Identification of Redox and Glucose-Dependent Txnip Protein Interactions

Thioredoxin-interacting protein (Txnip) acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S). Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip

Detoxification of Mitochondrial Oxidants and Apoptotic Signaling Are Facilitated by Thioredoxin-2 and Peroxiredoxin-3 during Hyperoxic Injury

Mitochondria play a fundamental role in the regulation of cell death during accumulation of oxidants. High concentrations of atmospheric oxygen (hyperoxia), used clinically to treat tissue hypoxia in premature newborns, is known to elicit oxidative stress and mitochondrial injury to pulmonary epithelial cells. A consequence of oxidative stress in mitochondria is the accumulation of peroxides which are detoxified by the dedicated mitochondrial thioredoxin system. This system is comprised of the oxidoreductase activities of peroxiredoxin-3 (Prx3), thioredoxin-2 (Trx2), and thioredoxin reductase-2 (TrxR2). The goal of this study was to understand the role of the mitochondrial thioredoxin system and mitochondrial injuries during hyperoxic exposure. Flow analysis of the redox-sensitive, mitochondrial-specific fluorophore, MitoSOX, indicated increased levels of mitochondrial oxidant formation in human adenocarcinoma cells cultured in 95% oxygen. Increased expression of Trx2 and TrxR2 in response to hyperoxia were not attributable to changes in mitochondrial mass, suggesting that hyperoxic upregulation of mitochondrial thioredoxins prevents accumulation of oxidized Prx3. Mitochondrial oxidoreductase activities were modulated through pharmacological inhibition of TrxR2 with auranofin and genetically through shRNA knockdown of Trx2 and Prx3. Diminished Trx2 and Prx3 expression was associated with accumulation of mitochondrial superoxide; however, only shRNA knockdown of Trx2 increased susceptibility to hyperoxic cell death and increased phosphorylation of apoptosis signal-regulating kinase-1 (ASK1). In conclusion, the mitochondrial thioredoxin system regulates hyperoxic-mediated death of pulmonary epithelial cells through detoxification of oxidants and regulation of redox-dependent apoptotic signaling

Consequences of a Maternal High-Fat Diet and Late Gestation Diabetes on the Developing Rat Lung.

RATIONALE:Infants born to diabetic or obese mothers are at risk of respiratory distress and persistent pulmonary hypertension of the newborn (PPHN), conceivably through fuel-mediated pathogenic mechanisms. Prior research and preventative measures focus on controlling maternal hyperglycemia, but growing evidence suggests a role for additional circulating fuels including lipids. Little is known about the individual or additive effects of a maternal high-fat diet on fetal lung development. OBJECTIVE:The objective of this study was to determine the effects of a maternal high-fat diet, alone and alongside late-gestation diabetes, on lung alveologenesis and vasculogenesis, as well as to ascertain if consequences persist beyond the perinatal period. METHODS:A rat model was used to study lung development in offspring from control, diabetes-exposed, high-fat diet-exposed and combination-exposed pregnancies via morphometric, histologic (alveolarization and vasculogenesis) and physiologic (echocardiography, pulmonary function) analyses at birth and 3 weeks of age. Outcomes were interrogated for diet, diabetes and interaction effect using ANOVA with significance set at p≤0.05. Findings prompted additional mechanistic inquiry of key molecular pathways. RESULTS:Offspring exposed to maternal diabetes or high-fat diet, alone and in combination, had smaller lungs and larger hearts at birth. High-fat diet-exposed, but not diabetes-exposed offspring, had a higher perinatal death rate and echocardiographic evidence of PPHN at birth. Alveolar mean linear intercept, septal thickness, and airspace area (D2) were not significantly different between the groups; however, markers of lung maturity were. Both diabetes-exposed and diet-exposed offspring expressed more T1α protein, a marker of type I cells. Diet-exposed newborn pups expressed less surfactant protein B and had fewer pulmonary vessels enumerated. Mechanistic inquiry revealed alterations in AKT activation, higher endothelin-1 expression, and an impaired Txnip/VEGF pathway that are important for vessel growth and migration. After 3 weeks, mortality remained highest and static lung compliance and hysteresis were lowest in combination-exposed offspring. CONCLUSION:This study emphasizes the effects of a maternal high-fat diet, especially alongside late-gestation diabetes, on pulmonary vasculogenesis, demonstrates adverse consequences beyond the perinatal period and directs attention to mechanistic pathways of interest. Findings provide a foundation for additional investigation of preventative and therapeutic strategies aimed at decreasing pulmonary morbidity in at-risk infants

Electron flux via Prx3 and Trx2 during hyperoxic injury.

<p>Schematic demonstrating mitochondrial electron flux via the Prx3 and Trx2 under control and hyperoxic conditions.</p

Protein expression of mitochondrial redoxins Prx3, Trx2, and TrxR2 during hyperoxia.

<p>Representative SDS-PAGE/immunoblots of cell lysates probed for Prx3, Trx2, and TrxR2 with β-actin as a loading control in (A) A549 and (B) H1299 human lung adenocarcinoma cell lines cultured for increasing days in hyperoxia. Images are representative of 3 independent biological replicates.</p

Trx2 and Prx3 oxidation during hyperoxia.

<p>(A,C) Differential Trx2 and Prx3 thiol labeling by AMS and NEM respectively, and (B,D) representative detection by SDS-PAGE/immunoblot in A549 cells after treatment with hyperoxia. Images are representative of 3–4 independent biological replicates.</p

Trx2 inhibition sensitizes cells to hyperoxic cell death.

<p>(A) Inhibition of TrxR activity in A549 cells cultured for 24 hours with 1 or 2.5 μM AFN. (B) Fold-change in MitoSOX red fluorescence intensity after 2 days hyperoxic culture and (C) TO-PRO-3 labeling after 3 days of hyperoxic culture with 1 or 2.5 μM AFN. (D) SDS-PAGE/immunoblot of A549 cell lysates for Trx2 and Prx3 protein expression 2 days following lentiviral delivery of non-targeting (NT) and Trx2- or Prx3-targeting shRNAs. (E) Fold-change MitoSOX red fluorescence intensity after 2 days of hyperoxia and (F) TO-PRO-3 labeling after 3 days hyperoxic culture following lentiviral transduction of NT, Trx2, or Prx3 shRNAs in A549 cells. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA. Statistical significance was defined as *p<0.05, **p<0.01, and †p<0.001 (n = 3).</p

Hyperoxia does not alter mitochondrial mass.

<p>Ratio of mitochondrial:nuclear Ct values for (A) <i>D-Loop</i> and (B) <i>COX1</i> after normalization to <i>β2M</i> quantified by qPCR in A549 during hyperoxic culture. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA.</p

Mitochondrial oxidants promote hyperoxic cell death.

<p>MitoSOX was used to quantify mitochondrial oxidants in A549 cells cultured in hyperoxia. (A) MitoSOX red fluorescence intensity and (B) fold-change after 2 days of hyperoxic culture and during a hyperoxic time course. (C) Hyperoxic cell death measured via flow cytometry using TOPRO-3. (D) Fold-change in MitoSOX red fluorescence intensity of A549 cells after 2 days of hyperoxic culture with increased concentrations of MitoTEMPO supplemented in the media. (E) Death of A549 cells cultured concurrently with 3 days hyperoxia and MitoTEMPO supplementation. Data are expressed as mean ± standard deviation of 3 biological replicates analyzed by one-way ANOVA. Statistical significance was defined as *p<0.05, **p<0.01, and †p<0.001.</p