Peter F. Vitiello

P21Cip1 Protects against Oxidative Stress by Suppressing ER Dependent Activation of Mitochondrial Death Pathways

Although it is well established that the cell cycle inhibitor p21 protects against genotoxic stress by preventing the replication of damaged DNA, recent studies have shown that the cytoplasmic form can also protect. It protects by delaying the loss of the antiapoptotic proteins Mcl-1 and Bcl-X(L); however, the mechanism of regulation is unknown. Utilizing hyperoxia as a model of chronic oxidative stress and DNA damage, p21 was detected in the nucleus and cytoplasm and cytoplasmic expression of p21 was sufficient for cytoprotection. p21 was enriched in a subcellular fraction containing mitochondria and endoplasmic reticulum (ER), suggesting that it may be coordinating ER and mitochondrial stress pathways. Consistent with this, p21 suppressed hyperoxic downregulation of BiP and subsequent activation of ER stress signaling, which affected Mcl-1, but not Bcl-X(L); though both inhibited hyperoxic cell death. Taken together, these data show that p21 integrates the DNA damage response with ER stress signaling, which then regulates mitochondrial death pathways during chronic genotoxic stress.

Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1.

Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death.

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.

Epithelial ablation of Bcl-XL increases sensitivity to oxygen without disrupting lung development.

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium. Because loss of Bcl-X(L) is embryonically lethal, Bcl-X(L) was ablated throughout the respiratory epithelium by mating mice with a floxed exon II of the Bcl-X gene with mice expressing Cre under control of the surfactant protein-C promoter. Interestingly, the loss of Bcl-X(L) in respiratory epithelium was perinatally lethal in approximately 50% of the expected offspring. However, some adult mice lacking the gene were obtained. The epithelial-specific ablation of Bcl-X(L) did not disrupt pulmonary function, the expression of epithelial cell-specific markers, or lung development. However, it shifted the lung toward a proapoptotic state, defined by a reduction in antiapoptotic Mcl-1, an increase in proapoptotic Bak, and increased sensitivity of the respiratory epithelium to hyperoxia. Intriguingly, increased 8-oxoguanine lesions seen during hyperoxia were also evident as lungs transitioned to room air at birth, a time when perinatal lethality in some mice lacking Bcl-X(L) was observed. These findings reveal that the epithelial-specific expression of Bcl-X(L) is not required for proper lung development, but functions to protect respiratory epithelial cells against oxygen-induced toxicity, such as during hyperoxia and the lungs first exposure to ambient air.

Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

ABSTRACT Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling. Summary: We identify genes that are differentially expressed in yeast lacking vacuolar cystine transporter Ers1p in order to find pathways, such as respiration and sulfur regulation, that are associated with cystine homeostasis.

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.

Differentiating Antiproliferative and Chemopreventive Modes of Activity for Electron-Deficient Aryl Isothiocyanates against Human MCF-7 Cells.

The consumption of Brassica vegetables provides beneficial effects through organic isothiocyanates (ITCs), products of the enzymatic hydrolysis of glucosinolate secondary metabolites. The ITC l‐sulforaphane (l‐SFN) is the principle agent in broccoli that demonstrates several modes of anticancer action. While the anticancer properties of ITCs like l‐SFN have been extensively studied and l‐SFN has been the subject of multiple human clinical trials, the scope of this work has largely been limited to those derivatives found in nature. Previous studies have demonstrated that structural changes in an ITC can lead to marked differences in a compounds potency to 1) inhibit the growth of cancer cells, and 2) alter cellular transcriptional profiles. This study describes the preparation of a library of non‐natural aryl ITCs and the development of a bifurcated screening approach to evaluate the dose‐ and time‐dependence on antiproliferative and chemopreventive properties against human MCF‐7 breast cancer cells. Antiproliferative effects were evaluated using a commercial MTS cell viability assay. Chemopreventive properties were evaluated using an antioxidant response element (ARE)‐promoted luciferase reporter assay. The results of this study have led to the identification of 1) several key structure–activity relationships and 2) lead ITCs for continued development.

Creating a Reliable, Cost-Effective ELISA Simulation

Abstract The enzyme-linked immunosorbent assay (ELISA) is a fundamental laboratory technique with direct applications across scientific research and clinical diagnostics as well as everyday life. Unfortunately, many challenges exist that inhibit both its introduction and implementation in the high school biology classroom. We present a reliable yet inexpensive way of effectively simulating this assay, allowing student exposure to several advanced topics, including immunodetection, clinical diagnostics, and qualitative and quantitative colorimetric analysis.