Sue Buckley

ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L714-L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-d-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1β-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-d-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 μM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.

TACE is required for fetal murine cardiac development and modeling.

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.

Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

Combined Effects of Corticosteroid, Thyroid Hormones, and β-Agonist on Surfactant, Pulmonary Mechanics, and β-Receptor Binding in Fetal Lamb Lung

ABSTRACT: We studied the interactions of corticosteroids, thyroid hormones, and β-agonist on surfactant phospholipids, pulmonary mechanics, and β-receptor binding in fetal lambs. We infused cortisol (450 μg/h for 48 h), thyrotropin-releasing hormone (TRH) (25 μg/h for 48 h), and ritodrine (1.3 μg/kg/min for 24 h) independently, and in double (cortisol plus β-agonist, cortisol plus TRH), and in triple (cortisol plus TRH plus β-agonist) combinations into chronically catheterized fetal lambs between 0.88 and 0.90 gestation. Infusion of the triple combination of cortisol plus TRH plus β-agonist resulted in a 20.9-fold increase in the saturated phosphatidylcholine content of fetal lung lavage, in a 5.8-fold increase in the saturated phosphatidylcholine content of whole fetal lung, and in a 13.3-fold increase in the saturated phosphatidylcholine content of fetal tracheal fluid. In addition, lung stability to inflation increased 3-fold, and lung stability to deflation increased 8-fold. The increases in the saturated phosphatidylcholine content of fetal lung lavage and tracheal fluid were greater than the effects of each hormone acting independently, or in the double combinations. The β-receptor maximal binding capacity was increased 30% by the combined infusion of cortisol and TRH. In addition, the maximal binding capacity after cortisol plus TRH plus β-agonist infusion was 54% greater than the maximal binding capacity after β-agonist infusion. We conclude that the triple combination of cortisol, TRH, and β-agonist increases fetal lamb lung surfactant phospholipids better than do any of the hormones acting either independently, or in double combinations, and also improves pulmonary mechanics better than single hormones or the double combination of TRH and cortisol (p < 0.01). We speculate that the interaction of cortisol and TRH enhances the capacity of the fetal lamb lung to respond to β-adrenergic stimulation, and that the triple combination of cortisol plus TRH plus β-agonist might prepare the fetal lung for air breathing more effectively than each of the hormones acting either independently, or in the double combinations.

Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia

Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of α-smooth muscle actin (α-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and α-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of α-SMA mRNA, fewer and more muscular arterioles (α-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of alpha-smooth muscle actin (alpha-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and alpha-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of alpha-SMA mRNA, fewer and more muscular arterioles (alpha-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.

Contractility, Transforming Growth Factor-|[beta]|, and Plasmin in Fetal Skin Fibroblasts: Role in Scarless Wound Healing

The early fetus responds to cutaneous wounds in a fundamentally different way from the adult; fetal wounds heal without scars. Wound contraction is a vital component of wound healing. The cytokine transforming growth factor(TGF)-β promotes wound contraction and can be activated by the serine protease plasmin. Herein, we explored whether murine skin fibroblast contractile properties, TGF-β, and plasmin formation are developmentally regulated. Our results showed that early fetal mouse embryonic day 15 skin fibroblasts contracted a collagen gel less, secreted less active and total TGF-β, and generated less plasmin than either late fetal (embryonic day 17) or adult skin fibroblasts. Furthermore, there was a slight positive correlation between the formation of plasmin and the level of activation of TGF-β. We conclude that early fetal mouse skin fibroblasts contract a collagen gel and secrete and activate TGF-β to a lesser extent than do late fetal and adult skin fibroblasts. We speculate that the fetal skin fibroblast undergoes a developmental transition that causes wounds in mouse to contract at or after embryonic day 17. Further, this developmental transition is influenced by growth factor-fibroblast interactions and coincides with the emergence of the skin fibroblasts ability to generate plasmin and activate TGF-β.

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1−/− lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1−/− lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1−/− lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.

Insulin-like growth factor II receptor, transforming growth factor-β, and Cdk4 expression and the developmental epigenetics of mouse palate morphogenesis and dysmorphogenesis

The B10/B10.A congenic mouse pair serves as a model for identifying specific genes related to morphogenesis and dysmorphogenesis of the embryonic palate and other organs. The present report describes our initial investigation of the Fraser‐Juriloff paradigm, which proposes that susceptibility to malformation results from genetically determined differences in normal developmental patterns. Specifically, we evaluated the relationship between Igf2r gene expression, transforming growth factor‐β (TGF‐β) activation, and cdk4 gene expression. By using in situ hybridization, RNase protection assays, indirect immunofluorescence, Western blots, and bioassays, we show 1) the presence of insulin‐like growth factor II (IGF‐II), IGF‐II receptor (IGF‐IIR), IGF‐IR, TGF‐β, plasminogen, plasminogen activators [urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA)], and Cdk4 in developing palates; 2) on embryonic day 14 (E14), which is a critical day for palatal growth, B10.A embryos have 82% greater IGF‐IIR mRNA than B10; 3) on E14, B10.A embryonic palates have a 57% greater level of active TGF‐β2 than B10, although the total TGF‐β2 is nearly identical; and 4) on E14, B10 embryonic palates have a 52% greater level of Cdk4 mRNA than B10.A palates, a measure of cell cycle progression. Because cellular activation of latent TGF‐β appears to require binding to the mannose‐6‐phosphate (M6P) binding site of the IGF‐IIR and is plasmin and plasminogen activator dependent, the positive correlation of IGF‐IIR levels and active TGF‐β2 levels seems to be key. Thus, the strain variation of TGF‐β2/IGF‐IIR‐mediated growth inhibition in late G1 phase would appear to account for the slower growth and development of B10.A palates relative to B10. Elevated corticosteroid (CORT) exposure in E14 B10.A embryos significantly increases TGF‐β levels, 87% of which is TGF‐β2, as well as the levels of active TGF‐β, 64% of which is TGF‐β2. Without exogenous CORT, B10.A embryos do not have clefts; hence, we present an outline of pathogenesis: slower growing B10.A embryos have an up‐regulation of IGF‐IIR, which serves to sequester IGF‐II from the growth‐promoting IGF‐IR and to bind more CORT‐up‐regulated, latent TGF‐β2 for subsequent plasmin‐dependent activation; higher levels of TGF‐β2 signaling down‐regulate Cdk4 and result in greater palatal growth inhibition at a critical stage of palatogenesis and, thus, cleft palate. We present an epigenetic model of information processing related to cell proliferation. The model is a dynamical network that uses continuous logic to learn its rules from changing conditions. Dev. Dyn. 1998;211:11–25.

Overexpression of transforming growth factor-β1 in fetal monkey lung results in prenatal pulmonary fibrosis

Altered transforming growth factor (TGF)-&bgr; expression levels have been linked to a variety of human respiratory diseases, including bronchopulmonary dysplasia and pulmonary fibrosis. However, a causative role for aberrant TGF-&bgr; in neonatal lung diseases has not been defined in primates. Exogenous and transient TGF-&bgr;1 overexpression in fetal monkey lung was achieved by transabdominal ultrasound-guided fetal intrapulmonary injection of adenoviral vector expressing TGF-&bgr;1 at the second or third trimester of pregnancy. The lungs were then harvested near term, and fixed for histology and immunohistochemistry. Lung hypoplasia was observed where TGF-&bgr;1 was overexpressed during the second trimester. The most clearly marked phenotype consisted of severe pulmonary and pleural fibrosis, which was independent of the gestational time point when TGF-&bgr;1 was overexpressed. Increased cell proliferation, particularly in &agr;-smooth muscle actin-positive myofibroblasts, was detected within the fibrotic foci. But epithelium to mesenchyme transdifferentiation was not detected. Massive collagen fibres were deposited on the inner and outer sides of the pleural membrane, with an intact elastin layer in the middle. This induced fibrotic pathology persisted even after adenoviral-mediated TGF-&bgr;1 overexpression was no longer evident. Therefore, overexpression of TGF-&bgr;1 within developing fetal monkey lung results in severe and progressive fibrosis in lung parenchyma and pleural membrane, in addition to pulmonary hypoplasia.