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1.
Redox Biol ; 67: 102866, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37703667

RESUMO

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 µM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 µU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control µU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 µM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.


Assuntos
Hemina , Xantina Desidrogenase , Animais , Camundongos , Xantina Desidrogenase/genética , Xantina Desidrogenase/metabolismo , Hemina/farmacologia , Ferro , NF-kappa B , Heme , Hepatócitos/metabolismo
2.
Redox Biol ; 62: 102636, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36906950

RESUMO

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 µmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).


Assuntos
Hemólise , Receptor 4 Toll-Like , Xantina Oxidase , Animais , Camundongos , Hemina , Fígado/metabolismo , Camundongos Knockout , Oxidantes , Xantina , Xantina Oxidase/metabolismo , Xantinas
3.
J Clin Invest ; 132(18)2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36106636

RESUMO

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.


Assuntos
Insuficiência Cardíaca , Miócitos Cardíacos , Animais , Morte Súbita Cardíaca , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Oxirredução , Volume Sistólico
4.
Obesity (Silver Spring) ; 30(7): 1420-1429, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35610936

RESUMO

OBJECTIVE: Obesity has emerged as a prominent risk factor for multiple serious disease states, including a variety of cancers, and is increasingly recognized as a primary contributor to preventable cancer risk. However, few studies of leukemia have been conducted in animal models of obesity. This study sought to characterize the impact of obesity, diet, and sex in a murine model of acute promyelocytic leukemia (APL). METHODS: Male and female C57BL/6J.mCG+/PR mice, genetically predisposed to sporadic APL development, and C57BL/6J (wild type) mice were placed on either a high-fat diet (HFD) or a low-fat diet (LFD) for up to 500 days. RESULTS: Relative to LFD-fed mice, HFD-fed animals displayed increased disease penetrance and shortened disease latency as indicated by accelerated disease onset. In addition, a diet-responsive sex difference in APL penetrance and incidence was identified, with LFD-fed male animals displaying increased penetrance and shortened latency relative to female counterparts. In contrast, both HFD-fed male and female mice displayed 100% disease penetrance and insignificant differences in disease latency, indicating that the sexual dimorphism was reduced through HFD feeding. CONCLUSIONS: Obesity and obesogenic diet promote the development of APL in vivo, reducing sexual dimorphisms in disease latency and penetrance.


Assuntos
Leucemia Promielocítica Aguda , Caracteres Sexuais , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Penetrância
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