Nuclear heme oxygenase-1 (HO-1) modulates subcellular distribution and activation of Nrf2, impacting metabolic and anti-oxidant defenses.

With oxidative injury as well as in some solid tumors and myeloid leukemia cells, heme oxygenase-1 (HO-1), the anti-oxidant, anti-inflammatory, and anti-apoptotic microsomal stress protein, migrates to the nucleus in a truncated and enzymatically inactive form. However, the function of HO-1 in the nucleus is not completely clear. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor and master regulator of numerous antioxidants and anti-apoptotic proteins, including HO-1, also accumulates in the nucleus with oxidative injury and in various types of cancer. Here we demonstrate that in oxidative stress, nuclear HO-1 interacts with Nrf2 and stabilizes it from glycogen synthase kinase 3ß (GSK3ß)-mediated phosphorylation coupled with ubiquitin-proteasomal degradation, thereby prolonging its accumulation in the nucleus. This regulation of Nrf2 post-induction by nuclear HO-1 is important for the preferential transcription of phase II detoxification enzymes such as NQO1 as well as glucose-6-phosphate dehydrogenase (G6PDH), a regulator of the pentose phosphate pathway. Using Nrf2 knock-out cells, we further demonstrate that nuclear HO-1-associated cytoprotection against oxidative stress depends on an HO-1/Nrf2 interaction. Although it is well known that Nrf2 induces HO-1 leading to mitigation of oxidant stress, we propose a novel mechanism by which HO-1, by modulating the activation of Nrf2, sets an adaptive reprogramming that enhances antioxidant defenses.

IκBß-mediated NF-κB activation confers protection against hyperoxic lung injury.

Supplemental oxygen is frequently used in an attempt to improve oxygen delivery; however, prolonged exposure results in damage to the pulmonary endothelium and epithelium. Although NF-κB has been identified as a redox-responsive transcription factor, whether NF-κB activation exacerbates or attenuates hyperoxic lung injury is unclear. We determined that sustained NF-κB activity mediated by IκBß attenuates lung injury and prevents mortality in adult mice exposed to greater than 95% O2. Adult wild-type mice demonstrated evidence of alveolar protein leak and 100% mortality by 6 days of hyperoxic exposure, and showed NF-κB nuclear translocation that terminated after 48 hours. Furthermore, these mice showed increased expression of NF-κB-regulated proinflammatory and proapoptotic cytokines. In contrast, mice overexpressing the NF-κB inhibitory protein, IκBß (AKBI), demonstrated significant resistance to hyperoxic lung injury, with 50% surviving through 8 days of exposure. This was associated with NF-κB nuclear translocation that persisted through 96 hours of exposure. Although induction of NF-κB-regulated proinflammatory cytokines was not different between wild-type and AKBI mice, significant up-regulation of antiapoptotic proteins (BCL-2, BCL-XL) was found exclusively in AKBI mice. We conclude that sustained NF-κB activity mediated by IκBß protects against hyperoxic lung injury through increased expression of antiapoptotic genes.

Heme oxygenase-1 regulates postnatal lung repair after hyperoxia: role of ß-catenin/hnRNPK signaling.

In the newborn, alveolarization continues postnatally and can be disrupted by hyperoxia, leading to long-lasting consequences on lung function. We wanted to better understand the role of heme oxygenase (HO)-1, the inducible form of the rate-limiting enzyme in heme degradation, in neonatal hyperoxic lung injury and repair. Although it was not observed after 3 days of hyperoxia alone, when exposed to hyperoxia and allowed to recover in air (O2/air recovered), neonatal HO-1 knockout (KO) mice had enlarged alveolar spaces and increased lung apoptosis as well as decreased lung protein translation and dysregulated gene expression in the recovery phase of the injury. Associated with these changes, KO had sustained low levels of active ß-catenin and lesser lung nuclear heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein levels, whereas lung nuclear hnRNPK was increased in transgenic mice over-expressing nuclear HO-1. Disruption of HO-1 may enhance hnRNPK-mediated inhibition of protein translation and subsequently impair the ß-catenin/hnRNPK regulated gene expression required for coordinated lung repair and regeneration.

Nuclear factor-κB (NF-κB) inhibitory protein IκBß determines apoptotic cell death following exposure to oxidative stress.

The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBß in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBß would prevent apoptotic cell death. Using IκBß knock-in mice (AKBI), in which the IκBα gene is replaced with the IκBß cDNA, we show that IκBß overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-κB subunits in the nucleus and maintenance of NF-κB activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing IκBß with simultaneous IκBα knockdown, whereas IκBß overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of IκBß in preventing oxidant stress-induced cell death.