mice injected with PGF2α. However, the impaired steroidogenesis and PGF2α-induced JNK-dependent apoptosis were rescued by the addition of the antioxidant N-acetyl-L-cysteine (NAC). This is the first study to demonstrate that Prx2 deficiency ultimately accelerated the PGF2α-induced luteal regression through activation of the ROS-dependent JNK pathway. These findings suggest that Prx2 plays a crucial role in preventing accelerated luteal regression via inhibition of the ROS/JNK pathway.


Assuntos
Corpo Lúteo/fisiologia , Luteólise/fisiologia , Peroxirredoxinas/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Células Cultivadas , Corpo Lúteo/patologia , Dinoprosta/metabolismo , Feminino , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/metabolismo
15.
Biochem J ; 473(17): 2603-10, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27334111

RESUMO

XBP1 (X-box-binding protein 1) is activated in cancer and has a pivotal role in tumorigenesis and progression of human cancer. In particular, the XBP1 transcriptional regulatory network is well known to drive cancer development, but little is known about whether the stability of XBP1 is regulated and, if so, what controls the stability of XBP1. In the present study we show that PIN1 prolyl isomerase interacts with the active form of XBP1 (XBP1s) in a phosphorylation-dependent manner and promotes XBP1s-induced cell proliferation and transformation through the regulation of XBP1 stability. By contrast, depletion of Pin1 in cancer cells reduced XBP1s expression, which subsequently inhibits cell proliferation and transformation. Interestingly, XBP1s activates multiple oncogenic pathways including NF-κB (nuclear factor κB), AP1 (activator protein 1) and Myc, and down-regulates PIN1 transcription via a negative-feedback mechanism through p53 induction. Ultimately, reciprocal regulation of Pin1 and XBP1s is associated with the activation of oncogenic pathways, and the relationship of PIN1 and XBP1 may be an attractive target for novel therapy in cancers.


Assuntos
Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias/patologia , Fosforilação
16.
Food Chem Toxicol ; 93: 5-12, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27117918

RESUMO

Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells.


Assuntos
Adipócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Chalconas/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Western Blotting , Hipoglicemiantes/metabolismo , Imunoprecipitação , Lipídeos/química , Camundongos , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Life Sci ; 146: 184-91, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26739509

RESUMO

AIMS: Leydig cells are characterized by their ability to produce testosterone. When the Leydig cells are unable to produce enough testosterone, spermatogenesis fails completely. Considering this, it is of great interest to investigate whether the expressions of steroidogenic enzymes are affected by testicular heat stress. This study aimed to demonstrate that heat induced ER-stress significantly influences steroidogenic enzyme expression and testosterone production in the Leydig cells. MAIN METHODS: C57BL/6 mice were subjected to repetitive testicular heat-treatment at 42 °C for 15 min per day, and heat-treated mLTC-1 cells following hCG treatment for 1h. The protein and RNA expressions were measured by Western blot, RT-PCR. The testosterone and progesterone levels were detected by EIA. The histological and pathological characteristics using hematoxylin and eosin (H&E) and antibody stains. KEY FINDINGS: The 3ß-HSD expression was decreased by heat-stress and hCG treatment. While the GRP78/BiP and CHOP levels were increased by ER-stress inducers, those of the steroidogenic enzyme and progesterone were decreased. In contrast, an ER-stress inhibitor rescued the testosterone levels, even under heat-stress conditions. Moreover, the Leydig cells were randomly scattered, and severely damaged upon repetitive testicular heat-treatment. Additionally, immunohistochemical analyses revealed that cleaved caspase-3 was elevated in the testicular Leydig cells, and rescued by TUDCA. Thus, repetitive testicular heat-treatment in mice promotes excessive ER-stress, thereby leading to apoptosis of the Leydig cells and thus, decreased testosterone production. SIGNIFICANCE: Our findings help to provide an ER-stress mediate mechanistic explanation to the impairment of spermatogenesis upon elevation of the testicular temperature.


Assuntos
Estresse do Retículo Endoplasmático , Hipertermia Induzida , Tumor de Células de Leydig/metabolismo , Testosterona/biossíntese , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Gonadotropina Coriônica/farmacologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Progesterona/biossíntese , RNA/biossíntese , Esteroides/biossíntese , Fator de Transcrição CHOP/biossíntese
18.
Mol Cells ; 38(12): 1071-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26486164

RESUMO

Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.


Assuntos
Gonadotropina Coriônica/administração & dosagem , Gonadotropinas Equinas/administração & dosagem , Ovário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Superovulação/efeitos dos fármacos , Animais , Gonadotropina Coriônica/farmacologia , Combinação de Medicamentos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/farmacologia , Proteínas de Homeodomínio/metabolismo , Camundongos , Especificidade de Órgãos , Ovário/metabolismo , Superovulação/genética , Superovulação/metabolismo
19.
Toxicology ; 337: 39-46, 2015 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-26318285

RESUMO

The accumulation of iron in neurons has been proposed to contribute to the pathology of numerous neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, insufficient research has been conducted on the precise mechanism underlying iron toxicity in neurons. In this study, we investigated mitochondrial dynamics in hippocampal HT-22 neurons exposed to ferric ammonium citrate (FAC) as a model of iron overload and neurodegeneration. Incubation with 150 µM FAC for 48 h resulted in decreased cell viability and apoptotic death in HT-22 cells. The FAC-induced iron overload triggered mitochondrial fragmentation, which was accompanied by Drp1(Ser637) dephosphorylation. Iron chelation with deferoxamine prevented the FAC-induced mitochondrial fragmentation and apoptotic cell death by inhibiting Drp1(Ser637) dephosphorylation. In addition, a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, protected against the FAC-induced mitochondrial fragmentation and neuronal apoptosis. FK506 and cyclosporine A, inhibitors of calcineurin activation, determined that calcineurin was associated with the iron-induced changes in mitochondrial morphology and the phosphorylation levels of Drp1. These results indicate that the FAC-induced dephosphorylation of Drp1-dependent mitochondrial fragmentation was rescued by the inhibition of calcineurin activation. Therefore, these findings suggest that calcineurin-mediated phosphorylation of Drp1(Ser637) acts as a key regulator of neuronal cell loss by modulating mitochondrial dynamics in iron-induced toxicity. These results may contribute to the development of novel therapies for treatment of neurodegenerative disorders related to iron toxicity.


Assuntos
Calcineurina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Sobrecarga de Ferro/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Animais , Apoptose/efeitos dos fármacos , Calcineurina/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dinaminas/efeitos dos fármacos , Camundongos , Mitocôndrias/ultraestrutura , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos
20.
Life Sci ; 113(1-2): 60-7, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25108065

RESUMO

AIMS: We examined whether the three unfolded protein response (UPR) signaling pathways, which are activated in response to endoplasmic reticulum (ER)-stress, are involved in progesterone production in the luteal cells of the corpus luteum (CL) during the mouse estrous cycle. MAIN METHODS: The luteal phase of C57BL/6 female mice (8 weeks old) was divided into two stages: the functional stage (16, 24, and 48 h) and the regression stage (72 and 96 h). Western blotting and reverse transcription (RT)-PCR were performed to analyze UPR protein/gene expression levels in each stage. We investigated whether ER stress affects the progesterone production by using Tm (0.5 µg/g BW) or TUDCA (0.5 µg/g BW) through intra-peritoneal injection. KEY FINDINGS: Our results indicate that expressions of Grp78/Bip, p-eIF2α/ATF4, p50ATF6, and p-IRE1/sXBP1 induced by UPR activation were predominantly maintained in functional and early regression stages of the CL. Furthermore, the expression of p-JNK, CHOP, and cleaved caspase3 as ER-stress mediated apoptotic factors increased during the regression stage. Cleaved caspase3 levels increased in the late-regression stage after p-JNK and CHOP expression in the early-regression stage. Additionally, although progesterone secretion and levels of steroidogenic enzymes decreased following intra-peritoneal injection of Tunicamycin, an ER stress inducer, the expression of Grp78/Bip, p50ATF6, and CHOP dramatically increased. SIGNIFICANCE: These results suggest that the UPR signaling pathways activated in response to ER stress may play important roles in the regulation of the CL function. Furthermore, our findings enhance the understanding of the basic mechanisms affecting the CL life span.


Assuntos
Corpo Lúteo/metabolismo , Perfilação da Expressão Gênica , Fase Luteal , Progesterona/biossíntese , Resposta a Proteínas não Dobradas , Fator 4 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Estro , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo
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