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AIMS: Vascular calcification is strongly linked to the development of major adverse cardiovascular events, but effective treatments are lacking. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are an emerging category of oral hypoglycemic drugs that have displayed marked effects on metabolic and cardiovascular diseases, including recently reported vascular medial calcification. However, the roles and underlying mechanisms of SGLT2 inhibitors in vascular calcification have not been fully elucidated. Thus, we aimed to further determine whether SGLT2 inhibitors protect against vascular calcification and to investigate the mechanisms involved. METHODS AND RESULTS: A computed tomography angiography investigation of coronary arteries from 1554 patients with type 2 diabetes revealed that SGLT2 inhibitor use was correlated with a lower Agatston calcification score. In the vitamin D3 overdose, 5/6 nephrectomy chronic kidney disease-induced medial calcification and Western diet-induced atherosclerotic intimal calcification models, dapagliflozin (DAPA) substantially alleviated vascular calcification in the aorta. Furthermore, we showed that DAPA reduced vascular calcification via Runx2-dependent osteogenic transdifferentiation in vascular smooth muscle cells (VSMCs). Transcriptome profiling revealed that thioredoxin domain containing 5 (TXNDC5) was involved in the attenuation of vascular calcification by DAPA. Rescue experiments showed that DAPA-induced TXNDC5 downregulation in VSMCs blocked the protective effect on vascular calcification. Furthermore, TXNDC5 downregulation disrupted protein folding-dependent Runx2 stability and promoted subsequent proteasomal degradation. Moreover, DAPA downregulated TXNDC5 expression via amelioration of oxidative stress and ATF6-dependent endoplasmic reticulum stress. Consistently, the class effects of SGLT2 inhibitors on vascular calcification were validated with empagliflozin in intimal and medial calcification models. CONCLUSIONS: SGLT2 inhibitors ameliorate vascular calcification through blocking endoplasmic reticulum stress-dependent TXNDC5 upregulation and promoting subsequent Runx2 proteasomal degradation, suggesting that SGLT2 inhibitors are potentially beneficial for vascular calcification treatment and prevention.
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Glucósidos , Osteogénesis , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Calcificación Vascular , Calcificación Vascular/metabolismo , Calcificación Vascular/tratamiento farmacológico , Calcificación Vascular/patología , Calcificación Vascular/etiología , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Animales , Humanos , Osteogénesis/efectos de los fármacos , Ratones , Glucósidos/farmacología , Masculino , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Compuestos de Bencidrilo/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Ratas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , FemeninoRESUMEN
Thioredoxin domain containing 5 (TXNDC5) is a protein disulfide isomerase involved in several diseases related to oxidative stress, energy metabolism and cellular inflammation. In a previous manuscript, a negative association between fatty liver development and hepatic Txndc5 expression was observed. To study the role of TXNDC5 in the liver, we generated Txndc5-deficient mice. The absence of the protein caused an increased metabolic need to gain weight along with a bigger and fatter liver. RNAseq was performed to elucidate the putative mechanisms, showing a substantial liver overexpression of serum amyloid genes (Saa1, Saa2) with no changes in hepatic protein, but discrete plasma augmentation by the gene inactivation. Higher levels of malonyldialdehyde, apolipoprotein A1 and platelet activating factor-aryl esterase activity were also found in serum from Txndc5-deficient mice. However, no difference in the distribution of high-density lipoproteins (HDL)-mayor components and SAA was found between groups, and even the reactive oxygen species decreased in HDL coming from Txndc5-deficient mice. These results confirm the relation of this gene with hepatic steatosis and with a fasting metabolic derive remedying an acute phase response. Likewise, they pose a new role in modulating the nature of HDL particles, and SAA-containing HDL particles are not particularly oxidized.
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Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress, compared to the pcDNA3.1(+) transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress.
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Disulfuros , Gastrópodos , Insulinas , Tiorredoxinas , Secuencia de Aminoácidos , Animales , Gastrópodos/genética , Regulación de la Expresión Génica , Listeria monocytogenes , Novirhabdovirus , Moléculas de Patrón Molecular Asociado a Patógenos , Filogenia , Tiorredoxinas/genética , Vibrio parahaemolyticusRESUMEN
BACKGROUND & AIMS: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention. METHODS: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease. RESULTS: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease. CONCLUSIONS: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD. LAY SUMMARY: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.
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The thioredoxin domain containing 5 (TXNDC5) is a recently discovered member of the protein disulfide isomerase family (PDI), which is mainly involved in the proper folding of and the correct formation of disulfide bonds in newly synthesized proteins via its disulfide isomerase and chaperone activities. Although the structural and functional features of mammalian TXNDC5 have been explored in previous studies, no studies have reported the functional characteristics of TXNDC5 in teleost fish. In this study, we report the identification and characterization of TXNDC5 from big-belly seahorse (Hippocampus abdominalis) (ShTXNDC5) accompanied by functional studies. The in-silico analysis revealed that the gene encodes a 433 amino acid (aa) long polypeptide chain with a predicted molecular weight of 49.3 kDa. According to homology analysis, ShTXNDC5 shares more than 55% sequence similarity with other teleost TXNDC5 proteins, and the alignment of the gene sequence convincingly reflects the accepted phylogeny of teleost. Analysis of the spatial distribution of ShTXNDC5 expression showed that its highest expression was observed in the ovary, gill, and pouch of seahorses. Moreover, significant upregulation of ShTXNDC5 transcription was noted in seahorse blood and kidney tissues in a time-dependent manner upon viral and bacterial immune challenges. Furthermore, considerable NADPH turnover, insulin reduction ability and significant cell survival effects of ShTXNDC5 were determined by the functional assay, revealing its capability to overcome cellular oxidative stress. Altogether, these findings expand our understanding of TXNDC5 at the molecular and functional levels, and its putative role in seahorse immunity.
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Edwardsiella tarda/fisiología , Infecciones por Enterobacteriaceae/inmunología , Proteínas de Peces/genética , Ovario/metabolismo , Smegmamorpha/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus iniae/fisiología , Tiorredoxinas/genética , Animales , Células Cultivadas , Disulfuros , Femenino , Proteínas de Peces/metabolismo , Inmunomodulación , Estrés Oxidativo , Filogenia , Proteína Disulfuro Isomerasas/genética , Alineación de Secuencia , Tiorredoxinas/metabolismo , Transcriptoma , Regulación hacia ArribaRESUMEN
Sulfiredoxin (Srx) reduces hyperoxidized 2-cysteine-containing peroxiredoxins (Prxs) and protects cells against oxidative stress. Previous studies have shown that Srx is highly expressed in primary specimens of lung cancer patients and plays a pivotal role in lung tumorigenesis and cancer progression. However, the oncogenic mechanisms of Srx in cancer are incompletely understood. In this study, we found that Srx knockdown sensitizes lung cancer cells to endoplasmic reticulum (ER) stress-induced cell death. Through MS analysis, we determined that Srx forms a complex with the ER-resident protein thioredoxin domain-containing protein 5 (TXNDC5). Using reciprocal co-immunoprecipitation, immunofluorescence imaging, subcellular fractionation, and domain-mapping assays with site-specific mutagenesis and purified recombinant proteins, we further characterized the Srx-TXNDC5 interaction. In response to ER stress but not to oxidative stress, Srx exhibits an increased association with TXNDC5, facilitating the retention of Srx in the ER. Of note, TXNDC5 knockdown in lung cancer cells inhibited cell proliferation and repressed anchorage-independent colony formation and migration, but increased cell invasion and activation of mitogen-activated protein kinases. Using immunohistochemical staining, we demonstrate that TXNDC5 is highly expressed in patient-derived lung cancer specimens. Bioinformatics analysis of publicly available data sets revealed that those with high Srx levels have significantly shorter survival and that those with high TXNDC5 levels have longer survival. We conclude that the cellular levels of Srx and TXNDC5 may be useful as biomarkers to predict the survival of individuals with lung cancer.
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Estrés del Retículo Endoplásmico , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Células A549 , Apoptosis/efectos de los fármacos , Sitios de Unión , Proliferación Celular , Biología Computacional/métodos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Espectrometría de Masas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Péptidos/análisis , Péptidos/química , Unión Proteica , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tunicamicina/farmacologíaRESUMEN
Thioredoxin domain-containing 5 (TXNDC5) is overexpressed in a number of human carcinomas. However, the involvement of TXNDC5 in gastric adenocarcinoma remains unclear. In the present study, the immunohistochemical expression and clinicopathological significance of TXNDC5 in gastric adenocarcinoma was investigated. The immunohistochemical expression of TXNDC5 was detected in 54 gastric adenocarcinoma specimens, and the correlation between TXNDC5 and the clinicopathological features was investigated. Of the 54 gastric adenocarcinoma specimens, 30 samples (55.6%) exhibited high TXNDC5 expression. In the adenocarcinoma specimens exhibiting high TXNDC5 expression, the proportion of poorly-differentiated adenocarcinomas was significantly higher than that in specimens exhibiting low TXNDC5 expression (P<0.05). Lymph node metastasis and the depth of tumor invasion in the specimens exhibiting high TXNDC5 expression were significantly higher than that in specimens exhibiting low TXNDC5 expression (P<0.05). The results of a survival analysis revealed that the prognosis of patients exhibiting high TXNDC5 expression was significantly poorer than that of patients exhibiting low TXNDC5 expression (P<0.05). Therefore, the expression of TXNDC5 may correlate with the differentiation, invasion and metastasis of gastric adenocarcinoma. Thus, TXNDC5 may be a tumor-enhancing gene that is involved in gastric cancer.
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Thioredoxin domain-containing protein-5 (TXNDC5) has been found to be associated with cancer development and growth. We investigated whether TXNDC5 gene polymorphisms are associated with hepatocellular carcinoma (HCC) in a Korean male population. Seven SNPs were selected based on minor allelic frequency (≥5%). The SNPs consisted of three exonic SNPs (rs8643, rs7764128 and rs1043784) and four intronic SNPs (rs1225944, rs1225943, rs1225945 and rs1225958). We selected and assessed these SNPs in 160 patients with HCC and 178 controls. Genetic data were analyzed using SNPAnalyzer Pro, SNPStats, and Haploview programs. Two SNPs of the TXNDC5 gene were found to be associated with the risk of HCC development. The genotypic frequency of rs1225944 was associated with HCC in the recessive model [CC/CT vs. TT, p=0.43, Fisher's exact test p=0.032; odds ratio (OR)=0.54, 95% confidence interval (CI)=0.11-2.71]. The genotypic frequency of rs1225943 was associated with HCC in the co-dominant 2 (AA vs. CC, p=0.07; Fisher's exact test p=0.001, OR=0.23, 95% CI=0.05-1.10), recessive (AA/AC vs. CC, p=0.044, Fisher's exact test p=0.001, OR=0.25, 95% CI=0.05-1.17), and log-additive models (p=0.08, Fisher's exact test p=0.002, OR=0.68, 95% CI=0.44-1.05). The haplotype CA and TC of rs1229544 and rs1225943, demonstrated a significant association with HCC. Our results suggest that TXNDC5 polymorphisms could be related to the development of HCC in the Korean male population.