HIF and ER stress are involved in TGFß1-mediated wound closure of alveolar epithelial cells.

Purpose: Alveolar epithelium dysfunction is associated with a very large spectrum of disease and an abnormal repair capacity of the airway epithelium has been proposed to explain the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Following epithelium insult, the damaged cells will activate pathways implicated in the repair process, including proliferation and acquisition of migratory capacities to cover the denuded basement membrane. Induction of Endoplasmic Reticulum stress may be implicated in this process. Interestingly, ER stress excessive activation has been proposed as a central event associated with aberrant repair process and cellular dysfunction observed in IPF. Methods: We study by wound healing assay the molecular targets associated with Alveolar Epithelial Cells (AEC) repair. Results: We demonstrate that the wound recovery of AEC is associated with TGF-ß1 signaling and increased transcriptional activity of ER stress and HIF-dependent genes. We further demonstrated that inhibition of TGF-ß1 signaling, CHOP expression or HIF-1 expression, limits AECs wound closure. Conclusion: the use of pharmacological drugs targeting the ER/HIF-1 axis could be an attractive approach to limit AEC dysregulation in pathological condition, and confirmed a critical role of theses factor in response to alveolar injury.

ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease of unknown origin. Alveolar epithelial cells (AECs) play an important role in the fibrotic process as they undergo sustained endoplasmic reticulum (ER) stress, and may acquire a mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT), two phenomena that could be induced by localized alveolar hypoxia. Here we investigated the potential links between hypoxia, ER stress and EMT in AECs. METHODS: ER stress and EMT markers were assessed by immunohistochemistry, western blot and qPCR analysis, both in vivo in rat lungs exposed to normoxia or hypoxia (equivalent to 8% O2) for 48 h, and in vitro in primary rat AECs exposed to normoxia or hypoxia (1.5% O2) for 2⁻6 days. RESULTS: Hypoxia induced expression of mesenchymal markers, pro-EMT transcription factors, and the activation of ER stress markers both in vivo in rat lungs, and in vitro in AECs. In vitro, pharmacological inhibition of ER stress by 4-PBA limited hypoxia-induced EMT. Calcium chelation or hypoxia-inducible factor (HIF) inhibition also prevented EMT induction under hypoxic condition. CONCLUSIONS: Hypoxia and intracellular calcium are both involved in EMT induction of AECs, mainly through the activation of ER stress and HIF signaling pathways.

Mesenchymal stem cells reduce hypoxia-induced apoptosis in alveolar epithelial cells by modulating HIF and ROS hypoxic signaling.

Distal lung diseases, such as pulmonary fibrosis or acute lung injury, are commonly associated with local alveolar hypoxia that may be deleterious through the stimulation of alveolar epithelial cell (AEC) apoptosis. In various murine models of alveolar injury, administration of allogenic human mesenchymal stem cells (hMSCs) exerts an overall protective paracrine effect, limiting lung inflammation and fibrosis. However, the precise mechanisms on lung cells themselves remain poorly understood. Here, we investigated whether hMSC-conditioned medium (hMSC-CM) would protect AECs from hypoxia-induced apoptosis and explored the mechanisms involved in this cytoprotective effect. Exposure of rat primary AECs to hypoxia (1.5% O2 for 24 h) resulted in hypoxia-inducible factor (HIF)-1α protein stabilization, partly dependent on reactive oxygen species (ROS) accumulation, and in a twofold increase in AEC apoptosis that was prevented by the HIF inhibitor 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole and the antioxidant drug N-acetyl cysteine. Incubation of AECs with hMSC-CM significantly reduced hypoxia-induced apoptosis. hMSC-CM decreased HIF-1α protein expression, as well as ROS accumulation through an increase in antioxidant enzyme activities. Expression of Bnip3 and CHOP, two proapoptotic targets of HIF-1α and ROS pathways, respectively, was suppressed by hMSC-CM, while Bcl-2 expression was restored. The paracrine protective effect of hMSC was partly dependent on keratinocyte growth factor and hepatocyte growth factor secretion, preventing ROS and HIF-1α accumulation.

Mesenchymal stem cells protect from hypoxia-induced alveolar epithelial-mesenchymal transition.

Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-ß1 mRNA expression and the secretion of active TGF-ß1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-ß1 expression and in TGF-ß1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-ß1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxia-induced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF.

Cycloheximide and lipopolysaccharide downregulate αENaC mRNA via different mechanisms in alveolar epithelial cells.

Active Na(+) transport mediated by epithelial Na(+) channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.

Induction of nitric oxide synthase expression by lipopolysaccharide is mediated by calcium-dependent PKCα-ß1 in alveolar epithelial cells.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na⁺ transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca²âº chelation and protein kinase C (PKCα-ß1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca²âº-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca²âº/PKCα-ß1 pathway independent of ATP signaling.

Chronic pulmonary fibrosis alters the functioning of the respiratory neural network.

Some patients with idiopathic pulmonary fibrosis present impaired ventilatory variables characterised by low forced vital capacity values associated with an increase in respiratory rate and a decrease in tidal volume which could be related to the increased pulmonary stiffness. The lung stiffness observed in pulmonary fibrosis may also have an effect on the functioning of the brainstem respiratory neural network, which could ultimately reinforce or accentuate ventilatory alterations. To this end, we sought to uncover the consequences of pulmonary fibrosis on ventilatory variables and how the modification of pulmonary rigidity could influence the functioning of the respiratory neuronal network. In a mouse model of pulmonary fibrosis obtained by 6 repeated intratracheal instillations of bleomycin (BLM), we first observed an increase in minute ventilation characterised by an increase in respiratory rate and tidal volume, a desaturation and a decrease in lung compliance. The changes in these ventilatory variables were correlated with the severity of the lung injury. The impact of lung fibrosis was also evaluated on the functioning of the medullary areas involved in the elaboration of the central respiratory drive. Thus, BLM-induced pulmonary fibrosis led to a change in the long-term activity of the medullary neuronal respiratory network, especially at the level of the nucleus of the solitary tract, the first central relay of the peripheral afferents, and the Pre-Bötzinger complex, the inspiratory rhythm generator. Our results showed that pulmonary fibrosis induced modifications not only of pulmonary architecture but also of central control of the respiratory neural network.

KCNK3 channel is important for the ventilatory response to hypoxia in rats.

To clarify the contribution of KCNK3/TASK-1 channel chemoreflex in response to hypoxia and hypercapnia, we used a unique Kcnk3-deficient rat. We assessed ventilatory variables using plethysmography in Kcnk3-deficient and wild-type rats at rest in response to hypoxia (10% O2) and hypercapnia (4% CO2). Immunostaining for C-Fos, a marker of neuronal activity, was performed to identify the regions of the respiratory neuronal network involved in the observed response.Under basal conditions, we observed increased minute ventilation in Kcnk3-deficient rats, which was associated with increased c-Fos positive cells in the ventrolateral region of the medulla oblongata. Kcnk3-deficient rats show an increase in ventilatory response to hypoxia without changes in response to hypercapnia. In Kcnk3-deficient rats, linked to an increased hypoxia response, we observed a greater increase in c-Fos-positive cells in the first central relay of peripheral chemoreceptors and Raphe Obscurus. This study reports that KCNK3/TASK-1 deficiency in rats induces an inadequate peripheral chemoreflex, alternating respiratory rhythmogenesis, and hypoxic chemoreflex.

PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones.

All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.

In Transgenic Erythropoietin Deficient Mice, an Increase in Respiratory Response to Hypercapnia Parallels Abnormal Distribution of CO2/H+-Activated Cells in the Medulla Oblongata.

Erythropoietin (Epo) and its receptor are expressed in central respiratory areas. We hypothesized that chronic Epo deficiency alters functioning of central respiratory areas and thus the respiratory adaptation to hypercapnia. The hypercapnic ventilatory response (HcVR) was evaluated by whole body plethysmography in wild type (WT) and Epo deficient (Epo-TAgh) adult male mice under 4%CO2. Epo-TAgh mice showed a larger HcVR than WT mice because of an increase in both respiratory frequency and tidal volume, whereas WT mice only increased their tidal volume. A functional histological approach revealed changes in CO2/H+-activated cells between Epo-TAgh and WT mice. First, Epo-TAgh mice showed a smaller increase under hypercapnia in c-FOS-positive number of cells in the retrotrapezoid nucleus/parafacial respiratory group than WT, and this, independently of changes in the number of PHOX2B-expressing cells. Second, we did not observe in Epo-TAgh mice the hypercapnic increase in c-FOS-positive number of cells in the nucleus of the solitary tract present in WT mice. Finally, whereas hypercapnia did not induce an increase in the c-FOS-positive number of cells in medullary raphe nuclei in WT mice, chronic Epo deficiency leads to raphe pallidus and magnus nuclei activation by hyperacpnia, with a significant part of c-FOS positive cells displaying an immunoreactivity for serotonin in the raphe pallidus nucleus. All of these results suggest that chronic Epo-deficiency affects both the pattern of ventilatory response to hypercapnia and associated medullary respiratory network at adult stage with an increase in the sensitivity of 5-HT and non-5-HT neurons of the raphe medullary nuclei leading to stimulation of f R for moderate level of CO2.

A New Model of Acute Exacerbation of Experimental Pulmonary Fibrosis in Mice.

RATIONALE: idiopathic pulmonary fibrosis (IPF) is the most severe form of fibrosing interstitial lung disease, characterized by progressive respiratory failure leading to death. IPF's natural history is heterogeneous, and its progression unpredictable. Most patients develop a progressive decline of respiratory function over years; some remain stable, but others present a fast-respiratory deterioration without identifiable cause, classified as acute exacerbation (AE). OBJECTIVES: to develop and characterize an experimental mice model of lung fibrosis AE, mimicking IPF-AE at the functional, histopathological, cellular and molecular levels. METHODS: we established in C57BL/6 male mice a chronic pulmonary fibrosis using a repetitive low-dose bleomycin (BLM) intratracheal (IT) instillation regimen (four instillations of BLM every 2 weeks), followed by two IT instillations of a simple or double-dose BLM challenge to induce AE. Clinical follow-up and histological and molecular analyses were done for fibrotic and inflammatory lung remodeling analysis. MEASUREMENTS AND MAIN RESULTS: as compared with a low-dose BLM regimen, this AE model induced a late burst of animal mortality, worsened lung fibrosis and remodeling, and superadded histopathological features as observed in humans IPF-AE. This was associated with stronger inflammation, increased macrophage infiltration of lung tissue and increased levels of pro-inflammatory cytokines in lung homogenates. Finally, it induced in the remodeled lung a diffuse expression of hypoxia-inducible factor 1α, a hallmark of tissular hypoxia response and a major player in the progression of IPF. CONCLUSION: this new model is a promising model of AE in chronic pulmonary fibrosis that could be relevant to mimic IPF-AE in preclinical trials.

Cytoprotective effects of erythropoietin: What about the lung?

Erythropoietin (Epo) is a pleiotropic cytokine, essential for erythropoiesis. Epo and its receptor (Epo-R) are produced by several tissues and it is now admitted that Epo displays other physiological functions than red blood cell synthesis. Indeed, Epo provides cytoprotective effects, which consist in prevention or fight against pathological processes. This perspective article reviews the various protective effects of Epo in several organs and tries to give a proof of concept about its effects in the lung. The tissue-protective effects of Epo could be a promising approach to limit the symptoms of acute and chronic lung diseases.

Sleep Apnea in Idiopathic Pulmonary Fibrosis: A Molecular Investigation in an Experimental Model of Fibrosis and Intermittent Hypoxia.

BACKGROUND: High prevalence of obstructive sleep apnea (OSA) is reported in incident and prevalent forms of idiopathic pulmonary fibrosis (IPF). We previously reported that Intermittent Hypoxia (IH), the major pathogenic element of OSA, worsens experimental lung fibrosis. Our objective was to investigate the molecular mechanisms involved. METHODS: Impact of IH was evaluated on C57BL/6J mice developing lung fibrosis after intratracheal instillation of Bleomycin (BLM). Mice were Pre-exposed 14 days to IH before induction of lung fibrosis or Co-challenged with IH and BLM for 14 days. Weight loss and survival were daily monitored. After experimentations, lungs were sampled for histology, and protein and RNA were extracted. RESULTS: Co-challenge or Pre-exposure of IH and BLM induced weight loss, increased tissue injury and collagen deposition, and pro-fibrotic markers. Major worsening effects of IH exposure on lung fibrosis were observed when mice were Pre-exposed to IH before developing lung fibrosis with a strong increase in sXBP1 and ATF6N ER stress markers. CONCLUSION: Our results showed that IH exacerbates BLM-induced lung fibrosis more markedly when IH precedes lung fibrosis induction, and that this is associated with an enhancement of ER stress markers.

Modulation of epithelial sodium channel activity by lipopolysaccharide in alveolar type II cells: involvement of purinergic signaling.

Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na(+) channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a approximately 60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y(2) purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

Cystic fibrosis transmembrane conductance regulator controls lung proteasomal degradation and nuclear factor-kappaB activity in conditions of oxidative stress.

Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-kappaB/IkappaB-alpha signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr(-/-)) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr(+/+)) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-kappaB inhibitor IkappaB-alpha. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-kappaB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl(-) channel by CFTR(inh-172) in the normal bronchial immortalized cell line 16HBE14o- increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-kappaB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl(-) channel activity is crucial for regulation of lung proteasomal degradation and NF-kappaB activity in conditions of oxidative stress.

[Na+ Transport in the lungs: differential impact of ENaC in the airways and alveoli]. / Transport du Na+ dans les poumons : impact différentiel du canal ENaC dans les voies aériennes et les alvéoles.

Na+ transport by airway epithelial cells, in conjunction with Cl- secretion is crucial for maintaining an adequate level of airway surface liquid (ASL) for an effective mucociliary clearance by the ciliated airway epithelial cells. It is also an important mechanism for lung liquid absorption at birth and oedema absorption during an acute respiratory distress syndrome (ARDS). The epithelial Na+ channel (ENaC) is the channel mostly involved in this process. The consequences of an imbalance in ENaC activity in the airways and in the distal lung are different. Experimental over expression of ENaC in the airways leads to a decrease in mucociliary clearance and inflammation similar to cystic fibrosis and chronic bronchitis. However, bacterial and viral pathogens, as well as pro-inflammatory cytokines present during lung infection downregulate ENaC expression and activity in airway and alveolar epithelial cells. ENaC downregulation by pathogens or inflammatory products could participate in the modulation of the severity of ARDS. Pharmacological strategies that modulate ENaC expression or activity could be important in the treatment of different lung diseases since it is actively involved in the lung innate defence mechanisms.

Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells: Potential mechanism for excessive IL-8 expression.

Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O(2)) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling.

Sodium 4-phenylbutyrate (4-PBA) has attracted a great deal of attention in cystic fibrosis (CF) pathology due to its capacity to traffic DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) to the cell membrane and restore CFTR chloride function at the plasma membrane of CF lung cells in vitro and in vivo. Using two different DeltaF508-CFTR lung epithelial cell lines (CFBE41o- and IB3-1 cells, characterized with DeltaF508-homozygous and heterozygous genotype, respectively) in vitro, 4-PBA induced an increase of proinflammatory cytokine interleukin (IL)-8 production in a concentration-dependent manner. This 4-PBA-induced IL-8 production was associated with a strong reduction of proteasome and nuclear factor-kappaB transcriptional activities in the two DeltaF508-CFTR lung cells either in a resting state or after tumor necrosis factor-alpha stimulation. In contrast, a strong increase of activator protein-1 transcriptional activity was observed. The inhibition of extracellular signal-regulated protein kinase 1/2 (ERK1/2) by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) and c-Jun-NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), respectively, was associated with a reduction (2-3.5-fold) of IL-8 production in both DeltaF508-CFTR lung cell lines treated with 4-PBA. No significant change of IL-8 production was observed after an inhibition of p38 MAPK with 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190). Therefore, we suggest that inhibition of both ERK1/2 and JNK signaling may be a means to strongly reduce 4-PBA-induced IL-8 production in combination with 4-PBA treatment to restore CFTR Cl(-) channel function in lung epithelial cells of patients with CF.

HIF-1α triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis.

Endoplasmic Reticulum (ER) stress of alveolar epithelial cells (AECs) is recognized as a key event of cell dysfunction in pulmonary fibrosis (PF). However, the mechanisms leading to AECs ER stress and ensuing unfolded protein response (UPR) pathways in idiopathic PF (IPF) remain unclear. We hypothesized that alveolar hypoxic microenvironment would generate ER stress and AECs apoptosis through the hypoxia-inducible factor-1α (HIF-1α). Combining ex vivo, in vivo and in vitro experiments, we investigated the effects of hypoxia on the UPR pathways and ER stress-mediated apoptosis, and consecutively the mechanisms linking hypoxia, HIF-1α, UPR and apoptosis. HIF-1α and the pro-apoptotic ER stress marker C/EBP homologous protein (CHOP) were co-expressed in hyperplastic AECs from bleomycin-treated mice and IPF lungs, not in controls. Hypoxic exposure of rat lungs or primary rat AECs induced HIF-1α, CHOP and apoptosis markers expression. In primary AECs, hypoxia activated UPR pathways. Pharmacological ER stress inhibitors and pharmacological inhibition or silencing of HIF-1α both prevented hypoxia-induced upregulation of CHOP and apoptosis. Interestingly, overexpression of HIF-1α in normoxic AECs increased UPR pathways transcription factors activities, and CHOP expression. These results indicate that hypoxia and HIF-1α can trigger ER stress and CHOP-mediated apoptosis in AECs, suggesting their potential contribution to the development of IPF.

Intermittent Hypoxia Increases the Severity of Bleomycin-Induced Lung Injury in Mice.

BACKGROUND: Severe obstructive sleep apnea (OSA) with chronic intermittent hypoxia (IH) is common in idiopathic pulmonary fibrosis (IPF). Here, we evaluated the impact of IH on bleomycin- (BLM-) induced pulmonary fibrosis in mice. METHODS: C57BL/6J mice received intratracheal BLM or saline and were exposed to IH (40 cycles/hour; FiO2 nadir: 6%; 8 hours/day) or intermittent air (IA). In the four experimental groups, we evaluated (i) survival; (ii) alveolar inflammation, pulmonary edema, lung oxidative stress, and antioxidant enzymes; (iii) lung cell apoptosis; and (iv) pulmonary fibrosis. RESULTS: Survival at day 21 was lower in the BLM-IH group (p < 0.05). Pulmonary fibrosis was more severe at day 21 in BLM-IH mice, as assessed by lung collagen content (p = 0.02) and histology. At day 4, BLM-IH mice developed a more severe neutrophilic alveolitis, (p < 0.001). Lung oxidative stress was observed, and superoxide dismutase and glutathione peroxidase expression was decreased in BLM-IH mice (p < 0.05 versus BLM-IA group). At day 8, pulmonary edema was observed and lung cell apoptosis was increased in the BLM-IH group. CONCLUSION: These results show that exposure to chronic IH increases mortality, lung inflammation, and lung fibrosis in BLM-treated mice. This study raises the question of the worsening impact of severe OSA in IPF patients.