Disrupted postnatal lung development in heme oxygenase-1 deficient mice

Abstract Background Heme oxygenase (HO) degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development. Methods Neonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex) till postnatal 14 days, and evaluated for lung development. Results Compared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment. Conclusions These experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development.</p

Hyperoxia-induced NF-κB activation occurs via a maturationally sensitive atypical pathway

NF-κB activation is exaggerated in neonatal organisms after oxidant and inflammatory insults, but the reason for this and the downstream effects are unclear. We hypothesized that specific phosphorylation patterns of IκBα could account for differences in NF-κB activation in hyperoxia-exposed fetal and adult lung fibroblasts. After exposure to hyperoxia (>95% O2), nuclear NF-κB binding increased in fetal, but not adult, lung fibroblasts. Unique to fetal cells, phosphorylation of IκBα on tyrosine 42, rather than serine 32/36 as seen in TNF-α-exposed cells, preceded NF-κB nuclear translocation. In fetal cells stably transfected with an NF-κB-driven luciferase reporter, hyperoxia significantly suppressed reporter activity, in contrast to increased reporter activity after TNF-α incubation. Targeted gene profiling analysis showed that hyperoxia resulted in decreased expression of multiple genes, including proapoptotic factors. Transfection with a dominant-negative IκBα (Y42F), which cannot be phosphorylated on tyrosine 42, resulted in upregulation of multiple proapoptotic genes. In support of this finding, caspase-3 activity and DNA laddering were specifically increased in fetal lung fibroblasts expressing Y42F after exposure to hyperoxia. These data demonstrate a unique pathway of NF-κB activation in fetal lung fibroblasts after exposure to hyperoxia, whereby these cells are protected against apoptosis. Activation of this pathway in fetal cells may prevent the normal pattern of fibroblast apoptosis necessary for normal lung development, resulting in aberrant lung morphology in vivo

Heme oxygenase-1 regulates postnatal lung repair after hyperoxia: Role of β-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 β-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 β-catenin/hnRNPK regulated gene expression required for coordinated lung repair and regeneration

The Joseph Rowntree Charitable Trust: A Study in Quaker Philanthropy and Adult Education 1904-1954

(A) Neurite formation in SY5Y cells in response to 13cRA. Representative phase contrast images depicting extension of neurites in response to 13cRA treatment after 6 days and 10 days. Long neurites were observed when cells were treated with 13cRA. (B) Statistical analysis of neurite outgrowth following 13cRA treatment in SY5Y cells. Length of neurites from three to five representative fields of 13cRA treated cells were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments, and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 208 kb

Additional file 4: Figure S4 of Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma

(A) Detection of neurites in NB69 cells in response to retinoic acid. Representative phase contrast images of NB69 cells showing long neurites upon 13cRA treatment for 6 and 10 days. (B) Statistical analysis of various neurites upon 13cRA treatment in NB69 cells. Length of neurites from various selected fields were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 296 kb

Additional file 2: Figure S2. of Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma

Effect of 13cRA on cell proliferation by SRB assay. SY5Y and NB69 cells were plated on 96 well plates and treated with or without 10 ΟM retinoic acid. Plates were harvested after 2, 5, and 7 days of 13cRA treatment. Cell viability was analyzed by an SRB assay measuring optical density (OD). Cell lines treated with 13cRA showed lower OD, indicating reduced cell number compared to untreated control cells. (DOC 46 kb

Additional file 5: Figure S5. of Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma

(A) Neurite inhibition by CEP-701 in SY5Y-TrkA cells. Representative phase contrast images of SY5Y-TrkA cells depicting extension of neurites in response to 13cRA treatment after 4 days, and subsequent neurite inhibition following 100 nM CEP-701 treatment (μm-micrometer) (B) Statistical analysis of neurite outgrowth following 13cRA as well as CEP-701 treatment of SY5Y-TrkA cells. Length of neurites (μm-micrometer) from three to five representative fields of 13cRA and CEP-701 treated cells were measured in μm and compared with neurites from untreated cells. P-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 148 kb