Lucille N. Meliton

Forkhead Box m1 transcription factor is required for perinatal lung function

The Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is an important positive regulator of DNA replication and mitosis in a variety of cell types. Global deletion of Foxm1 in Foxm1−/− mice is lethal in the embryonic period, causing multiple abnormalities in the liver, heart, lung, and blood vessels. In the present study, Foxm1 was deleted conditionally in the respiratory epithelium (epFoxm1−/−). Surprisingly, deletion of Foxm1 did not alter lung growth, branching morphogenesis, or epithelial proliferation but inhibited lung maturation and caused respiratory failure after birth. Maturation defects in epFoxm1−/− lungs were associated with decreased expression of T1-α and aquaporin 5, consistent with a delay of type I cell differentiation. Expression of surfactant-associated proteins A, B, C, and D was decreased by deletion of Foxm1. Foxm1 directly bound and induced transcriptional activity of the mouse surfactant protein B and A (Sftpb and Sftpa) promoters in vitro, indicating that Foxm1 is a direct transcriptional activator of these genes. Foxm1 is critical for surfactant homeostasis and lung maturation before birth and is required for adaptation to air breathing.

Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box m1 transcription factor

The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 −/− mouse line to demonstrate that Foxm1‐deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 −/− hearts were associated with a reduced expression of Cdk1‐activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk‐inhibitor p21Cip1 protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL‐1 cardiomyocytes. Endothelial‐specific deletion of the Foxm1 fl/fl allele in Tie2‐Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the −9,259/−9,288‐bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development. Developmental Dynamics 236:1000–1013, 2007.

Deletion of Forkhead Box M1 Transcription Factor from Respiratory Epithelial Cells Inhibits Pulmonary Tumorigenesis

The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm1−/− mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Decreased lung tumorigenesis in epFoxm1−/− mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2α (TOPO-2α), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2α mRNA and protein levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2α promoter region, indicating that TOPO-2α is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO-2α expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy.

Secretory Group V Phospholipase A2 Regulates Acute Lung Injury and Neutrophilic Inflammation Caused by LPS in Mice

We investigated the regulatory role of 14-kDa secretory group V phospholipase A(2) (gVPLA(2)) in the development of acute lung injury (ALI) and neutrophilic inflammation (NI) caused by intratracheal administration of LPS. Experiments were conducted in gVPLA(2) knockout (pla2g5(-/-)) mice, which lack the gene, and gVPLA(2) wild-type littermate control (pla2g5(+/+)) mice. Indices of pulmonary injury were evaluated 24 h after intratracheal administration of LPS. Expression of gVPLA(2) in microsections of airways and mRNA content in lung homogenates were increased substantially in pla2g5(+/+) mice after LPS-administered compared with saline-treated pla2g5(+/+) mice. By contrast, expression of gVPLA(2) was neither localized in LPS- nor saline-treated pla2g5(-/-) mice. LPS also caused 1) reduced transthoracic static compliance, 2) lung edema, 3) neutrophilic infiltration, and 4) increased neutrophil myeloperoxidase activity in pla2g5(+/+) mice. These events were attenuated in pla2g5(-/-) mice exposed to LPS or in pla2g5(+/+) mice receiving MCL-3G1, a neutralizing MAb directed against gVPLA(2), before LPS administration. Our data demonstrate that gVPLA(2) is an inducible protein in pla2g5(+/+) mice but not in pla2g5(-/-) mice within 24 h after LPS treatment. Specific inhibition of gVPLA(2) with MCL-3G1 or gene-targeted mice lacking gVPLA(2) blocks ALI and attenuates NI caused by LPS.

Forkhead Box F1 Is Essential for Migration of Mesenchymal Cells and Directly Induces Integrin-Beta3 Expression

ABSTRACT The Forkhead box f1 (Foxf1) transcription factor is expressed in mesenchymal cells of the lung, liver, and gallbladder. Although Foxf1 deficiency causes severe abnormalities in the development of these organs, the molecular mechanisms underlying Foxf1 function remain uncharacterized. In this study we inactivated Foxf1 function in lung mesenchymal cells and mouse embryonic fibroblasts (MEFs) by use of either short interfering RNA duplexes or a membrane-transducing Foxf1 dominant negative (DN) mutant protein (Foxf1 DN), the latter of which is fused to the human immunodeficiency virus TAT protein transduction domain. Although Foxf1 did not influence DNA replication or cell survival, Foxf1 depletion severely diminished mesenchyme migration. Foxf1 deficiency in mesenchymal cells was associated with reduced expression of the integrin-beta3 (Itgβ3) subunit. Furthermore, we generated transgenic mice containing a tetracycline-inducible Foxf1 DN transgene. Adenovirus-mediated activation of Foxf1 DN in transgenic MEFs caused diminished cell migration and reduced Itgβ3 expression. A chromatin immunoprecipitation assay demonstrated that Foxf1 protein binds to the bp −871 to −815 region of the mouse Itgβ3 promoter. Deletion of the −871 to −815 Itgβ3 promoter region completely abolished the ability of Foxf1 to activate transcription of the Itgβ3 promoter in cotransfection experiments, indicating that the mouse Itgβ3 is a direct transcriptional target of Foxf1 protein. Foxf1 plays an essential role in mesenchyme migration by transcriptionally regulating Itgβ3.

Pulmonary Mastocytosis and Enhanced Lung Inflammation in Mice Heterozygous Null for the Foxf1 Gene

The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl(4) and butylated hydroxytoluene (BHT) injury models. Foxf1(+/-) mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation after injury. Pulmonary inflammation in Foxf1(+/-) mice was associated with diminished expression of Foxf1, increased mast cell tryptase, and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of CXCL12 were observed in Foxf1(+/-) mice. During lung development, Foxf1(+/-) embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of a doxycycline-inducible Foxf1 dominant-negative transgene in primary cultures of lung endothelial cells increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation after chemically induced or allergen-mediated lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.

Mechanical induction of group V phospholipase A2 causes lung inflammation and acute lung injury

Ventilation at high tidal volume may cause lung inflammation and barrier dysfunction that culminates in ventilator-induced lung injury (VILI). However, the mechanisms by which mechanical stimulation triggers the inflammatory response have not been fully elucidated. This study tested the hypothesis that onset of VILI is triggered by activation of secretory group V phospholipase A(2) (gVPLA2) in pulmonary vascular endothelium exposed to excessive mechanical stretch. High-magnitude cyclic stretch (18% CS) increased expression and surface exposure of gVPLA2 in human pulmonary endothelial cells (EC). CS-induced gVPLA2 activation was required for activation of ICAM-1 expression and polymorphonuclear neutrophil (PMN) adhesion to CS-preconditioned EC. By contrast, physiological CS (5% CS) had no effect on gVPLA2 activation or EC-PMN adhesion. CS-induced ICAM-1 expression and EC-PMN adhesion were attenuated by the gVPLA2-blocking antibody (MCL-3G1), general inhibitor of soluble PLA2, LY311727, or siRNA-induced EC gVPLA2 knockdown. In vivo, ventilator-induced lung leukocyte recruitment, cell and protein accumulation in the alveolar space, and total lung myeloperoxidase activity were strongly suppressed in gVPLA2 mouse knockout model or upon administration of MCL-3G1. These results demonstrate a novel role for gVPLA2 as the downstream effector of pathological mechanical stretch leading to an inflammatory response associated with VILI.

Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro

BackgroundCytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of β2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro.MethodsMigration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining.ResultsIL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane.ConclusionWe demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.

TAT-Protein Blockade during Ischemia/Reperfusion Reveals Critical Role for p85 PI3K-PTEN Interaction in Cardiomyocyte Injury

Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-Δp110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-ΔPTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.

Group V Phospholipase A2 Increases Pulmonary Endothelial Permeability Through Direct Hydrolysis of the Cell Membrane

Acute lung injury (ALI) is characterized by inflammatory disruption of the alveolar—vascular barrier, resulting in severe respiratory compromise. Inhibition of the intercellular messenger protein, Group V phospholipase A2 (gVPLA2), blocks vascular permeability caused by LPS both in vivo and in vitro. In this investigation we studied the mechanism by which recombinant gVPLA2 increases permeability of cultured human pulmonary endothelial cells (EC). Exogenous gVPLA2 (500 nM), a highly hydrolytic enzyme, caused a significant increase in EC permeability that began within minutes and persisted for >10 hours. However, the major hydrolysis products of gVPLA2 (Lyso-PC, Lyso-PG, LPA, arachidonic acid) did not cause EC structural rearrangement or loss of barrier function at concentrations >10 μM. Higher concentrations (≥ 30 μM) of these membrane hydrolysis products caused some increased permeability but were associated with EC toxicity (measured by propidium iodide incorporation) that did not occur with barrier disruption by gVPLA2 (500 nM). Pharmacologic inhibition of multiple intracellular signaling pathways induced by gVPLA2 activity (ERK, p38, PI3K, cytosolic gIVPLA2) also did not prevent EC barrier disruption by gVPLA2. Finally, pretreatment with heparinase to prevent internalization of gVPLA2 did not inhibit EC barrier disruption by gVPLA2. Our data thus indicate that gVPLA2 increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. Disruption of EC barrier function does not depend upon membrane hydrolysis products, gVPLA2 internalization, or upregulation of downstream intracellular signaling.