Expression Level and Subcellular Localization of Heme Oxygenase-1 Modulates Its Cytoprotective Properties in Response to Lung Injury: A Mouse Model

Abstract

Premature infants exposed to hyperoxia suffer acute and long-term pulmonary consequences. Nevertheless, neonates survive hyperoxia better than adults. The factors contributing to neonatal hyperoxic tolerance are not fully elucidated. In contrast to adults, heme oxygenase (HO)-1, an endoplasmic reticulum (ER)-anchored protein, is abundant in the neonatal lung but is not inducible in response to hyperoxia. The latter may be important, because very high levels of HO-1 overexpression are associated with significant oxygen cytotoxicity in vitro. Also, in contrast to adults, HO-1 localizes to the nucleus in neonatal mice exposed to hyperoxia. To understand the mechanisms by which HO-1 expression levels and subcellular localization contribute to hyperoxic tolerance in neonates, lung-specific transgenic mice expressing high or low levels of full-length HO-1 (cytoplasmic, HO-1-FL(H) or HO-1-FL(L)) or C-terminally truncated HO-1 (nuclear, Nuc-HO-1-TR) were generated. In HO-1-FL(L), the lungs had a normal alveolar appearance and lesser oxidative damage after hyperoxic exposure. In contrast, in HO-1-FL(H), alveolar wall thickness with type II cell hyperproliferation was observed as well worsened pulmonary function and evidence of abnormal lung cell hyperproliferation in recovery from hyperoxia. In Nuc-HO-1-TR, the lungs had increased DNA oxidative damage, increased poly (ADP-ribose) polymerase (PARP) protein expression, and reduced poly (ADP-ribose) (PAR) hydrolysis as well as reduced pulmonary function in recovery from hyperoxia. These data indicate that low cytoplasmic HO-1 levels protect against hyperoxia-induced lung injury by attenuating oxidative stress, whereas high cytoplasmic HO-1 levels worsen lung injury by increasing proliferation and decreasing apoptosis of alveolar type II cells. Enhanced lung nuclear HO-1 levels impaired recovery from hyperoxic lung injury by disabling PAR-dependent regulation of DNA repair. Lastly both high cytoplasmic and nuclear expression of HO-1 predisposed to long-term abnormal lung cellular proliferation. To maximize HO-1 cytoprotective effects, therapeutic strategies must account for the specific effects of its subcellular localization and expression levels.

Heme Oxygenase in Neonatal Lung Injury and Repair

“…In neonatal HO-1 null mutant mice and WT littermates exposed to hyperoxia for 3 days and allowed to recover in room air for 11 days, six DNA damage-response genes were down-regulated in the WT; whereas these were up-regulated many-fold in the knockout, suggesting that HO-1 disruption modifies DNA repair pathways which are important in tumorigenesis (115). We also show that transgenic mice with cytoplasmic over-expression of HO-1 in type II cells exposed to 3 days of hyperoxia as neonates had increased numbers of multinucleated hyper-proliferating type II cells and foamy macrophages but no evidence of fibrosis or inflammation (69). This correlated with lung lesions on MRI with enhanced p-ERK (which is seen in early tumorigenesis) and PCNA staining (69).…”

“…Despite the cytoprotective effects of moderate HO-1 overexpression, we have shown that at high levels, HO-1 may be deleterious (108), resulting in enhanced oxidative stress and apoptosis and decreased cell proliferation in vitro (99) and, when targeted to type II cells in vivo, this leads to a maladaptive over-proliferation of type II cells and perhaps a failure to differentiate to type I cells which are important for recapitulation of the normal alveolar epithelium. This then manifests as increased alveolar thickness and decreased lung function (69). In neonatal rats exposed to hyperoxia, no significant increase in lung HO-1 mRNA was seen in contrast to similarly exposed adults (21).…”

“…Corroborating this, mouse HO-1 null mutant MEFs with stable over-expression of nuclear HO-1 show decreased cellular proliferation and are relatively tolerant to 24 h of hyperoxia compared with MEFs expressing cytoplasmic HO-1 or empty vector controls (Fernando, unpublished observations). Intriguingly, transgenic mice over-expressing nuclear HO-1 in type II cells initially showed improved alveolarization with (3 days) hyperoxic exposure but had increased oxidative DNA damage and abnormal lung histology and pulmonary function tests as adults (69), raising the possibility that the duration of nuclear HO-1 protein signaling is key to cytoprotective responses to injury and repair.…”

“…To this effect, lung (Type II cell)-specific transgenic mice expressing high or low levels of fulllength HO-1 (cytoplasmic) or C-terminally truncated HO-1 (nuclear) were generated (69). Mice were exposed to hyperoxia for 3 days as neonates and then allowed to recover in room air for approximately 8 weeks.…”

“…This demonstrates that low cytoplasmic levels of HO-1 protect against hyperoxiainduced lung injury by attenuating oxidative stress, whereas high cytoplasmic levels worsen lung injury by increasing type II cell proliferation, alveolar wall thickness, thereby impeding gas exchange. Enhanced lung nuclear HO-1 impairs recovery by disabling poly (ADP-ribose)-dependent regulation of DNA repair (69). Interestingly, when HO-1 null mutant mice exposed to hyperoxia as neonates were evaluated after 11 days of recovery in room air, these mice exhibited significant changes in lung alveolarization and altered expression of genes which were important in cell proliferation and DNA damage (115).…”