Exploring redox vulnerabilities in JAK2V617F-positive cellular models

ABSTRACT Background: In Philadelphia chromosome-negative myeloproliferative neoplasm (MPN) models, reactive oxygen species (ROS) are elevated and have been implicated in genomic instability, JAK2/STAT signaling amplification, and disease progression. Although the potential effects of ROS on the MPN phenotype, the effects of ruxolitinib treatment on ROS regulation have been poorly explored. Herein, we have reported the impact of ruxolitinib on redox signaling transcriptional network, and the effects of diphenyleneiodonium (DPI), a pan NOX inhibitor, in JAK2V617F-driven cellular models. Method: Redox signaling-related genes were investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (GEO accession GSE69827). SET2 and HEL cells, which represent JAK2V617F-positive MPN cellular models with distinct sensitivity to apoptosis induced by ruxolitinib, were used. Cell viability was evaluated by MTT, apoptosis by annexin V/PI and flow cytometry, and cell signaling by quantitative PCR and Western blot. Main results: Ruxolitinib impacted on a network composed of redox signaling-related genes, and DUOX1 and DUOX2 were identified as potential modulators of ruxolitinib response. In SET2 and HEL cells, DPI reduced cell viability and, at low doses, it significantly potentiated ruxolitinib-induced apoptosis. In the molecular scenario, DPI inhibited STAT3, STAT5 and S6 ribosomal protein phosphorylation and induced PARP1 cleavage in JAK2V617F-positive cells. DPI combined with ruxolitinib increased PARP1 cleavage in SET2 cells and potentiated ruxolitinib-reduced STAT3, STAT5 and S6 ribosomal protein in HEL cells. Conclusion: Our study reveals a potential adaptation mechanism for resistance against ruxolitinib by transcriptionally reprogramming redox signaling in JAK2V617F cells and exposes redox vulnerabilities with therapeutic value in MPN cellular models.

Mini review: immunologic functions of dual oxidases in mucosal systems of vertebrates / Minirrevisão: funções imunológicas de oxidases duplas em sistemas mucosos de vertebrados

Abstract Mucosal epithelial cells act as the first immunologic barrier of organisms, and contact directly with pathogens. Therefore, hosts must have differential strategies to combat pathogens efficiently. Reactive oxygen species (ROS), as a kind of oxidizing agents, participates in the early stage of killing pathogens quickly. Recent reports have revealed that dual oxidase (DUOX) plays a key role in mucosal immunity. And the DUOX is a transmembrane protein which produces ROS as their primary enzymatic products. This process is an important pattern for eliminating pathogens. In this review, we highlight the DUOX immunologic functions in the respiratory and digestive tract of vertebrates.

Resumo As células epiteliais da mucosa atuam como a primeira barreira imunológica dos organismos e entram em contato direto com os patógenos. Portanto, os hospedeiros devem ter estratégias diferenciadas para combater os patógenos de forma eficiente. Trabalhos recentes revelaram que a oxidase dupla (DUOX) desempenha um papel fundamental para a imunidade da mucosa. A DUOX é uma proteína transmembrana geradora de espécies reativas de oxigênio (EROs) como seus principais produtos enzimáticos. Nesta revisão, apresentaremos as funções imunológicas da DUOX no trato respiratório e digestivo dos vertebrados.

The role of vitamin C in the gene expression of oxidative stress markers in fibroblasts from burn patients

Abstract Purpose: To assess the action of vitamin C on the expression of 84 oxidative stress related-genes in cultured skin fibroblasts from burn patients. Methods: Skin samples were obtained from ten burn patients. Human primary fibroblasts were isolated and cultured to be distributed into 2 groups: TF (n = 10, fibroblasts treated with vitamin C) and UF (n = 10, untreated fibroblasts). Gene expression analysis using quantitative polymerase chain reaction array was performed for comparisons between groups. Results: The comparison revealed 10 upregulated genes as follows: arachidonate 12-lipoxygenase (ALOX12), 24-dehydrocholesterol reductase (DHCR24), dual oxidase 1 (DUOX1), glutathione peroxidase 2 (GPX2), glutathione peroxidase 5 (GPX5), microsomal glutathione S-transferase 3 (MGST3), peroxiredoxin 4 (PRDX4), phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1 (P-REX1), prostaglandin-endoperoxide synthase 1 (PTGS1), and ring finger protein 7 (RNF7). Conclusion: Cultured fibroblasts obtained from burn patients and treated with vitamin C resulted in 10 differentially expressed genes, all overexpressed, with DUOX1, GPX5, GPX2 and PTGS1 being of most interest.

Variant analysis of a patient with dyshormonogenesis due to congenital hypothyroidism / 中华医学遗传学杂志

OBJECTIVE@#To carry out variant analysis for a Chinese boy featuring dyshormonogenesis due to congenital hypothyroidism.@*METHODS@#DNA of the patient and his parents was extracted and sequenced by high-throughput sequencing. The results were validated with Sanger sequencing and analyzed with Bioinformatics software.@*RESULTS@#Sequencing result showed that the patient has carried compound variants of c.2654G>T(p.Arg885Leu) and c.943G>T(p.Gly315X) of the DUOX2 gene, which were inherited respectively from his mother and father.@*CONCLUSION@#The missense mutation c.2654G>T and nonsense mutation c.943G>T probably underlie the disease in this child.

Hipotiroidismo congénito transitorio por defectos bialélicos del genDUOX2. Dos casos clínicos / Transient congenital hypothyroidism due to biallelic defects of DUOX2 gene. Two clinical cases

El hipotiroidismo congénito afecta a 1:2000-3000 recién nacidos detectados por pesquisa neonatal. Las oxidasas duales, DUOX1 y 2, generan agua oxigenada, lo que constituye un paso crítico en la síntesis hormonal. Se han comunicado mutaciones en el gen DUOX2 en casos de hipotiroidismo congénito transitorio y permanente. Se describen dos hermanos con hipotiroidismo congénito detectados por pesquisa neonatal, con glándula tiroides eutópica y tiroglobulina elevada. Recibieron levotiroxina hasta su reevaluación en la infancia con suspensión del tratamiento. Su función tiroidea fue normal y se consideró el cuadro como transitorio por un posible defecto de organificación. Ambos pacientes eran heterocigotos compuestos para una mutación en el exón 9 del alelo paterno (c.1057_1058delTT, p.F353PfsX36 o p.F353fsX388) y otra en el exón 11 del alelo materno (c.1271T>G, p.Y425X) del gen DUOX2. Nuestro hallazgo confirma que la magnitud del defecto de DUOX2 no se relaciona con el número de alelos afectados, lo que sugiere mecanismos compensadores en la generación de peróxido.

Congenital hypothyroidism affects 1:2000-3000 newborns detected by neonatal screening programs. Dual oxidases, DUOX1 and 2, generate hydrogen peroxide needed for the thyroid hormone synthesis. Mutations in the DUOX2 gene have been described in transient and permanent congenital hypothyroidism. Two brothers with congenital hypothyroidism detected by neonatal screening with eutopic gland and elevated thyroglobulin are described. They were treated with levothyroxine until it could be suspended in both during childhood, assuming the picture as transient. Organification disorder was confirmed. Both patients were compounds heterozygous for a mutation in exon 9 of the paternal allele (c.1057_1058delTT, p.F353PfsX36 or p.F353fsX388) and in exon 11 of the maternal allele (c.1271T > G, p.Y425X) of DUOX2 gene. Our finding confirms that the magnitude of the defect of DUOX2 is not related to the number of inactivated alleles, suggesting compensatory mechanisms in the peroxide supply

DUOX2 mutations in children with congenital hypothyroidism / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the features of DUOX2 mutations and genotype-phenotype relationship in children with congenital hypothyroidism (CH), in order to provide evidence for gene diagnosis and gene treatment of CH.</p><p><b>METHODS</b>Blood samples were collected from 10 CH children with thyromegaly. Genomic DNA was extracted from peripheral blood leukocytes. All exons of DUOX2 gene were analyzed using PCR and direct sequencing.</p><p><b>RESULTS</b>G3632A mutation in the exon 28 of DUOX2 that may result in arginine to histidine substitution at codon 1211 was found in one patient. T2033C mutation in the exon 17 of DUOX2 that may result in histidine to arginine substitution at codon 678 was found in three patients. They were all heterozygous mutations.</p><p><b>CONCLUSIONS</b>Heterozygous mutations in DUOX2 may affect protein function and cause CH. The relationship between DUOX2 genotypes and clinical phenotypes is unclear and needs further studies.</p>

Primary mechanism of the role of dual oxidase-1 causing airway allergic diseases in human bronchial epithelium / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the role of dual oxidase-1 (DUOX-1) inducing airway hyperresponsiveness in human bronchial epithelium.</p><p><b>METHODS</b>The human bronchial epithelial cells were divided into several groups: control group, tumor necrosis factor-α (TNF-α) group, methyl-β-cyclodextrin (M-β-CD)+TNF-α group, desipramine (DES)+ TNF-α group, diphenylene iodonium (DPI) + TNF-α group and apocynin (APO)+TNF-α group. Fractionation was performed by sucrose gradient centrifugation and the protein DUOX-1 was measured by western blotting. The lipid raft clusters and its colocalization with DUOX-1 were confocal analysed. The intracellular reactive oxygen species (ROS) accumulation was measured by fluorescence of reactive oxygen probe of intracellular measurement. Sigmastat 3.02 software was used to analyze the data.</p><p><b>RESULTS</b>(1) Detection of ROS, control group: 1.00 ± 0.00; TNF-α group: 1.95 ± 0.16; M-β-CD+TNF-α group: 0.91 ± 0.16; DES+TNF-α group: 1.49 ± 0.20; DPI+TNF-α group: 1.03 ± 0.16; APO+TNF-α group: 1.47 ± 0.26. The difference was statistically significant (F = 3.83, P < 0.05). (2) Extracts in rafts to lipid rafts region represents the ratio of total protein, protein content DUOX-1 each group, control group: 0.21 ± 0.02; TNF-α group: 0.49 ± 0.04; M-β-CD+TNF-α group: 0.08 ± 0.02; DES+TNF-α group: 0.09 ± 0.03; the difference was statistically significant (F = 3.96, P < 0.05). (3) DUOX-1 protein fluorescence values, control group: 1.72 ± 0.21; TNF-α group: 8.11 ± 1.23; M-β-CD+TNF-α group: 1.51 ± 0.32; DES+TNF-α group: 1.43 ± 0.11; the difference was statistically significant (F = 4.87, P < 0.05). (4) DUOX-1 gene detection, control group: 1.00 ± 0.00 ScrRNA+TNF-α group: 1.75 ± 0.04; DUOX-1siRNA+TNF-αgroup: 1.15 ± 0.02; the difference was statistically significant (F = 4.19, P < 0.05).</p><p><b>CONCLUSION</b>TNF-α can induce DUOX-1 expression increasing in lipid raft, then the DUOX-1 can be activated to increase reactive oxygen species level; acidic sphingomyelinase inhibitor desipramine can inhibit this process, the results disclose that the process will depend on the ceramide of lipid raft.</p>

DUOX2 gene mutation in patients with congenital goiter with hypothyroidism / 中华儿科杂志

<p><b>OBJECTIVE</b>To identify DUOX2 gene mutation in patients with congenital goiter with hypothyroidism.</p><p><b>METHOD</b>Five patients who had transit congenital hypothyroidism with goiter were enrolled. The exons of DUOX2 gene were amplified and sequenced.</p><p><b>RESULT</b>A heterozygous missense mutation C1329T in the exon 10 of the DUOX2 gene was found in one patient, predicted to result in a Tryptophan to Arginine substitution at codon 376. However no mutation was detected in the other patients.</p><p><b>CONCLUSION</b>p.Arg376Trp mutation in DUOX2 was found in newborns of congenital hypothyroidism. The alleles frequency of this mutation may contribute to the function loss of congenital hypothyroidism.</p>