Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Regulates LPS-Induced Inflammation and Alveolar Remodeling in the Developing Lung.

In premature infants, sepsis is associated with alveolar simplification manifesting as bronchopulmonary dysplasia. The redox-dependent mechanisms underlying sepsis-induced inflammation and alveolar remodeling in the immature lung remain unclear. We developed a neonatal mouse model of sepsis-induced lung injury to investigate whether nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) regulates Toll-like receptor (TLR)-mediated inflammation and alveolar remodeling. Six-day-old NOX2+/+ and NOX2-/- mice were injected with intraperitoneal LPS to induce sepsis. Lung inflammation and canonical TLR signaling were assessed 24 hours after LPS. Alveolar development was examined in 15-day-old mice after LPS on Day 6. The in vivo efficacy of a NOX2 inhibitor (NOX2-I) on NOX2 complex assembly and sepsis-induced lung inflammation were examined. Lung cytokine expression and neutrophil influx induced with sepsis in NOX2+/+ mice was decreased by >50% in NOX2-/- mice. LPS-induced TLR4 signaling evident by inhibitor of NF-κB kinase-ß and mitogen-activated protein kinase phosphorylation, and nuclear factor-κB/AP-1 translocation were attenuated in NOX2-/- mice. LPS increased matrix metalloproteinase 9 while decreasing elastin and keratinocyte growth factor levels in NOX2+/+ mice. An LPS-induced increase in matrix metalloproteinase 9 and decrease in fibroblast growth factor 7 and elastin were not evident in NOX2-/- mice. An LPS-induced reduction in radial alveolar counts and increased mean linear intercepts were attenuated in NOX2-/- mice. LPS-induced NOX2 assembly evident by p67phox/gp91phox coimmunoprecipitation was disrupted with NOX2-I. NOX2-I also mitigated LPS-induced cytokine expression, TLR pathway signaling, and alveolar simplification. In a mouse model of neonatal sepsis, NOX2 regulates proinflammatory TLR signaling and alveolar remodeling induced by a single dose of LPS. Our results provide mechanistic insight into the regulation of sepsis-induced alveolar remodeling in the developing lung.

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation.

BACKGROUND: Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFß receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts--a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known. METHODS: TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis. RESULTS: The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFß ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis. CONCLUSIONS: TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.

TRIP-1 regulates TGF-ß1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-ß receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-ß type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-ß1 action. TGF-ß1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-ß1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-ß1 induction of EMT. Mechanistically, a pathway involving increased TGF-ß type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death.

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

A PI-3 kinase-dependent, Stat1-independent signaling pathway regulates interferon-stimulated monocyte adhesion.

Type I interferon (IFN)-alpha/beta and type II IFN-gamma induce the expression of early response genes through activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Stat) pathway. Although IFNs regulate a variety of other signaling cascades, little is known about how they contribute to the biological activities of these cytokines. In this study, we demonstrate that IFN-beta or IFN-gamma induces the phosphorylation of the serine/threonine kinase Akt in primary human peripheral blood monocytes. Abrogation of the IFN-stimulated Akt activation by phosphatidylinositol-3 kinase (PI-3K) inhibitors prevents IFN-induced adhesion in these cells, and IFN activation of the Stat1-dependent guanylate-binding protein (GBP) gene is not affected. Importantly, Stat1-deficient bone marrow macrophages displayed a similar level of IFN-gamma-stimulated adhesion compared with macrophages derived from wild-type littermates. These findings demonstrate for the first time that IFN stimulation of a PI-3K signaling cascade modulates the ability of these cytokines to regulate monocyte adhesion, and this process does not require the expression of Stat1, a primary mediator of IFN-gamma signaling.