A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung

<p>Abstract</p> <p>Background</p> <p>Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung.</p> <p>Results</p> <p>We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia.</p> <p>Conclusions</p> <p>When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.</p

PARK2 Depletion Connects Energy and Oxidative Stress to PI3K/Akt Activation via PTEN S-Nitrosylation

© 2017 The Authors. PARK2 is a gene implicated in disease states with opposing responses in cell fate determination, yet its contribution in pro-survival signaling is largely un-known. Here we show that PARK2is altered in over a third of all human cancers, and its depletion results in enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and increased vulnerability to PI3K/Akt/mTOR inhibitors. PARK2 depletion contributes to AMPK-mediated activation of endothelial nitricoxide synthase (eNOS), enhanced levels of reactiveoxygen species, and a concomitant increase inoxidized nitric oxide levels, thereby promoting theinhibition of PTEN by S-nitrosylation and ubiquitination. Notably, AMPK activation alone is sufficient to induce PTEN S-nitrosylation in the absence of PARK2 depletion. Park2 loss and Pten loss also display striking cooperativity to promote tumorigenesis in vivo. Together, our findings reveal an important missing mechanism that might account for PTEN suppression in PARK2-deficient tumors, and they highlight the importance of PTEN S-nitrosylationin supporting cell survival and proliferation under conditions of energy deprivation.NIH P01-CA120964 (J.M.A. and L.C.C.) and R01-GM041890; Ministry of Education, Culture and Sport under the Program for Promoting and Hiring of Talent and its Employability (Subprogram for Mobility) of the Spanish Government; ICR; MRC grant MC_UP_1202/1

Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs

<div><p>Background</p><p>The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.</p><p>Methodology</p><p>We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2^-/- and VEGFTG x NOS3^+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated.</p><p>Results</p><p>VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2^−/− and VEGFTG x NOS3^+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.</p><p>Conclusion</p><p>Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.</p></div

A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-beta1-mediated injury in the developing murine lung

Abstract Background Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung. Results We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia. Conclusions When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway

A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung.

BackgroundTransforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung.ResultsWe used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia.ConclusionsWhen viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway

Effect of VEGF overexpression on pulmonary phenotype and caspase 3 mRNA expression in the BPD murine model.

<p>NB WT and VEGF TG+ mice were exposed to room air or hyperoxia to induce the murine model of BPD (hyperoxia from PN1-4; room air from PN5-PN14) and were killed on PN14. All received DOX water from PN day 5 to 14. (<b>A</b>) Representative microphotographs from H&E-stained lung sections showed alveolar size, as confirmed by chord length measurements (<b>B</b>), demonstrated the increased simplification in the NB VEGF TG+ mice as compared to VEGF TG- (WT) in hyperoxia-induced murine model of BPD. (<b>C</b>) The mRNA expression of caspase 3 was also increased in VEGF TG+ lung in NB BPD lungs. Each bar represents the mean ± SEM for a minimum of four animals. Each bar represents the mean ± SEM for a minimum of four animals. *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01 and ***<i>P</i> ≤ 0.001, 2-way ANOVA followed by Tukey test. VEGF: vascular endothelial growth factor; BPD: bronchopulmonary dysplasia; NB: newborn; WT: wild type; NOS: nitric oxide synthase; TG+: transgene positive; TG-: transgene negative; PN: postnatal; DOX: doxycycline.</p

Role of NOS isoforms in VEGF overexpressed lung on angiogenic markers.

<p>NB VEGF TG-, VEGF TG+ and VEGF-TG/ NOS3^+/− mice were sacrificed at PN7. All received DOX water from PN day1 to 7. (<b>A</b> and <b>B</b>) Ang1 and NOTCH2 proteins, with β-actin as controls, were detected by western blotting and analyzed by densitometry. (<b>C</b>) Ang1 and NOTCH2 proteins, with β-actin were detected by western blotting (and densitometry) in L-NIO (NOS1 inhibitor) treated VEGF TG mice. (<b>D—F</b>) Protein levels (Western blot and densitometry) of Collagen IV, VE-cadherin, Ang2, NOS1, NOS2 and NOS3 are performed in indicated groups of PN7 mouse lungs. The figure is representative of n = 3 mice per group, *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001 and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; NB: newborn; TG-: transgene negative; TG+: transgene positive; PN: postnatal; DOX: doxycycline; Ang: angiopoietin.</p

Role of NOS isoforms in VEGF overexpressed lung phenotype.

<p>VEGF TG+ and VEGF TG- mice were received DOX water from PN day1 to 7. (<b>A</b>) NB lungs from a VEGF TG+ showed increased NOS1, NOS2 and NOS3 staining of the endothelial and inflammatory cells was observed in the PN7 lung, compared with control lung samples. The figures are illustrative of a minimum of 5 animals in each group. (<b>B</b> and <b>C</b>) Alveolar size, as measured by chord length, confirmed features noted on lung histology in VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− (<b>D</b> and <b>E</b>) Similarly, chord length of VEGF TG-, VEGF TG+ and VEGF-TG/ NOS3^+/− and (<b>F</b>) chord lengths of VEGF TG+/LNIO groups. Each bar represents the mean ± SEM for a minimum of four animals, *<i>P</i> < 0.05 and **<i>P</i> ≤ 0.01, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; DOX: doxycycline; NB: newborn; PN: postnatal.</p

Role of NOS isoforms in VEGF overexpressed lung cell proliferation.

<p>(<b>A-C</b>) Representative examples of Ki67 (brown) staining in lungs from 7-day-old VEGF TG-, VEGF TG+, VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/−. All received DOX water from PN day1 to 7. Decreased percentage of Ki67 staining area in VEGF TG+, which is increased in VEGF-TG/ NOS2^−/− and VEGF-TG/ NOS3^+/− lungs. Each bar represents the mean ± SEM for a minimum of three animals. *<i>P</i> < 0.05, **<i>P</i> ≤ 0.01, and ****<i>P</i> ≤ 0.0001, 2-way ANOVA followed by Tukey test. NOS: nitric oxide synthase; VEGF: vascular endothelial growth factor; TG-: transgene negative; TG+: transgene positive; DOX: doxycycline; PN: postnatal.</p