Acute depletion of telomerase components DKC1 and NOP10 induces oxidative stress and disrupts ribosomal biogenesis via NPM1 and activation of the P53 pathway.

Mutations in DKC1, NOP10, and TINF2 genes, coding for proteins in telomerase and shelterin complexes, are responsible for diverse diseases known as telomeropathies and ribosomopathies, including dyskeratosis congenita (DC, ORPHA 1775). These genes contribute to the DC phenotype through mechanisms that are not completely understood. We previously demonstrated in models of DC that oxidative stress is an early and independent event that occurs prior to telomere shortening. To clarify the mechanisms that induce oxidative stress, we silenced genes DKC1, NOP10, and TINF2 with siRNA technology. With RNA array hybridisation, we found several altered pathways for each siRNA model. Afterwards, we identified common related genes. The silenced cell line with the most deregulated genes and pathways was siNOP10, followed by siDKC1, and then by siTINF2 to a lesser extent. The siDKC1 and siNOP10 models shared altered expression of genes in the p53 pathway, while siNOP10 and siTINF2 had the adherens junction pathway in common. We also observed that depletion of DKC1 and NOP10 H/ACA ribonucleoprotein produced ribosomal biogenesis impairment which, in turn, promoted p53 pathway activation. Finally, we found that those enzymes responsible for GSH synthesis were down-regulated in models of siDKC1 and siNOP10. In contrast, the silenced cells for TINF2 showed no disruption of ribosomal biogenesis or oxidative stress and did not produce p53 pathway activation. These results indicate that depletion of DKC1 and NOP10 promotes oxidative stress and disrupts ribosomal biogenesis which, in turn, activates the p53 pathway.

Extracellular histones activate autophagy and apoptosis via mTOR signaling in human endothelial cells.

Circulating histones have been proposed as targets for therapy in sepsis and hyperinflammatory symptoms. However, the proposed strategies have failed in clinical trials. Although different mechanisms for histone-related cytotoxicity are being explored, those mediated by circulating histones are not fully understood. Extracellular histones induce endothelial cell death, thereby contributing to the pathogenesis of complex diseases such as sepsis and septic shock. Therefore, the comprehension of cellular responses triggered by histones is capital to design effective therapeutic strategies. Here we report how extracellular histones induce autophagy and apoptosis in a dose-dependent manner in cultured human endothelial cells. In addition, we describe how histones regulate these pathways via Sestrin2/AMPK/ULK1-mTOR and AKT/mTOR. Furthermore, we evaluate the effect of Toll-like receptors in mediating autophagy and apoptosis demonstrating how TLR inhibitors do not prevent apoptosis and/or autophagy induced by histones. Our results confirm that histones and autophagic pathways can be considered as novel targets to design therapeutic strategies in endothelial damage.

Role of p16INK4a and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3-6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased ß-galactosidase activity and increased lysil oxidase expression, which is mediated by p16INK4a pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.

Oxidative stress and antioxidant response in fibroblasts from Werner and atypical Werner syndromes.

Werner Syndrome (WS, ICD-10 E34.8, ORPHA902) and Atypical Werner Syndrome (AWS, ICD-10 E34.8, ORPHA79474) are very rare inherited syndromes characterized by premature aging. While approximately 90% of WS individuals have any of a range of mutations in theWRN gene, there exists a clinical subgroup in which the mutation occurs in the LMNA/C gene in heterozygosity. Although both syndromes exhibit an age-related pleiotropic phenotype, AWS manifests the onset of the disease during childhood, while major symptoms in WS appear between the ages of 20 and 30. To study the molecular mechanisms of progeroid diseases provides a useful insight into the normal aging process. Main changes found were the decrease in Cu/Zn and Mn SOD activities in the three cell lines. In AWS, both mRNA SOD and protein levels were also decreased. Catalase and glutathione peroxidases decrease, mainly in AWS. Glutaredoxin (Grx) and thioredoxin (Trx) protein expression was lower in the three progeroid cell lines. Grx and Trx were subjected to post-transcriptional regulation, because protein expression was reduced although mRNA levels were not greatly affected in WS. Low antioxidant defense and oxidative stress occur simultaneously in these rare genetic instability disorders at the onset of progeroid disease.

Glutathione and cellular redox control in epigenetic regulation.

Epigenetics is defined as the mitotically/meiotically heritable changes in gene expression that are not due to changes in the primary DNA sequence. Over recent years, growing evidence has suggested a link between redox metabolism and the control of epigenetic mechanisms. The effect of the redox control, oxidative stress, and glutathione (GSH) on the epigenetic mechanisms occur at different levels affecting DNA methylation, miRNAs expression, and histone post-translational modifications (PTMs). Furthermore, a number of redox PTMs are being described, so enriching the histone code. Pioneer works showed how oxidized GSH inhibits the activity of S-adenosyl methionine synthetase, MAT1A, a key enzyme involved in the synthesis of S-adenosyl methionine (SAM), which is used by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). Alteration in NAD /NADH ratio affects the activity of class III histone deacetylases (HDACs) and poly-ADP ribosyltransferases (PARPs). Furthermore, the iron redox state of the catalytic center of key enzymes influences the activity of HDACs and the activity of Tet methylcytosine dioxygenases (DNA demetylases) and JmjC histone demethylases. In this communication, we will show the intricate mechanisms that participate in the redox control of the epigenetic mechanisms. We specially focus our work in the characterization of new PTMs in histones, such as histone carbonylation and glutathionylation. Demonstrating how GSH influences the epigenetic mechanisms beyond a mere regulation of SAM levels. The mechanisms described in this communication place GSH and redox control in the landscape of the epigenetic regulation. The results shown underscore the relevant role that oxidative stress and GSH play as key factors in epigenetics, opening a new window for understating the underlying mechanisms that control cell differentiation, proliferation, development, and disease.