Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila

The vertebrate Apolipoprotein D (ApoD) is a lipocalin secreted from subsets of neurons and glia during neural development and aging . A strong correlation exists between ApoD overexpression and numerous nervous system pathologies as well as obesity, diabetes, and many forms of cancer . However, the exact relationship between the function of ApoD and the pathophysiology of these diseases is still unknown. We have generated loss-of-function Drosophila mutants for the Glial Lazarillo (GLaz) gene , a homolog of ApoD in the fruit fly, mainly expressed in subsets of adult glial cells. The absence of GLaz reduces the organism's resistance to oxidative stress and starvation and shortens male lifespan. The mutant flies exhibit a smaller body mass due to a lower amount of neutral lipids stored in the fat body. Apoptotic neural cell death increases in aged flies or upon paraquat treatment, which also impairs neural function as assessed by behavioral tests. The higher sensitivity to oxidative stress and starvation and the reduced fat storage revert to control levels when a GFP-GLaz fusion protein is expressed under the control of the GLaz natural promoter. Finally, GLaz mutants have a higher concentration of lipid peroxidation products, pointing to a lipid peroxidation protection or scavenging as the mechanism of action for this lipocalin. In agreement with Walker et al. (, in this issue of Current Biology), who analyze the effects of overexpressing GLaz, we conclude that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration.     

Drosophila lacking a homologue of mammalian ALDH2 have multiple fitness defects

Little is known about the roles of aldehyde dehydrogenases in non-vertebrate animals. We recently showed that in Drosophila melanogaster, an enzyme with ～70% amino acid identity to mammalian ALDH2 is necessary for detoxification of dietary ethanol. To investigate other functions of this enzyme, DmALDH, encoded by the gene Aldh, we compared two strains homozygous for Aldh-null mutations to two closely related wild type strains in measures of fitness and stress resistance in the absence of ethanol. Aldh-null strains have lower total reproductive rate, pre-adult viability, resistance to starvation, and possibly longevity than wild-type strains. When maintained under hyperoxia, Aldh nulls die more quickly and accumulate higher levels of protein carbonyls than wild-types, thereby providing evidence that DmALDH is important for detoxifying reactive aldehydes generated by lipid peroxidation. However no effect of Aldh was seen on protein carbonyl levels in flies maintained under normoxia. It is possible that Aldh nulls experience elevated rates of protein carbonylation under normoxia, but this is compensated (at a fitness cost) by increased rates of degradation of the defective proteins. Alternatively, the fitness defects of Aldh nulls under normoxia may result from the absence of one or more other functions of DmALDH, unrelated to protection against protein carbonylation.     

Single and multigenerational responses of body mass to atmospheric oxygen concentrations in Drosophila melanogaster : evidence for roles of plasticity and evolution

Greater oxygen availability has been hypothesized to be important in allowing the evolution of larger invertebrates during the Earth’s history, and across aquatic environments. We tested for evolutionary and developmental responses of adult body size of Drosophila melanogaster to hypoxia and hyperoxia. Individually reared flies were smaller in hypoxia, but hyperoxia had no effect. In each of three oxygen treatments (hypoxia, normoxia or hyperoxia) we reared three replicate lines of flies for seven generations, followed by four generations in normoxia. In hypoxia, responses were due primarily to developmental plasticity, as average body size fell in one generation and returned to control values after one to two generations of normoxia. In hyperoxia, flies evolved larger body sizes. Maximal fly mass was reached during the first generation of return from hyperoxia to normoxia. Our results suggest that higher oxygen levels could cause invertebrate species to evolve larger average sizes, rather than simply permitting evolution of giant species.     

Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz

Lazarillo (Laz) is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein first characterized in the developing nervous system of the grasshopper Schistocerca americana. It belongs to the Lipocalins, a functionally diverse family of mostly secreted proteins. In this work we test whether the protective capacity known for Laz homologs in flies and vertebrates (NLaz, GLaz and ApoD) is evolutionarily conserved in grasshopper Laz, and can be exerted from the plasma membrane in a cell-autonomous manner. First we demonstrate that extracellular forms of Laz have autocrine and paracrine protecting effects for oxidative stress-challenged Drosophila S2 cells. Then we assay the effects of overexpressing GPI-linked Laz in adult Drosophila and whether it rescues both known and novel phenotypes of NLaz null mutants. Local effects of GPI-linked Laz inside and outside the nervous system promote survival upon different stress forms, and extend lifespan and healthspan of the flies in a cell-type dependent manner. Outside the nervous system, expression in fat body cells but not in hemocytes results in protection. Within the nervous system, glial cell expression is more effective than neuronal expression. Laz actions are sexually dimorphic in some expression domains. Fat storage promotion and not modifications in hydrocarbon profiles or quantities explain the starvation-desiccation resistance caused by Laz overexpression. This effect is exerted when Laz is expressed ubiquitously or in dopaminergic cells, but not in hemocytes. Grasshopper Laz functionally restores the loss of NLaz, rescuing stress-sensitivity as well as premature accumulation of aging-related damage, monitored by advanced glycation end products (AGEs). However Laz does not rescue NLaz courtship behavioral defects. Finally, the presence of two new Lipocalins with predicted GPI-anchors in mosquitoes shows that the functional advantages of GPI-linkage have been commonly exploited by Lipocalins in the arthropodan lineage.     

Responses of Drosophila melanogaster to atypical oxygen atmospheres

We examined physiological phenotypes of Drosophila melanogaster in hypoxic to hyperoxic atmospheres. We performed measurements on life span or behavioural function in 5, 21, 40, 60, and 80% O₂, and combined this with literature data for 2% and 100% O₂. O₂ incubation resulted in a concentration-dependent reduction of life span in both hypoxia and hyperoxia, though different measures of life span were affected differently. We also examined how behavioural and metabolic functions were affected by exposure to hyperoxia (up to 60% O₂). Climbing behaviour was measured as a fast (4 s) and slow (55 s) response in a negative geotaxis assay. In normoxia, both measures of climbing response declined exponentially until disappearing completely. Interestingly, survivorship was very high until the loss of climbing ability, after which it dropped rapidly. This pattern appeared accelerated in 40% O₂. However, while flies in 60% O₂ also apparently lost their fast climbing ability immediately prior to the drop in survivorship, they maintained considerable climbing ability over the longer trial. Metabolism, measured by CO₂ release, did not change with age in normoxic flies, but was significantly lower in flies exposed to hyperoxia, particularly as the flies aged. There was, however, a slight increase in water loss rate with age in normoxia, while in hyperoxia, water loss was reduced. Uniquely, the water loss rates of flies in 60% O₂ doubled immediately prior to the end of their life span. Because ageing results in generally irreversible functional declines, we examined if functional declines in hyperoxia (60% O₂) were also irreversible, or whether some functioning could recover after a return to normoxia. After 7 days of recovery, water loss rates decreased, CO₂ exhalation slightly increased, and climbing ability was partially recovered. Therefore, the effect of O₂ on D. melanogaster function is non-linear, may be reversible, and may include unique phenotypes that arise at some O₂ concentrations, and not others.     

Loss of Gadd45a does not modify the pulmonary response to oxidative stress

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.     

Hemodynamics of local cerebral blood flow induced by somatosensory stimulation under normoxia and hyperoxia in rats.

We observed changes in the local cerebral blood flow (LCBF), red blood cell (RBC) concentration and RBC velocity in alpha-chloralose anesthetized rats using laser-Doppler flowmetry during activation of the somatosensory cortex following electrical stimulation of the hind paw under hyperoxia (PaO(2)=513.5+/-48.4 mmHg; mean+/-S.D.) and normoxia (PaO(2)=106.4+/-8.4 mmHg). Electrical stimuli of 5 and 10 Hz (pulse width 0.1 ms) with an intensity of 1.5 mA were applied for 5 s (n=13 at 5 Hz, n=9 at 10 Hz). Baseline levels of LCBF and RBC concentration under hyperoxia were, respectively, 5.6+/-3.3 and 8.8+/-3.0% lower than those under normoxia (P<0.05), and that of RBC velocity under hyperoxia was slightly higher than that under normoxia (NS), suggesting mild vasoconstriction at rest under hyperoxia. At 5 Hz stimulation, after normalization to each baseline level, normalized response magnitudes of LCBF, RBC concentration and RBC velocity under hyperoxia were, respectively, 68.2+/-48.0, 71.1+/-65.5 and 66.0+/-56.3% greater than those under normoxia (P<0.05). At 10-Hz stimulation, normalized response magnitudes of LCBF and RBC concentration under hyperoxia were, respectively, 44.6+/-32.0 and 55.9+/-43.5% greater than those under normoxia (P<0.05), although a significant difference in the normalized response magnitude of RBC velocity was not detected between both conditions. The evoked LCBF under hyperoxia increased earlier, by approximately 0.15 s, than that under normoxia regardless of the stimulus frequency (P<0.05). These results suggest the involvement of oxygen interaction on the regulation of LCBF during neuronal activation.     

Effects of continuous and episodic hyperoxia on stress and hepatic glutathione levels in one-summer-old rainbow trout (Oncorhynchus mykiss)

One-summer-old rainbow trout ( Oncorhynchus mykiss ) were exposed to continuous hyperoxia (173 ± 24%) and three hyperoxic/normoxic treatments for 14 days. Hepatic glutathione status as the indicator of oxidative stress, as well as classical stress indicators such as hemoglobin, hematocrit and plasma cortisol levels, were measured during normoxic, constantly hyperoxic and the following episodically hyperoxic oxygen treatment regimes: 12 h hyperoxia:12 h normoxia (12 HYP:12 NOR), 24 HYP:24 NOR and 48 HYP:24 NOR. Constant hyperoxia tended to shrink erythrocytes, but the 12 HYP:12 NOR treatment increased the number of erythrocytes and thus enhanced the oxygen carrying capacity of blood. Similarly, a trend toward an elevation in plasma cortisol concentrations was detected in 12 HYP:12 NOR treatment group. The finding that elevated hepatic glutathione (GSH) levels (P < 0.01), indicative of enhanced potential of the liver tissue to resist oxidative stress, coincided with elevated cortisol levels might suggest that in the 12 HYP:12 NOR treatment physiological processes were recruited to increase oxygen carrying capacity in blood and to elevate protection against oxyradicals. However, none of the episodic hyperoxia treatments or continuous hyperoxia caused mortality or resulted in better growth. These data indicate that continuous hyperoxia (173 ± 24%) and hyperoxic-normoxic treatments may be applied in intensive culture of rainbow trout provided that fish have at least 24 h in normoxia prior to the next bout of hyperoxia. Shorter recovery periods, like in a 12 HYP:12 NOR treatment, may result in the increased need of oxygen in tissues followed by an activation of glutathione dependent defence system against an increased oxygen load.     

The role of cholinergic networks of the anterior basal and inferior frontal lobes in the predatory behaviour of Sepia officinalis

Hyperoxia reduces the hemodynamic latency and enhances the response magnitude of the evoked local cerebral blood flow (LCBF). The objective of this study was to test the hypothesis that a change in the production of nitric oxide (NO) is involved in a unique change in evoked LCBF during hyperoxia. We measured LCBF in alpha-chloralose-anesthetized rats by laser-Doppler flowmetry. Systemic administration of the NO synthase inhibitor N(omega)-nitro-L-arginine (LNA) caused a decline in the baseline level of LCBF (P<0.01). The LNA intravenous injection during hyperoxia (hyperoxia with LNA) reduced the normalized evoked LCBF (normalization with respect to the baseline level of LCBF) in response to somatosensory stimulation by approximately 37% when compared under normal conditions (normoxia without LNA) (P<0.01), although that during normoxia (normoxia with LNA) did not cause a significant difference in the normalized evoked LCBF. The integrated neuronal activity under hyperoxia with LNA was approximately 11% lower than that under normoxia without LNA (P<0.05), although there was no significant difference in integrated neuronal activity between normoxia with LNA and normoxia without LNA. These results do not support our hypothesis and suggest the existence of another interaction mechanism involving oxygen for the enhancement of evoked LCBF under hyperoxia.     

Evidence for an age-related attenuation of cerebral microvascular antioxidant response to oxidative stress

Effects of aging and oxidative stress were studied in cerebral microvessels and microvessel-depleted brain from 6-, 18-, and 24-month-old C57Bl/6J mice exposed to normoxia, 24 or 48 h hyperoxia, or 24 h hyperoxia followed by 24 h normoxia. Microvessels lacked smooth muscle and consisted predominantly of endothelium. Following exposure and isolation of microvessel and parenchymal proteins, Western blot analysis was performed for detection of cytosolic thioredoxin 1 (TRx 1) and mitochondrial thioredoxin 2 (TRx 2), protein carbonyl, and mitochondrial superoxide dismutase (MnSOD). Both microvessel and parenchymal TRx 1 levels were increased by hyperoxia; however, the microvascular response was limited and delayed in comparison to that of the parenchymal fraction. Whereas TRx 2 levels in microvessels were increased in older mice, irrespective of exposure condition, hyperoxia per se had little or no apparent effect. Parenchymal cells showed no age-related increase in TRx 2 level under normoxic conditions, but showed increased levels following hyperoxia. Microvessel MnSOD was lower than that in parenchymal cells, but increased with age under normoxia, and also was correlated with the duration of hyperoxia. Although hyperoxia augmented MnSOD levels in young (6 months) and middle-aged (18 months) animals, the response was less pronounced in microvessels from senescent, 24-month-old mice. Unlike microvessels, which showed a sustained age-related increase in MnSOD level under each exposure condition, parenchymal cells from normoxic mice showed no increase, and hyperoxia-induced elevations declined with prolonged 48 h exposure. These results indicate that the microvessel endothelium is (1) subjected to a more intense oxidative environment than neurons and glia and (2) is limited by aging in its ability to respond to oxidative insult.     

Oxygen-induced changes in hemoglobin expression in Drosophila

The hemoglobin gene 1 (dmeglob1) of the fruit fly Drosophila melanogaster is expressed in the tracheal system and fat body, and has been implicated in hypoxia resistance. Here we investigate the expression levels of dmeglob1 and lactate dehydrogenase (a positive control) in embryos, third instar larvae and adult flies under various regimes of hypoxia and hyperoxia. As expected, mRNA levels of lactate dehydrogenase increased under hypoxia. We show that expression levels of dmeglob1 are decreased under both short- and long-term hypoxia, compared with the normoxic (21% O2) control. By contrast, a hypoxia/reoxygenation regime applied to third instar larvae elevated the level of dmeglob1 mRNA. An excess of O2 (hyperoxia) also triggered an increase in dmeglob1 mRNA. The data suggest that Drosophila hemoglobin may be unlikely to function merely as a myoglobin-like O2 storage protein. Rather, dmeglob1 may protect the fly from an excess of O2, either by buffering the flux of O2 from the tracheoles to the cells or by degrading noxious reactive oxygen species.     

Human 8-oxoguanine DNA glycosylase increases resistance to hyperoxic cytotoxicity in lung epithelial cells and involvement with altered MAPK activity

It is unknown whether base excision DNA repair (BER) proteins interact with mitogen-activated protein kinases (MAPK) under oxidation. Here, we explored roles of BER proteins in signaling transduction involving MAPK during hyperoxia. We demonstrated that ERK1/2 phosphorylation in A549 cells was increased in 95% O(2). p38 activity in A549 cells was also increased by exposure to 95% O(2). To evaluate regulatory roles of MAPK, we have transduced A549 cells and primary alveolar epithelial type II cells (AECII) to overexpress 8-oxoguanine DNA glycosylase (hOgg1). Overexpression of hOgg1 reduced hyperoxic toxicity in A549 and AECII cells. Furthermore, protection by BER against hyperoxia appeared to involve an upregulation of ERK1/2 and downregulation of p38. These observations demonstrate, for the first time, that reduction of hyperoxic toxicity by BER proteins may be involved with MAPK activity, thereby impacting cell survival. Furthermore, our studies suggest that modulation of MAPK may be used in combination with BER proteins to counteract hyperoxic toxicity.     

Enhancing macroautophagy protects against ischemia/reperfusion injury in cardiac myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x(L) were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x(L) binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.     

Distinct role of Hsp70 in Drosophila hemocytes during severe hypoxia

Severe hypoxia can lead to injury and mortality in vertebrate or invertebrate organisms. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. In this study, we employed the UAS-Gal4 system to dissect the protective role of Hsp70 in specific tissues in vivo under severe hypoxia. In contrast to overexpression in tissues such as muscles, heart, and brain, we found that overexpression of Hsp70 in hemocytes of flies provides a remarkable survival benefit to flies exposed to severe hypoxia for days. Furthermore, these flies were tolerant not only to severe hypoxia but also to other stresses such as oxidant stress (e.g., paraquat feeding or hyperoxia). Interestingly we observed that the better survival with Hsp70 overexpression in hemocytes under hypoxia or oxidant stress is causally linked to reactive oxygen species (ROS) reduction in whole flies. We also show that hemocytes are a major source of ROS generation, leading to injury during hypoxia, and their elimination results in a better survival under hypoxia. Hence, our study identified a protective role for Hsp70 in Drosophila hemocytes, which is linked to ROS reduction in the whole flies and thus helps in their remarkable survival during oxidant or hypoxic stress.     

Oxygen-limited thermal tolerance in Antarctic fish investigated by MRI and (31)P-MRS

The hypothesis of an oxygen-limited thermal tolerance was tested in the Antarctic teleost Pachycara brachycephalum. With the use of flow-through respirometry, in vivo (31)P-NMR spectroscopy, and MRI, we studied energy metabolism, intracellular pH (pH(i)), blood flow, and oxygenation between 0 and 13 degrees C under normoxia (PO(2): 20.3 to 21.3 kPa) and hyperoxia (PO(2): 45 kPa). Hyperoxia reduced the metabolic increment and the rise in arterial blood flow observed under normoxia. The normoxic increase of blood flow leveled off beyond 7 degrees C, indicating a cardiovascular capacity limitation. Ventilatory effort displayed an exponential rise in both groups. In the liver, blood oxygenation increased, whereas in white muscle it remained unaltered (normoxia) or declined (hyperoxia). In both groups, the slope of pH(i) changes followed the alpha-stat pattern below 6 degrees C, whereas it decreased above. In conclusion, aerobic scope declines around 6 degrees C under normoxia, marking the pejus temperature. By reducing circulatory costs, hyperoxia improves aerobic scope but is unable to shift the breakpoint in pH regulation or lethal limits. Hyperoxia appears beneficial at sublethal temperatures, but no longer beyond when cellular or molecular functions become disturbed.     

Phenoloxidase activity and pathogen resistance in yellow dung flies Scathophaga stercoraria

Phenoloxidase (PO) is an important component of the insect immune system and is frequently used to measure an individual's immune defence ability. However, evidence documenting positive correlations between the immune assay and resistance against pathogens is scarce and contradictory. We used replicate lines of yellow dung flies Scathophaga stercoraria (L.) with different PO levels to investigate whether PO levels affect resistance against parasitic mites and entomopathogenic fungi. Prevalence of flies exposed to pathogens was the same in all selection regimes, although pathogens clearly negatively affected fitness. PO measurements alone therefore do not necessarily predict overall resistance against pathogens. Furthermore, under starvation lines selected for high PO levels did not survive longer than those selected for low PO levels, irrespective of exposure to pathogens. This suggests that even if elevated immune levels increase an individual's ability to combat pathogens, the benefits may not outweigh the costs of increased investment in immunity.     

Effect of normobaric hyperoxia on antioxidant defenses of HeLa and CHO cells

The effect of increased intracellular oxygen activation on cellular antioxidant defenses in CHO and HeLa cells was studied. In both cell types, hyperoxic exposure (up to 4 days, 600-700 mm Hg O2) and in CHO cells menadione (up to 3 days, 15 microM) failed to affect the enzymatic antioxidant defenses Mn-containing superoxide dismutase (Mn-SOD), CuZn-SOD, catalase and glutathione peroxidase. The markedly increased antioxidant enzyme activities observed in a recently obtained oxygen-tolerant CHO variant persisted under normoxia. These data suggest that the synthesis of antioxidant enzymes is constitutive. Glutathione levels of HeLa cells did not respond to hyperoxia whereas in CHO cells hyperoxia and menadione exposure resulted in a 2- and 7-fold increase in glutathione contents, respectively. However, considering the large variations in glutathione contents observed under normal culture conditions, it is uncertain whether this increase is to be considered as a true adaptive response.     

Selection for resistance to a fungal pathogen in Drosophila melanogaster

An artificial selection experiment designed to explore the evolution of resistance to a fungal pathogen, Beauveria bassiana, in Drosophila melanogaster is reported here. The experiment was designed to test whether there is sufficient additive genetic variation in this trait for increased resistance to evolve, and, if so, whether there are correlated responses that might represent a cost to defence. After 15 generations of selection, flies from selected lines did not have higher overall fitness after infection compared with control lines. The response to selection for resistance against this pathogen is thus much weaker than against other species, in particular, parasitoids. There was, however, evidence for increased late-life fecundity in selected lines, which may indicate evolved tolerance of fungal infection. This increase was accompanied by reduced early-life fitness, which may reflect the well-known trade-off between early and late reproduction. In the absence of fungal infection, selected flies had lower fitness than control flies, and the possibility that this is also a trade-off with increased tolerance is explored.     

Wnt3a Mediates the Inhibitory Effect of Hyperoxia on the Transdifferentiation of AECIIs to AECIs

The aim of this study is to investigate the effect of Wnt3a in the transdifferentiation of type II alveolar epithelial cells (AECIIs) to type I alveolar epithelial cells (AECIs) under hyperoxia condition. In the in vivo study, preterm rats were exposed in hyperoxia for 21 days. In the in vitro study, primary rat AECIIs were subjected to a hyperoxia and normoxia exposure alternatively every 24 hr for 7 days. siRNA-mediated knockout of Wnt3a and exogenous Wnt3a were used to investigate the effect of Wnt3a on transdifferentiation of AECIIs to AECIs. Wnt5a-overexpressed AECIIs were also used to investigate whether Wnt3a could counteract the effect of Wnt5a. The results showed that hyperoxia induced alveolar damage in the lung of preterm born rats, as well as an increased expression of Wnt3a and nuclear accumulation of β-catenin. In addition, Wnt3a/β-catenin signaling was activated in isolated AECIIs after hyperoxia exposure. Wnt3a knockout blocked the inhibition of the transdifferentiation induced by hyperoxia, and Wnt3a addition exacerbated this inhibition. Furthermore, Wnt3a addition blocked the transdifferentiation-promoting effect of Wnt5a in hyperoxia-exposed Wnt5a-overexpressed AECIIs. In conclusion, our results demonstrate that the activated Wnt3a/β-catenin signal may be involved in the hyperoxia-induced inhibition of AECIIs’ transdifferentiation to AECIs.