Developmental Regulation of p66Shc Is Altered by Bronchopulmonary Dysplasia in Baboons and Humans
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美国呼吸和危急护理医学 2005年第6期
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California
the Neonatology Division, Departments of Pediatrics and Pathology, University of Southern California/Los Angeles County Medical Center, Los Angeles, California
Department of Pediatrics, Golisano Children's Hospital at Strong, University of Rochester Medical Center, Rochester, New York
Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Rationale: The p66Shc adapter protein antagonizes mitogen-activated protein, or MAP, kinase, mediates oxidative stress, and is developmentally regulated in fetal mouse lungs. Objectives: To determine if p66Shc is similarly regulated in primates and in bronchopulmonary dysplasia (BPD), which results from oxidative injury to immature lungs. Methods: Normal and injured lungs from humans and baboons were evaluated by Western analysis and immunohistochemistry. Measurements and Main Results: In baboons, p66Shc decreased 80% between 125 and 175 days' gestation (p = 0.025), then doubled after term delivery at 185 days (p = 0.0013). In the hyperoxic 140-day fetal baboon BPD model, p66Shc expression persisted, and its localization shifted from the epithelium of gestational controls to the mesenchyme of diseased lungs, coincident with expression of proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase, a marker of apoptosis. Treatment with the antibombesin antibody 2A11 attenuated BPD, reduced cell proliferation, increased p66Shc expression 10.5-fold, and preserved epithelial p66Shc localization. p66Shc also decreased during normal human lung development, falling 87% between 18 and 24 weeks' gestation (p = 0.02). p66Shc was expressed throughout 18-week human lungs, became restricted to scattered epithelial cells by 24 weeks, and localized to isolated mesenchymal cells after term delivery. In contrast, p66Shc remained prominent in the epithelium of lungs with acute injury or mild BPD, and in the mesenchyme of lungs with severe disease. p66Shc localized to tissues expressing proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase. Conclusions: p66Shc expression, cell proliferation, and apoptosis are concomitantly altered during lung development and in BPD.
Key Words: fetal development lung MAP kinases ShcA protein
Lung development is regulated by interactions between epithelial and mesenchymal cells. These interactions are typically mediated by growth factors that bind cognate receptors to initiate intracellular phosphorylation cascades. The developmental expression of these factors controls important aspects of lung morphogenesis. However, signaling components within targeted cells may also be developmentally regulated. Growth factor receptors typically phosphorylate adaptor proteins that combine with other proteins to activate downstream effectors. Important examples include the Shc protein family, which comprises three isoforms with different functions. On tyrosine phosphorylation, the ubiquitous 52-kD isoform (p52Shc) complexes with Grb2 and Sos and translocates to the plasma membrane where it encounters and activates Ras, thus initiating the mitogen-activated protein (MAP) kinase cascade, culminating in cell proliferation (1). A 46-kD alternative translation product appears to function similarly (2). Although Shc is a canonic substrate of tyrosine kinase receptors, such as that of epidermal growth (3) factor and insulin (4), it is also associated with signaling by G-proteineCcoupled receptors (5) and integrins (6).
A third isoform of 66 kD (p66Shc) results from an alternative splice reaction that appends 109 amino acids to p52Shc. Unlike the other isoforms, p66Shc antagonizes Ras activation (2) and mediates oxidative stresseCinduced apoptosis (7). We have reported that, although p52Shc is highly expressed at all stages of fetal mouse lung development, p66Shc is highly expressed only through the 18th day of gestation, after which its expression decreases precipitously (8). This expression pattern may be of particular interest in the preterm human lung, which is uniquely exposed to wide fluctuations in oxidative stress loads and highly susceptible to bronchopulmonary dysplasia (BPD), a chronic disease of the immature lung. Moreover, p66Shc primarily localizes to mesenchymal cells adjoining the basement membranes of large and intermediate airways within the fetal mouse lung. Mesenchymal cell proliferation is characteristic of severe BPD. We therefore hypothesized that p66Shc is developmentally regulated in normal fetal human and baboon lungs, and that this regulation is distorted by the pathogenesis of BPD.
Two animal models of BPD were evaluated. The original characterization of BPD (9) emphasized areas of atelectasis interspersed with regions of compensatory emphysema, interstitial fibrosis, epithelial metaplasia, and chronic reactive airways disease with peribronchial smooth muscle hypertrophy. This severely fibrotic BPD was reproduced using baboons delivered at 140 days' gestation and maintained in 100% oxygen (10), and is attenuated by intravenous infusion of antibodies against bombesin-like peptides (BLPs) (11). With advances in neonatal therapy, the clinical presentation of BPD has evolved and is currently characterized by reduced interstitial fibrosis, chronic reactive airways disease, and attenuated alveolar septation within more premature lungs (12). This mildly fibrotic form of BPD was modeled using baboons delivered at 125 days' gestation and benefiting from lower oxygen concentrations, intrauterine corticosteroids, and exogenous surfactant. Our results in both animal models and in postmortem human specimens suggest that p66Shc expression in the primate lung is developmentally regulated in isoform-specific fashion, and that the normal ontogeny of Shc isoform expression is disrupted by the onset of BPD. Some of the results of these studies have been previously reported in the form of an abstract (13).
METHODS
Normal Baboon Tissues
Fetal baboon tissue was provided by the Southwest Foundation for Biomedical Research under protocols approved by the Animal Care Committee of that institution. The National Research Council Guide for the Care and Use of Laboratory Animals was strictly adhered to. Normal lungs were obtained at 125, 140, 160, and 175 days' gestation. At least four fetuses were analyzed for each time point. Four specimens were obtained 1, 2, and 3 days after term delivery at 185 days and pooled as the postpartum cohort.
Baboon BPD Models
Mildly fibrotic BPD was induced in baboons delivered at 125 days' gestation and treated with intrauterine betamethasone, exogenous surfactant, endotracheal intubation, minimal respiratory support required to maintain arterial carbon dioxide between 45 and 55 mm Hg, and minimal oxygen necessary to maintain arterial oxygen between 55 and 70 mm Hg (14). Severely fibrotic BPD was induced in animals delivered at 140 days and exposed to 10 days of 100% oxygen (15). BPD in this model is attenuated by adsorbing circulating BLPs. Animals received intravenous infusions of either anti-BLP antibody (2A11) or nonspecific control antibody (MOPC-21; Sigma, St. Louis, MO) (11). Histologic specimens were fixed in 4% paraformaldehyde. For Western analysis, left-lower-lobe lung sections were snap-frozen in liquid nitrogen. At least four animals were analyzed for each experimental condition.
Human Tissues
Human tissues were obtained under protocols approved by the Institutional Review Boards of Advanced Bioscience Resources, Strong Memorial Hospital, and the University of Southern California/Los Angeles County Medical Center. Snap-frozen lung tissues from 12 normal fetuses were provided by Advanced Bioscience Resources (Alameda, CA). Pathologic specimens represent 12 infants who died in the intensive care nursery of Strong Memorial Hospital (Rochester, NY). Clinical synopses are provided in Table 1. Patients were clinically diagnosed with BPD based on delivery before 32 weeks' gestation, intubation for neonatal respiratory distress syndrome, and mechanical ventilation with supplemental oxygen for at least 13 days. Pathologic diagnoses of BPD were based on epithelial metaplasia, mesenchymal thickening, and alveolar dysplasia. Specimens were also obtained from infants succumbing to nonpulmonary disorders and whose autopsies indicated normal lung histology. Samples were processed within 6 hours of death.
Western Analysis
Western analyses were performed as described (8) using an anti-Shc antibody (Transduction Laboratories, Lexington, KY). Samples were equalized by total protein and equivalent loading confirmed by glyceraldehyde phosphate dehydrogenase (Chemicon, Temecula, CA) reprobe and/or colloidal gold staining (Bio-Rad, Hercules, CA). Quantitation was performed with an Image Station 1000 (Eastman Kodak, New Haven CT). Statistical analyses were performed using Excel (Microsoft, Redmond, WA) and SPSS (SPSS, Inc., Chicago, IL).
Immunohistochemistry
After rehydration, specimens were microwaved for 8 minutes at 300 W in 1 M sodium citrate, pH 6.4. Sections were probed with anti-p66Shc, antieCproliferating cell nuclear antigen (PCNA; Zymed, South San Francisco, CA), anticleaved poly(adenyl ribose) polymerase (PARP; Cell Signaling Technologies, Beverly, MA), antieCpan-cytokeratin (Sigma), and/or antieC-smooth muscle actin (Sigma) antibodies followed by Alexa 488eCconjugated antirabbit and Alexa 594eCconjugated antimouse IgG antibodies (Molecular Probes, Eugene, OR) (8). Autofluorescence was quenched in 10 mM CuSO4, 50 mM CH3COONH4. To confirm specificity, sections were probed with anti-p66Shc antibody preadsorbed to immunizing peptide at 1:20 wt/wt.
RESULTS
Normal Baboon Lung Development
To determine whether Shc isoforms are independently regulated in the fetal primate lung, specimens of normal baboon fetal lung were assessed by Shc Western analysis (Figure 1A and Figure E1 on the online supplement). Baboon lung homogenates contain proteins recognized by antibodies specific for the human and murine Shc SH2 domains, and whose migration corresponded to human and murine p66Shc, p52Shc, and p46Shc isoforms. Two additional bands migrating between p52Shc and p66Shc were also detected, and may represent alternative Shc gene products unique to baboons. The p52Shc and p46Shc isoforms were more highly expressed than the p66Shc isoform. The expression of p66Shc decreased significantly between 125 and 160 days' gestation (p = 0.011 by one-way analysis of variance (ANOVA; Figure 1B) (16). Relative to Day 125, average p66Shc protein content decreased 74% by Day 140, 71% by Day 160, and 80% by Day 175. Four specimens obtained 1 to 3 days after term delivery at 185 days were pooled for analysis (newborn, NB). Between 175 days' gestation and the immediate postpartum period, p66Shc expression increased by a factor of 2.02 (p = 0.034 by t test, assuming equal variances) (17), suggesting upregulation of p66Shc expression with term delivery. Similarly, average p46Shc expression decreased 67% from 125 to 175 days' gestation (p = 0.00008 by ANOVA). Expression of p52Shc decreased 31% between 125 and 160 days' gestation, but this change did not achieve statistical significance.
The localization of p66Shc was evaluated in early fetal baboon lung using an isoform-specific antibody directed against the p66Shc CH2 domain (8). Because p66Shc putatively antagonizes cell proliferation, sections were also assessed for expression of PCNA, an indicator of cell replication (18). In the 60-day lung, p66Shc was highly expressed in both epithelial and mesenchymal cells (Figure 2A). PCNA expression was widespread among epithelial cells and relatively sparse in the mesenchyme. By 90 days, overall p66Shc expression had decreased considerably (Figure 2B). Although most cells expressed some p66Shc, high-level expression was restricted to the mesenchyme directly adjacent to the incipient airways and moderate expression was present in the apical cytoplasm of epithelial cells. PCNA expression persisted in the epithelium and was moderately increased in the mesenchyme. The expression of p66Shc continued to decrease through 125 days (Figure 2C), at which time p66Shc expression was largely mesenchymal with little epithelial localization. Subsequently, mesenchymal expression diminished, whereas epithelial expression became more prominent through 140 and 160 days' gestation. By 175 days (Figure 2D), overall p66Shc expression was restricted to isolated epithelial cells. PCNA expression concomitantly shifted from groups of epithelial cells to isolated epithelial and mesenchymal cells. Three days after term delivery at 185 days (Figure 2E), little p66Shc was evident and PCNA expression remained scattered through the lung.
Because p66Shc has also been described as mediating oxidative stresseCinduced cell death, its expression was also compared with that of cleaved PARP, a nuclear protein involved in DNA repair whose proteolysis is an early indicator of apoptosis (19). Although PARP cleavage is associated with p66Shc signaling (20), minimal PARP proteolysis was identified at any stage of baboon lung development despite robust early p66Shc expression (Figure E2).
These results suggest that Shc isoforms are independently regulated during baboon lung development, and that expression of p66Shc and p46Shc decrease with maturation. Localization of p66Shc also shifts during lung development. In the baboon lung, the saccular stage begins at approximately 125 days and proceeds to 160 days. Pulmonary p66Shc and p46Shc expression are therefore downregulated in the baboon lung at a developmental stage similar to that of the mouse lung. In the mesenchyme, p66Shc expression appeared to correlate inversely with PCNA expression, consistent with its putative function as a mitogenic inhibitor. However, epithelial PCNA expression persisted despite high p66Shc levels.
Mildly Fibrotic 125-Day Baboon BPD Model
Having demonstrated that Shc isoform expression is modulated during normal lung development, we hypothesized that this regulation may be altered by premature birth and the subsequent onset of neonatal chronic lung disease. To evaluate this postulate, specimens were obtained from baboons delivered prematurely at 125 days' gestation and maintained for up to 21 days under a protocol developed to replicate the mildly fibrotic BPD associated with extremely premature delivery and current clinical practice (14). In this model, oxygen was administered only as clinically indicated to maintain transcutaneous oxygen saturations at approximately 90%, and barotrauma was minimized. Shc expression was assessed by Western analysis (Figures 3A and 3B). Unlike the decrease in p66Shc and p46Shc content observed during normal lung development, p66Shc and p46Shc expression increased in the 21 days after premature delivery. The expression of p66Shc and p46Shc increased by factors of 3.6 (p = 0.025 by one-way ANOVA) and 3.3 (p = 0.0000024 by one-way ANOVA), respectively. In contrast, the expression of p52Shc over the same period was remarkably consistent, varying by less than 7%. Isoform-specific immunohistochemistry also demonstrated a change in p66Shc localization. In normal 125-day fetal baboons, p66Shc was distributed throughout the lung, with higher expression in mesenchymal cells (Figure 3C), whereas PCNA was primarily expressed in the epithelium. In contrast, animals maintained for 14 days ex utero expressed p66Shc most strongly in the epithelium (Figure 3D). PCNA was evident throughout the lungs of these animals. Cleaved PARP expression was also evaluated but not detected (Figure E3). Because epithelial and mesenchymal cells may become interspersed with BPD, p66Shc localization was confirmed by colocalization with cytokeratin and with eCsmooth muscle actin (Figure E3). Interestingly, p66Shc tended not to colocalize with eCsmooth muscle actin, even when strongly expressed in adjacent mesenchymal cells. Mesenchymal p66Shc labeling was most intense in the endothelium of developing vessels. Modest parenchymal thickening was evident, but fibrotic changes were minimal. Intracellular distribution was cytoplasmic, consistent with previous descriptions of Shc intracellular localization (21).
Severely Fibrotic 140-Day Baboon BPD Model
Shc expression was also evaluated in a model of severely fibrotic BPD in which baboons are delivered prematurely at 140 days' gestation and maintained on 100% oxygen for 10 days (140 days/100% oxygen). Animals subjected to this protocol develop severe BPD similar to that originally described by Northway and colleagues (9). In this model, the clinical and pathologic severity of lung injury is attenuated by intravenous infusions of 2A11, an anti-BLP antibody (11). To determine whether Shc isoform expression is altered by 2A11 treatment in parallel with BPD abrogation, Shc content was assessed by Western analysis (Figure 4A). Samples were equalized by total protein content, and equivalent loading subsequently confirmed by glyceraldehyde phosphate dehydrogenase Western analysis (Figure 4B). Relative to 140-day gestational control animals, p66Shc expression was 4.8-fold higher in 10-day-old 140-day/100% oxygen severely fibrotic BPD baboons treated with MOPC-21 nonimmune control antibody (Figure 4C). This difference approached statistical significance (p = 0.059 by t test after log transformation). In contrast, otherwise identically treated animals receiving 2A11 had dramatically increased absolute and relative p66Shc expression. Although animals receiving 2A11 respectively yielded 6.2- and 7.1-fold higher p52Shc and p46Shc expression relative to 140-day/100% oxygen MOPC-21 baboons (p < 0.002 by t test for both), p66Shc expression increased 10.5-fold (p = 0.0026 by t test). Regulation of p66Shc differed significantly from that of the other isoforms (p = 0.0073 by two-way ANOVA) (16). These findings suggest that 2A11 attenuates clinical and pathologic signs of BPD while disproportionately increasing p66Shc expression.
Tissue localization of p66Shc, PCNA, and cleaved PARP were also evaluated in this model. In the normal 140-day fetal baboon lung (Figure 5A), p66Shc and PCNA were expressed in both epithelial and mesenchymal cells. Cleaved PARP expression was sparse and localized to isolated epithelial cells, coincident with high p66Shc expression (Figure 6A). Animals exposed to supplemental oxygen for 10 days only as clinically indicated developed minimal fibrosis and yielded patterns of p66Shc, PCNA, and cleaved PARP localization similar to those of 140-day gestational control animals (Figures 5B and 6B). In contrast, 140-day animals exposed to 100% oxygen and infused with either no antibody (not shown) or with MOPC-21 expressed p66Shc, PCNA (Figure 5C), and cleaved PARP (Figure 6C) in clusters of mesenchymal cells within fibrotic interalveolar septa. Cleaved PARP tended to localize to mesenchymal cells with low p66Shc content. Cells expressing p66Shc were not derived from vascular endothelium, as inferred by a lack of coexpressed platelet endothelial cell adhesion molecule-1 (data not shown). This shift in p66Shc localization did not occur in otherwise identical animals treated with 100% oxygen and 2A11 anti-BLP antibody. In these animals, p66Shc, PCNA (Figure 5D), and cleaved PARP (Figure 6D) continued to be expressed in the epithelium. Moreover, PCNA expression and PARP proteolysis were both diminished by 2A11 treatment. In the normal 160-day baboon lung, p66Shc was also localized to epithelial cells (Figure 5E); cells with strong PCNA expression were rare, and PARP proteolysis was undetectable. Tissue localization of p66Shc was further confirmed by coimmunolocalization with cytokeratin and with eCsmooth muscle actin (Figures E4 and E5, respectively). Again, p66Shc tended not to colocalize with eCsmooth muscle actin, even when strongly expressed in the mesenchyme. These findings suggest that p66Shc, PCNA, and cleaved PARP localization shift from the epithelium to the mesenchyme with induction of classical BPD, and that this shift is prevented by adsorption of circulating BLPs.
Normal Human Lung Development
To determine whether isoform-specific Shc regulation is also relevant to human lung development, snap-frozen samples were obtained from the lungs of 12 normal human fetuses whose gestational ages ranged from 18 to 24 weeks. Isoform-specific Shc regulation was assessed by Western analysis (Figure 7A). Samples were equalized by total protein content and equivalent loading confirmed by colloidal gold staining and glyceraldehyde phosphate dehydrogenase reprobe (Figure E6). The expression of p52Shc and p46Shc was higher than that of p66Shc, necessitating longer chemiluminescence exposure times to evaluate p66Shc expression. The highest levels of p66Shc expression were observed before 21 weeks' gestation, after which p66Shc content fell dramatically (Figure 7B; p = 0.02 by one-way ANOVA). Average p66Shc expression was maximal and nearly identical at 18 and 20 weeks. Subsequently, expression decreased 12% by 21 weeks, 42% by 22 weeks, and 87% by 24 weeks. In contrast, p52Shc and p46Shc expression were relatively consistent across all gestational ages, with all values within 27% of the mean.
Normal human fetal lung specimens were also evaluated by p66Shc immunostaining, which demonstrated widespread expression of p66Shc throughout the 18-week lung (Figure 8A). p66Shc was particularly prominent within epithelial cells lining the primitive airspaces. PCNA and p66Shc were widely coexpressed in the epithelium, similar to the normal 125-day baboon lung (Figure 2C). At 20 weeks (Figure 8B), the contrast between the epithelium and the mesenchyme became progressively more distinct, reflecting diminished mesenchymal expression. Moreover, within highly expressing cells, p66Shc labeling was less homogeneous and assumed a more apical distribution. PCNA was again expressed in most epithelial cells and in isolated mesenchymal cells.
Subsequently, the proportion of epithelial cells expressing high levels of p66Shc immunostaining decreased with maturation. Although most cells in the 22-week lung exhibited low concentrations of p66Shc, high-level expression was restricted to occasional cells in both the epithelial and mesenchymal compartments (Figure 8C). Epithelial PCNA expression diminished in parallel with p66Shc expression but continued to be expressed in isolated mesenchymal cells. By 24 weeks, much of the mesenchyme had involuted (Figure 8D). Relatively few epithelial cells continued to express high levels of p66Shc, and some mesenchymal cell populations expressed no detectable p66Shc, as is evident from the dark regions scattered throughout the parenchyma. PCNA became further restricted to isolated epithelial and mesenchymal cells.
The progressive restriction of p66Shc expression to epithelial cells during human lung development recapitulates p66Shc immunolocalization in 125- and 140-day baboon lungs, and the quantitative decrease in p66Shc expression demonstrated by Western analysis correlates with decreasing epithelial p66Shc localization. PCNA expression in developing human pulmonary epithelium decreased in parallel with p66Shc expression, also recapitulating the pattern observed in the developing baboon lung. However, the maturational increase in mesenchymal PCNA expression observed in the baboon lung was not apparent in humans.
Tissue localization of p66Shc was confirmed by coimmunolocalization of cytokeratin (Figure E7) and eCsmooth muscle actin (Figure E8). Again, mesenchymal cells expressing p66Shc did not simultaneously express eCsmooth muscle actin.
p66Shc was also coimmunolocalized with cleaved PARP in these samples. PARP proteolysis was sparse at all gestational ages (Figure E9). When present, cleaved PARP tended to be expressed in mesenchymal cells with low p66Shc content.
Human Postmortem BPD Lungs
To evaluate p66Shc expression in the prematurely delivered human lung, autopsy specimens were evaluated by p66Shc, PCNA, and cleaved PARP immunolocalization. Autopsies were performed within 6 hours of death, and clinical synopses are listed in Table 1. Postmortem lung specimens were selected from four cases whose final pathologic diagnoses included bronchopulmonary dysplasia, including one (Case 4) with a clinical history and postmortem findings characteristic of severe fibrotic BPD. For comparison, specimens were also obtained from term and premature infants succumbing to sepsis or other nonpulmonary causes, and whose histologic diagnoses did not include BPD or chronic lung disease. Most infants diagnosed with BPD were considerably older than the non-BPD cases at the time of death. Specimens from all three categories demonstrated p66Shc expression in most cells, with varying populations of highly expressing cells. The distribution and the proportions of highly expressing cells were consistent within regions of similar tissue morphology.
In near-term newborn infants without lung disease (Figures 9A and 10A; Case 2, a 4-day-old exeC36-week infant), interalveolar septa were thin, with little mesenchyme. Although nearly all cells expressed some p66Shc, a population of intensely labeled cells were scattered throughout the mesenchyme. Within these cells, p66Shc localized to a nuclear or perinuclear distribution. This localization contrasts with that of the prenatal lung, in which epithelial expression is more prominent. PCNA expression was limited to scattered cells (predominantly epithelial), and PARP cleavage was undetected.
Newborn preterm infants had thicker septa with more mesenchyme (Figures 9B and 10B; Case 9, a 1-day-old exeC27-week infant) than term newborns. Moderate levels of p66Shc were present in most mesenchymal cells, and high-level expression was apparent in scattered epithelial cells. PCNA expression was limited to isolated epithelial cells, and PARP proteolysis was not observed. Older premature infants without clinical or pathologic BPD (Figures 9C and 10C; Case 10, a 32-day-old exeC28-week infant) had thinner interalveolar septa, but continued to exhibit moderate p66Shc labeling of most mesenchymal cells and increased expression within scattered epithelial cells. Cells expressing PCNA were sparse and mostly epithelial; again, PARP cleavage was undetectable.
In contrast, the lungs of a 13-day-old exeC28-week premature infant with early evolving BPD (Figures 9D and 10D, Case 7) had thickened septa with heterogeneously labeled mesenchymal cells. Most prominent, however, were continuous lines of strongly expressing epithelial cells. This expression pattern was reminiscent of the 18- to 22-week human fetal lung. Overall, the prevalence of highly expressing cells was greater than in lungs without BPD. However, PCNA expression and PARP cleavage remained low.
In an older patient with mildly fibrotic BPD (Figures 9E and 10E; Case 5, a 60-day-old exeC26-week infant), p66Shc localized to continuous lines of strongly expressing epithelial cells. As in early BPD, the mesenchymal compartment was heterogeneously labeled, with patches of strongly expressing cells adjoining areas of minimal expression. Highly labeled macrophages were also evident. PCNA expression and PARP proteolysis were both increased, and most prominent among epithelial cells. In contrast, the patient with histologic findings and a clinical course consistent with classical severe fibrotic BPD (Figures 9F and 10F; Case 4, a 97-day-old exeC27-week infant) expressed p66Shc primarily in the mesenchyme, with relatively little epithelial or endothelial expression. Although areas of highly expressing cells were scattered throughout the mesenchyme, most mesenchymal cells expressed moderate levels of p66Shc. PCNA expression was very low, and PARP cleavage was undetectable.
p66Shc tissue localization was again confirmed by coimmunolocalization with cytokeratin (Figure E10) and eCsmooth muscle actin (Figure E11). As in the baboon BPD model, mesenchymal cells expressing p66Shc did not simultaneously express eCsmooth muscle actin.
In both fibrotic and nonfibrotic BPD, intracellular p66Shc localization was cytoplasmic, and not perinuclear as in normal lungs or lungs with acute disease. The distribution of p66Shc in the patient with severe BPD was similar to that of baboons delivered at 140 days and treated with 100% oxygen for 10 days. Expression of p66Shc in the patients with mildly fibrotic BPD resembled that of baboons delivered at 125 days' gestation and maintained with oxygen only as needed. These findings indicate that, in both humans and baboons, p66Shc expression and localization change with premature birth and the pathogenesis of chronic lung disease.
DISCUSSION
Our findings support the hypothesis that pulmonary p66Shc expression is developmentally regulated in baboons and humans as well as in mice. For all three species, p66Shc expression is highest in the early fetal lung and decreases with maturation. In the mouse lung, p66Shc expression decreases just before birth at 18.5 days' gestation, during the early saccular stage of development (22). By comparison, p66Shc decreased through the saccular stage of baboon lung development (14) and during the late canalicular stage of human lung development (23). At the end of gestation, mice have saccular stage lungs, whereas term baboon and human infants have begun alveolarization. Thus, differences in the timing of p66Shc downregulation reflect differences in the ontogeny of lung development within each species. Conservation of the three Shc isoforms and their developmental regulation across taxonomic orders suggests that p66Shc regulates lung development in many mammalian species, and that attenuation of p66Shc before birth is adaptive.
After premature delivery, p66Shc expression in the premature lung correlated inversely to the degree of fibrosis. Relatively little p66Shc was expressed in 140-dayeCgestation baboons, and expression was mildly increased with induction of severely fibrotic BPD. Conversely, experimental attenuation of severe BPD using intravenous anti-BLP antibodies dramatically increased relative and absolute p66Shc expression. Moreover, in the 125-day mildly fibrotic baboon BPD model, p66Shc is high at birth and remains elevated for at least 21 days, rather than declining as it would in utero. Thus, in premature lungs, elevated p66Shc expression was associated with a mildly fibrotic BPD phenotype. We note that p66Shc consistently failed to colocalize with eCsmooth muscle actin, leading us to speculate that p66Shc expression is inversely correlated to the differentiation of myofibroblast populations implicated in the pathogenesis of BPD (24).
In both humans and baboons, mild or minimally fibrotic lung disease was associated with epithelial p66Shc localization. This change in p66Shc localization parallels the shift in expression seen during normal lung development In contrast, baboons and the human with severely fibrotic BPD expressed p66Shc primarily in the mesenchyme. Moreover, experimental attenuation of BPD in the 140-day/100% oxygen baboon BPD model using 2A11 anti-BLP antibody was associated with an epithelial p66Shc distribution as well as greatly increased p66Shc expression. Thus, loss of epithelial p66Shc expression appears to correlate with mesenchymal expansion and the development of severely fibrotic BPD in both premature baboons and humans. The persistence of mesenchymal p66Shc expression in the mildly fibrotic BPD model may reflect the degree to which normal lung development is disrupted.
The function of p66Shc in the developing lung is likely to be related to its previously defined signaling functions. On tyrosine phosphorylation, p66Shc binds and sequesters Grb2 (25), thereby antagonizing Ras activation by receptor tyrosine kinases. We speculate that expression of p66Shc may represent inhibitory feedback on mitogenic signaling through MAP kinases. Thus, p66Shc localization may indicate the proliferation of specific cell populations. A previous evaluation of lung cell proliferation in the mildly fibrotic 125-day baboon BPD model (26) supports this postulate. First, cell proliferation in the baboon decreases with maturation, falling 97% between Days 125 and 175. This drop parallels the decrease in p66Shc expression over the same period. Second, cell proliferation in the mildly fibrotic 125-day baboon BPD model remains elevated through at least 21 days after birth, again closely paralleling the expression of p66Shc in the same model. Finally, in the baboon BPD model, proliferating cells localize primarily to the epithelium through the first 14 days of life, coincident with p66Shc immunolocalization. In the current study, p66Shc consistently localized to the same tissues as PCNA, though often in different individual cells. We therefore speculate that p66Shc expression represents the inhibitory limb of a feedback loop regulating cell proliferation in the BPD lung. Its expression in the mesenchyme of fibrotic lungs may therefore relate to ongoing proliferation within this compartment. Conversely, epithelial p66Shc expression after 2A11 infusion appears to be independent of any initial increase in proliferation. In this population, Shc isoform expression may represent a mechanism by which bombesin signaling regulates epithelial cell mitogenesis.
p66Shc also mediates oxidative stress signaling on serine phosphorylation, resulting in activation of the mitochondrial apoptosis pathway, culminating in caspase activation and cytochrome-c release (27, 28). In the current study, both cleaved PARP and p66Shc localized to the mesenchyme of 140-day baboons with BPD, and attenuation of BPD in the same model shifted both p66Shc and cleaved PARP expression to the epithelium. These findings are therefore broadly consistent with the putative role of p66Shc in mediating oxidative stress responses. However, the large increase in p66Shc expression associated with 2A11 treatment infers that its function in BPD is adaptive. In contrast, p66Shc has been characterized as playing a maladaptive role in oxidative lung disease. Mice deficient in p66Shc are resistant to oxidative injury from hydrogen peroxide and paraquat, and live 30% longer (7). Susceptibility to oxidative stress is restored by induced p66Shc expression. It is unclear how these findings may be reconciled; it is possible that p66Shc confers different functions in neonates and adults.
The expression and localization of p66Shc within the neonatal lung may therefore identify a population of cells that is both rapidly proliferating and at increased risk of oxidative stresseCinduced cell death. The shift of p66Shc expression from the epithelium to the mesenchyme in severely fibrotic BPD may directly regulate both cell populations. We note that many of the signaling pathways converging on Shc have been implicated in the regulation of lung cell proliferation and survival, including those of epidermal growth factor (29), insulin (4), and fibroblast growth factors (30). The elucidation of the mechanisms governing Shc isoform expression may provide additional insight into the determinants of pulmonary fibrosis after premature birth. Moreover, the signaling pathways converging on Shc may be manipulated to determine how mitogenic and oxidative stress signaling interact to regulate fibrogenic precursors.
Acknowledgments
Key R-10 support personnel include Vicki Winters and the pathology staff at University of Texas Health Sciences Center at San Antonio, and the Neonatal Intensive Care Unit and animal production staffs at the Southwest Foundation for Biomedical Research. Histology technical support was provided by Pablo Bringas Jr. and Valentino Santos of the University of Southern California Center for Craniofacial Molecular Biology.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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the Neonatology Division, Departments of Pediatrics and Pathology, University of Southern California/Los Angeles County Medical Center, Los Angeles, California
Department of Pediatrics, Golisano Children's Hospital at Strong, University of Rochester Medical Center, Rochester, New York
Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Rationale: The p66Shc adapter protein antagonizes mitogen-activated protein, or MAP, kinase, mediates oxidative stress, and is developmentally regulated in fetal mouse lungs. Objectives: To determine if p66Shc is similarly regulated in primates and in bronchopulmonary dysplasia (BPD), which results from oxidative injury to immature lungs. Methods: Normal and injured lungs from humans and baboons were evaluated by Western analysis and immunohistochemistry. Measurements and Main Results: In baboons, p66Shc decreased 80% between 125 and 175 days' gestation (p = 0.025), then doubled after term delivery at 185 days (p = 0.0013). In the hyperoxic 140-day fetal baboon BPD model, p66Shc expression persisted, and its localization shifted from the epithelium of gestational controls to the mesenchyme of diseased lungs, coincident with expression of proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase, a marker of apoptosis. Treatment with the antibombesin antibody 2A11 attenuated BPD, reduced cell proliferation, increased p66Shc expression 10.5-fold, and preserved epithelial p66Shc localization. p66Shc also decreased during normal human lung development, falling 87% between 18 and 24 weeks' gestation (p = 0.02). p66Shc was expressed throughout 18-week human lungs, became restricted to scattered epithelial cells by 24 weeks, and localized to isolated mesenchymal cells after term delivery. In contrast, p66Shc remained prominent in the epithelium of lungs with acute injury or mild BPD, and in the mesenchyme of lungs with severe disease. p66Shc localized to tissues expressing proliferating cell nuclear antigen and cleaved poly(adenyl ribose) polymerase. Conclusions: p66Shc expression, cell proliferation, and apoptosis are concomitantly altered during lung development and in BPD.
Key Words: fetal development lung MAP kinases ShcA protein
Lung development is regulated by interactions between epithelial and mesenchymal cells. These interactions are typically mediated by growth factors that bind cognate receptors to initiate intracellular phosphorylation cascades. The developmental expression of these factors controls important aspects of lung morphogenesis. However, signaling components within targeted cells may also be developmentally regulated. Growth factor receptors typically phosphorylate adaptor proteins that combine with other proteins to activate downstream effectors. Important examples include the Shc protein family, which comprises three isoforms with different functions. On tyrosine phosphorylation, the ubiquitous 52-kD isoform (p52Shc) complexes with Grb2 and Sos and translocates to the plasma membrane where it encounters and activates Ras, thus initiating the mitogen-activated protein (MAP) kinase cascade, culminating in cell proliferation (1). A 46-kD alternative translation product appears to function similarly (2). Although Shc is a canonic substrate of tyrosine kinase receptors, such as that of epidermal growth (3) factor and insulin (4), it is also associated with signaling by G-proteineCcoupled receptors (5) and integrins (6).
A third isoform of 66 kD (p66Shc) results from an alternative splice reaction that appends 109 amino acids to p52Shc. Unlike the other isoforms, p66Shc antagonizes Ras activation (2) and mediates oxidative stresseCinduced apoptosis (7). We have reported that, although p52Shc is highly expressed at all stages of fetal mouse lung development, p66Shc is highly expressed only through the 18th day of gestation, after which its expression decreases precipitously (8). This expression pattern may be of particular interest in the preterm human lung, which is uniquely exposed to wide fluctuations in oxidative stress loads and highly susceptible to bronchopulmonary dysplasia (BPD), a chronic disease of the immature lung. Moreover, p66Shc primarily localizes to mesenchymal cells adjoining the basement membranes of large and intermediate airways within the fetal mouse lung. Mesenchymal cell proliferation is characteristic of severe BPD. We therefore hypothesized that p66Shc is developmentally regulated in normal fetal human and baboon lungs, and that this regulation is distorted by the pathogenesis of BPD.
Two animal models of BPD were evaluated. The original characterization of BPD (9) emphasized areas of atelectasis interspersed with regions of compensatory emphysema, interstitial fibrosis, epithelial metaplasia, and chronic reactive airways disease with peribronchial smooth muscle hypertrophy. This severely fibrotic BPD was reproduced using baboons delivered at 140 days' gestation and maintained in 100% oxygen (10), and is attenuated by intravenous infusion of antibodies against bombesin-like peptides (BLPs) (11). With advances in neonatal therapy, the clinical presentation of BPD has evolved and is currently characterized by reduced interstitial fibrosis, chronic reactive airways disease, and attenuated alveolar septation within more premature lungs (12). This mildly fibrotic form of BPD was modeled using baboons delivered at 125 days' gestation and benefiting from lower oxygen concentrations, intrauterine corticosteroids, and exogenous surfactant. Our results in both animal models and in postmortem human specimens suggest that p66Shc expression in the primate lung is developmentally regulated in isoform-specific fashion, and that the normal ontogeny of Shc isoform expression is disrupted by the onset of BPD. Some of the results of these studies have been previously reported in the form of an abstract (13).
METHODS
Normal Baboon Tissues
Fetal baboon tissue was provided by the Southwest Foundation for Biomedical Research under protocols approved by the Animal Care Committee of that institution. The National Research Council Guide for the Care and Use of Laboratory Animals was strictly adhered to. Normal lungs were obtained at 125, 140, 160, and 175 days' gestation. At least four fetuses were analyzed for each time point. Four specimens were obtained 1, 2, and 3 days after term delivery at 185 days and pooled as the postpartum cohort.
Baboon BPD Models
Mildly fibrotic BPD was induced in baboons delivered at 125 days' gestation and treated with intrauterine betamethasone, exogenous surfactant, endotracheal intubation, minimal respiratory support required to maintain arterial carbon dioxide between 45 and 55 mm Hg, and minimal oxygen necessary to maintain arterial oxygen between 55 and 70 mm Hg (14). Severely fibrotic BPD was induced in animals delivered at 140 days and exposed to 10 days of 100% oxygen (15). BPD in this model is attenuated by adsorbing circulating BLPs. Animals received intravenous infusions of either anti-BLP antibody (2A11) or nonspecific control antibody (MOPC-21; Sigma, St. Louis, MO) (11). Histologic specimens were fixed in 4% paraformaldehyde. For Western analysis, left-lower-lobe lung sections were snap-frozen in liquid nitrogen. At least four animals were analyzed for each experimental condition.
Human Tissues
Human tissues were obtained under protocols approved by the Institutional Review Boards of Advanced Bioscience Resources, Strong Memorial Hospital, and the University of Southern California/Los Angeles County Medical Center. Snap-frozen lung tissues from 12 normal fetuses were provided by Advanced Bioscience Resources (Alameda, CA). Pathologic specimens represent 12 infants who died in the intensive care nursery of Strong Memorial Hospital (Rochester, NY). Clinical synopses are provided in Table 1. Patients were clinically diagnosed with BPD based on delivery before 32 weeks' gestation, intubation for neonatal respiratory distress syndrome, and mechanical ventilation with supplemental oxygen for at least 13 days. Pathologic diagnoses of BPD were based on epithelial metaplasia, mesenchymal thickening, and alveolar dysplasia. Specimens were also obtained from infants succumbing to nonpulmonary disorders and whose autopsies indicated normal lung histology. Samples were processed within 6 hours of death.
Western Analysis
Western analyses were performed as described (8) using an anti-Shc antibody (Transduction Laboratories, Lexington, KY). Samples were equalized by total protein and equivalent loading confirmed by glyceraldehyde phosphate dehydrogenase (Chemicon, Temecula, CA) reprobe and/or colloidal gold staining (Bio-Rad, Hercules, CA). Quantitation was performed with an Image Station 1000 (Eastman Kodak, New Haven CT). Statistical analyses were performed using Excel (Microsoft, Redmond, WA) and SPSS (SPSS, Inc., Chicago, IL).
Immunohistochemistry
After rehydration, specimens were microwaved for 8 minutes at 300 W in 1 M sodium citrate, pH 6.4. Sections were probed with anti-p66Shc, antieCproliferating cell nuclear antigen (PCNA; Zymed, South San Francisco, CA), anticleaved poly(adenyl ribose) polymerase (PARP; Cell Signaling Technologies, Beverly, MA), antieCpan-cytokeratin (Sigma), and/or antieC-smooth muscle actin (Sigma) antibodies followed by Alexa 488eCconjugated antirabbit and Alexa 594eCconjugated antimouse IgG antibodies (Molecular Probes, Eugene, OR) (8). Autofluorescence was quenched in 10 mM CuSO4, 50 mM CH3COONH4. To confirm specificity, sections were probed with anti-p66Shc antibody preadsorbed to immunizing peptide at 1:20 wt/wt.
RESULTS
Normal Baboon Lung Development
To determine whether Shc isoforms are independently regulated in the fetal primate lung, specimens of normal baboon fetal lung were assessed by Shc Western analysis (Figure 1A and Figure E1 on the online supplement). Baboon lung homogenates contain proteins recognized by antibodies specific for the human and murine Shc SH2 domains, and whose migration corresponded to human and murine p66Shc, p52Shc, and p46Shc isoforms. Two additional bands migrating between p52Shc and p66Shc were also detected, and may represent alternative Shc gene products unique to baboons. The p52Shc and p46Shc isoforms were more highly expressed than the p66Shc isoform. The expression of p66Shc decreased significantly between 125 and 160 days' gestation (p = 0.011 by one-way analysis of variance (ANOVA; Figure 1B) (16). Relative to Day 125, average p66Shc protein content decreased 74% by Day 140, 71% by Day 160, and 80% by Day 175. Four specimens obtained 1 to 3 days after term delivery at 185 days were pooled for analysis (newborn, NB). Between 175 days' gestation and the immediate postpartum period, p66Shc expression increased by a factor of 2.02 (p = 0.034 by t test, assuming equal variances) (17), suggesting upregulation of p66Shc expression with term delivery. Similarly, average p46Shc expression decreased 67% from 125 to 175 days' gestation (p = 0.00008 by ANOVA). Expression of p52Shc decreased 31% between 125 and 160 days' gestation, but this change did not achieve statistical significance.
The localization of p66Shc was evaluated in early fetal baboon lung using an isoform-specific antibody directed against the p66Shc CH2 domain (8). Because p66Shc putatively antagonizes cell proliferation, sections were also assessed for expression of PCNA, an indicator of cell replication (18). In the 60-day lung, p66Shc was highly expressed in both epithelial and mesenchymal cells (Figure 2A). PCNA expression was widespread among epithelial cells and relatively sparse in the mesenchyme. By 90 days, overall p66Shc expression had decreased considerably (Figure 2B). Although most cells expressed some p66Shc, high-level expression was restricted to the mesenchyme directly adjacent to the incipient airways and moderate expression was present in the apical cytoplasm of epithelial cells. PCNA expression persisted in the epithelium and was moderately increased in the mesenchyme. The expression of p66Shc continued to decrease through 125 days (Figure 2C), at which time p66Shc expression was largely mesenchymal with little epithelial localization. Subsequently, mesenchymal expression diminished, whereas epithelial expression became more prominent through 140 and 160 days' gestation. By 175 days (Figure 2D), overall p66Shc expression was restricted to isolated epithelial cells. PCNA expression concomitantly shifted from groups of epithelial cells to isolated epithelial and mesenchymal cells. Three days after term delivery at 185 days (Figure 2E), little p66Shc was evident and PCNA expression remained scattered through the lung.
Because p66Shc has also been described as mediating oxidative stresseCinduced cell death, its expression was also compared with that of cleaved PARP, a nuclear protein involved in DNA repair whose proteolysis is an early indicator of apoptosis (19). Although PARP cleavage is associated with p66Shc signaling (20), minimal PARP proteolysis was identified at any stage of baboon lung development despite robust early p66Shc expression (Figure E2).
These results suggest that Shc isoforms are independently regulated during baboon lung development, and that expression of p66Shc and p46Shc decrease with maturation. Localization of p66Shc also shifts during lung development. In the baboon lung, the saccular stage begins at approximately 125 days and proceeds to 160 days. Pulmonary p66Shc and p46Shc expression are therefore downregulated in the baboon lung at a developmental stage similar to that of the mouse lung. In the mesenchyme, p66Shc expression appeared to correlate inversely with PCNA expression, consistent with its putative function as a mitogenic inhibitor. However, epithelial PCNA expression persisted despite high p66Shc levels.
Mildly Fibrotic 125-Day Baboon BPD Model
Having demonstrated that Shc isoform expression is modulated during normal lung development, we hypothesized that this regulation may be altered by premature birth and the subsequent onset of neonatal chronic lung disease. To evaluate this postulate, specimens were obtained from baboons delivered prematurely at 125 days' gestation and maintained for up to 21 days under a protocol developed to replicate the mildly fibrotic BPD associated with extremely premature delivery and current clinical practice (14). In this model, oxygen was administered only as clinically indicated to maintain transcutaneous oxygen saturations at approximately 90%, and barotrauma was minimized. Shc expression was assessed by Western analysis (Figures 3A and 3B). Unlike the decrease in p66Shc and p46Shc content observed during normal lung development, p66Shc and p46Shc expression increased in the 21 days after premature delivery. The expression of p66Shc and p46Shc increased by factors of 3.6 (p = 0.025 by one-way ANOVA) and 3.3 (p = 0.0000024 by one-way ANOVA), respectively. In contrast, the expression of p52Shc over the same period was remarkably consistent, varying by less than 7%. Isoform-specific immunohistochemistry also demonstrated a change in p66Shc localization. In normal 125-day fetal baboons, p66Shc was distributed throughout the lung, with higher expression in mesenchymal cells (Figure 3C), whereas PCNA was primarily expressed in the epithelium. In contrast, animals maintained for 14 days ex utero expressed p66Shc most strongly in the epithelium (Figure 3D). PCNA was evident throughout the lungs of these animals. Cleaved PARP expression was also evaluated but not detected (Figure E3). Because epithelial and mesenchymal cells may become interspersed with BPD, p66Shc localization was confirmed by colocalization with cytokeratin and with eCsmooth muscle actin (Figure E3). Interestingly, p66Shc tended not to colocalize with eCsmooth muscle actin, even when strongly expressed in adjacent mesenchymal cells. Mesenchymal p66Shc labeling was most intense in the endothelium of developing vessels. Modest parenchymal thickening was evident, but fibrotic changes were minimal. Intracellular distribution was cytoplasmic, consistent with previous descriptions of Shc intracellular localization (21).
Severely Fibrotic 140-Day Baboon BPD Model
Shc expression was also evaluated in a model of severely fibrotic BPD in which baboons are delivered prematurely at 140 days' gestation and maintained on 100% oxygen for 10 days (140 days/100% oxygen). Animals subjected to this protocol develop severe BPD similar to that originally described by Northway and colleagues (9). In this model, the clinical and pathologic severity of lung injury is attenuated by intravenous infusions of 2A11, an anti-BLP antibody (11). To determine whether Shc isoform expression is altered by 2A11 treatment in parallel with BPD abrogation, Shc content was assessed by Western analysis (Figure 4A). Samples were equalized by total protein content, and equivalent loading subsequently confirmed by glyceraldehyde phosphate dehydrogenase Western analysis (Figure 4B). Relative to 140-day gestational control animals, p66Shc expression was 4.8-fold higher in 10-day-old 140-day/100% oxygen severely fibrotic BPD baboons treated with MOPC-21 nonimmune control antibody (Figure 4C). This difference approached statistical significance (p = 0.059 by t test after log transformation). In contrast, otherwise identically treated animals receiving 2A11 had dramatically increased absolute and relative p66Shc expression. Although animals receiving 2A11 respectively yielded 6.2- and 7.1-fold higher p52Shc and p46Shc expression relative to 140-day/100% oxygen MOPC-21 baboons (p < 0.002 by t test for both), p66Shc expression increased 10.5-fold (p = 0.0026 by t test). Regulation of p66Shc differed significantly from that of the other isoforms (p = 0.0073 by two-way ANOVA) (16). These findings suggest that 2A11 attenuates clinical and pathologic signs of BPD while disproportionately increasing p66Shc expression.
Tissue localization of p66Shc, PCNA, and cleaved PARP were also evaluated in this model. In the normal 140-day fetal baboon lung (Figure 5A), p66Shc and PCNA were expressed in both epithelial and mesenchymal cells. Cleaved PARP expression was sparse and localized to isolated epithelial cells, coincident with high p66Shc expression (Figure 6A). Animals exposed to supplemental oxygen for 10 days only as clinically indicated developed minimal fibrosis and yielded patterns of p66Shc, PCNA, and cleaved PARP localization similar to those of 140-day gestational control animals (Figures 5B and 6B). In contrast, 140-day animals exposed to 100% oxygen and infused with either no antibody (not shown) or with MOPC-21 expressed p66Shc, PCNA (Figure 5C), and cleaved PARP (Figure 6C) in clusters of mesenchymal cells within fibrotic interalveolar septa. Cleaved PARP tended to localize to mesenchymal cells with low p66Shc content. Cells expressing p66Shc were not derived from vascular endothelium, as inferred by a lack of coexpressed platelet endothelial cell adhesion molecule-1 (data not shown). This shift in p66Shc localization did not occur in otherwise identical animals treated with 100% oxygen and 2A11 anti-BLP antibody. In these animals, p66Shc, PCNA (Figure 5D), and cleaved PARP (Figure 6D) continued to be expressed in the epithelium. Moreover, PCNA expression and PARP proteolysis were both diminished by 2A11 treatment. In the normal 160-day baboon lung, p66Shc was also localized to epithelial cells (Figure 5E); cells with strong PCNA expression were rare, and PARP proteolysis was undetectable. Tissue localization of p66Shc was further confirmed by coimmunolocalization with cytokeratin and with eCsmooth muscle actin (Figures E4 and E5, respectively). Again, p66Shc tended not to colocalize with eCsmooth muscle actin, even when strongly expressed in the mesenchyme. These findings suggest that p66Shc, PCNA, and cleaved PARP localization shift from the epithelium to the mesenchyme with induction of classical BPD, and that this shift is prevented by adsorption of circulating BLPs.
Normal Human Lung Development
To determine whether isoform-specific Shc regulation is also relevant to human lung development, snap-frozen samples were obtained from the lungs of 12 normal human fetuses whose gestational ages ranged from 18 to 24 weeks. Isoform-specific Shc regulation was assessed by Western analysis (Figure 7A). Samples were equalized by total protein content and equivalent loading confirmed by colloidal gold staining and glyceraldehyde phosphate dehydrogenase reprobe (Figure E6). The expression of p52Shc and p46Shc was higher than that of p66Shc, necessitating longer chemiluminescence exposure times to evaluate p66Shc expression. The highest levels of p66Shc expression were observed before 21 weeks' gestation, after which p66Shc content fell dramatically (Figure 7B; p = 0.02 by one-way ANOVA). Average p66Shc expression was maximal and nearly identical at 18 and 20 weeks. Subsequently, expression decreased 12% by 21 weeks, 42% by 22 weeks, and 87% by 24 weeks. In contrast, p52Shc and p46Shc expression were relatively consistent across all gestational ages, with all values within 27% of the mean.
Normal human fetal lung specimens were also evaluated by p66Shc immunostaining, which demonstrated widespread expression of p66Shc throughout the 18-week lung (Figure 8A). p66Shc was particularly prominent within epithelial cells lining the primitive airspaces. PCNA and p66Shc were widely coexpressed in the epithelium, similar to the normal 125-day baboon lung (Figure 2C). At 20 weeks (Figure 8B), the contrast between the epithelium and the mesenchyme became progressively more distinct, reflecting diminished mesenchymal expression. Moreover, within highly expressing cells, p66Shc labeling was less homogeneous and assumed a more apical distribution. PCNA was again expressed in most epithelial cells and in isolated mesenchymal cells.
Subsequently, the proportion of epithelial cells expressing high levels of p66Shc immunostaining decreased with maturation. Although most cells in the 22-week lung exhibited low concentrations of p66Shc, high-level expression was restricted to occasional cells in both the epithelial and mesenchymal compartments (Figure 8C). Epithelial PCNA expression diminished in parallel with p66Shc expression but continued to be expressed in isolated mesenchymal cells. By 24 weeks, much of the mesenchyme had involuted (Figure 8D). Relatively few epithelial cells continued to express high levels of p66Shc, and some mesenchymal cell populations expressed no detectable p66Shc, as is evident from the dark regions scattered throughout the parenchyma. PCNA became further restricted to isolated epithelial and mesenchymal cells.
The progressive restriction of p66Shc expression to epithelial cells during human lung development recapitulates p66Shc immunolocalization in 125- and 140-day baboon lungs, and the quantitative decrease in p66Shc expression demonstrated by Western analysis correlates with decreasing epithelial p66Shc localization. PCNA expression in developing human pulmonary epithelium decreased in parallel with p66Shc expression, also recapitulating the pattern observed in the developing baboon lung. However, the maturational increase in mesenchymal PCNA expression observed in the baboon lung was not apparent in humans.
Tissue localization of p66Shc was confirmed by coimmunolocalization of cytokeratin (Figure E7) and eCsmooth muscle actin (Figure E8). Again, mesenchymal cells expressing p66Shc did not simultaneously express eCsmooth muscle actin.
p66Shc was also coimmunolocalized with cleaved PARP in these samples. PARP proteolysis was sparse at all gestational ages (Figure E9). When present, cleaved PARP tended to be expressed in mesenchymal cells with low p66Shc content.
Human Postmortem BPD Lungs
To evaluate p66Shc expression in the prematurely delivered human lung, autopsy specimens were evaluated by p66Shc, PCNA, and cleaved PARP immunolocalization. Autopsies were performed within 6 hours of death, and clinical synopses are listed in Table 1. Postmortem lung specimens were selected from four cases whose final pathologic diagnoses included bronchopulmonary dysplasia, including one (Case 4) with a clinical history and postmortem findings characteristic of severe fibrotic BPD. For comparison, specimens were also obtained from term and premature infants succumbing to sepsis or other nonpulmonary causes, and whose histologic diagnoses did not include BPD or chronic lung disease. Most infants diagnosed with BPD were considerably older than the non-BPD cases at the time of death. Specimens from all three categories demonstrated p66Shc expression in most cells, with varying populations of highly expressing cells. The distribution and the proportions of highly expressing cells were consistent within regions of similar tissue morphology.
In near-term newborn infants without lung disease (Figures 9A and 10A; Case 2, a 4-day-old exeC36-week infant), interalveolar septa were thin, with little mesenchyme. Although nearly all cells expressed some p66Shc, a population of intensely labeled cells were scattered throughout the mesenchyme. Within these cells, p66Shc localized to a nuclear or perinuclear distribution. This localization contrasts with that of the prenatal lung, in which epithelial expression is more prominent. PCNA expression was limited to scattered cells (predominantly epithelial), and PARP cleavage was undetected.
Newborn preterm infants had thicker septa with more mesenchyme (Figures 9B and 10B; Case 9, a 1-day-old exeC27-week infant) than term newborns. Moderate levels of p66Shc were present in most mesenchymal cells, and high-level expression was apparent in scattered epithelial cells. PCNA expression was limited to isolated epithelial cells, and PARP proteolysis was not observed. Older premature infants without clinical or pathologic BPD (Figures 9C and 10C; Case 10, a 32-day-old exeC28-week infant) had thinner interalveolar septa, but continued to exhibit moderate p66Shc labeling of most mesenchymal cells and increased expression within scattered epithelial cells. Cells expressing PCNA were sparse and mostly epithelial; again, PARP cleavage was undetectable.
In contrast, the lungs of a 13-day-old exeC28-week premature infant with early evolving BPD (Figures 9D and 10D, Case 7) had thickened septa with heterogeneously labeled mesenchymal cells. Most prominent, however, were continuous lines of strongly expressing epithelial cells. This expression pattern was reminiscent of the 18- to 22-week human fetal lung. Overall, the prevalence of highly expressing cells was greater than in lungs without BPD. However, PCNA expression and PARP cleavage remained low.
In an older patient with mildly fibrotic BPD (Figures 9E and 10E; Case 5, a 60-day-old exeC26-week infant), p66Shc localized to continuous lines of strongly expressing epithelial cells. As in early BPD, the mesenchymal compartment was heterogeneously labeled, with patches of strongly expressing cells adjoining areas of minimal expression. Highly labeled macrophages were also evident. PCNA expression and PARP proteolysis were both increased, and most prominent among epithelial cells. In contrast, the patient with histologic findings and a clinical course consistent with classical severe fibrotic BPD (Figures 9F and 10F; Case 4, a 97-day-old exeC27-week infant) expressed p66Shc primarily in the mesenchyme, with relatively little epithelial or endothelial expression. Although areas of highly expressing cells were scattered throughout the mesenchyme, most mesenchymal cells expressed moderate levels of p66Shc. PCNA expression was very low, and PARP cleavage was undetectable.
p66Shc tissue localization was again confirmed by coimmunolocalization with cytokeratin (Figure E10) and eCsmooth muscle actin (Figure E11). As in the baboon BPD model, mesenchymal cells expressing p66Shc did not simultaneously express eCsmooth muscle actin.
In both fibrotic and nonfibrotic BPD, intracellular p66Shc localization was cytoplasmic, and not perinuclear as in normal lungs or lungs with acute disease. The distribution of p66Shc in the patient with severe BPD was similar to that of baboons delivered at 140 days and treated with 100% oxygen for 10 days. Expression of p66Shc in the patients with mildly fibrotic BPD resembled that of baboons delivered at 125 days' gestation and maintained with oxygen only as needed. These findings indicate that, in both humans and baboons, p66Shc expression and localization change with premature birth and the pathogenesis of chronic lung disease.
DISCUSSION
Our findings support the hypothesis that pulmonary p66Shc expression is developmentally regulated in baboons and humans as well as in mice. For all three species, p66Shc expression is highest in the early fetal lung and decreases with maturation. In the mouse lung, p66Shc expression decreases just before birth at 18.5 days' gestation, during the early saccular stage of development (22). By comparison, p66Shc decreased through the saccular stage of baboon lung development (14) and during the late canalicular stage of human lung development (23). At the end of gestation, mice have saccular stage lungs, whereas term baboon and human infants have begun alveolarization. Thus, differences in the timing of p66Shc downregulation reflect differences in the ontogeny of lung development within each species. Conservation of the three Shc isoforms and their developmental regulation across taxonomic orders suggests that p66Shc regulates lung development in many mammalian species, and that attenuation of p66Shc before birth is adaptive.
After premature delivery, p66Shc expression in the premature lung correlated inversely to the degree of fibrosis. Relatively little p66Shc was expressed in 140-dayeCgestation baboons, and expression was mildly increased with induction of severely fibrotic BPD. Conversely, experimental attenuation of severe BPD using intravenous anti-BLP antibodies dramatically increased relative and absolute p66Shc expression. Moreover, in the 125-day mildly fibrotic baboon BPD model, p66Shc is high at birth and remains elevated for at least 21 days, rather than declining as it would in utero. Thus, in premature lungs, elevated p66Shc expression was associated with a mildly fibrotic BPD phenotype. We note that p66Shc consistently failed to colocalize with eCsmooth muscle actin, leading us to speculate that p66Shc expression is inversely correlated to the differentiation of myofibroblast populations implicated in the pathogenesis of BPD (24).
In both humans and baboons, mild or minimally fibrotic lung disease was associated with epithelial p66Shc localization. This change in p66Shc localization parallels the shift in expression seen during normal lung development In contrast, baboons and the human with severely fibrotic BPD expressed p66Shc primarily in the mesenchyme. Moreover, experimental attenuation of BPD in the 140-day/100% oxygen baboon BPD model using 2A11 anti-BLP antibody was associated with an epithelial p66Shc distribution as well as greatly increased p66Shc expression. Thus, loss of epithelial p66Shc expression appears to correlate with mesenchymal expansion and the development of severely fibrotic BPD in both premature baboons and humans. The persistence of mesenchymal p66Shc expression in the mildly fibrotic BPD model may reflect the degree to which normal lung development is disrupted.
The function of p66Shc in the developing lung is likely to be related to its previously defined signaling functions. On tyrosine phosphorylation, p66Shc binds and sequesters Grb2 (25), thereby antagonizing Ras activation by receptor tyrosine kinases. We speculate that expression of p66Shc may represent inhibitory feedback on mitogenic signaling through MAP kinases. Thus, p66Shc localization may indicate the proliferation of specific cell populations. A previous evaluation of lung cell proliferation in the mildly fibrotic 125-day baboon BPD model (26) supports this postulate. First, cell proliferation in the baboon decreases with maturation, falling 97% between Days 125 and 175. This drop parallels the decrease in p66Shc expression over the same period. Second, cell proliferation in the mildly fibrotic 125-day baboon BPD model remains elevated through at least 21 days after birth, again closely paralleling the expression of p66Shc in the same model. Finally, in the baboon BPD model, proliferating cells localize primarily to the epithelium through the first 14 days of life, coincident with p66Shc immunolocalization. In the current study, p66Shc consistently localized to the same tissues as PCNA, though often in different individual cells. We therefore speculate that p66Shc expression represents the inhibitory limb of a feedback loop regulating cell proliferation in the BPD lung. Its expression in the mesenchyme of fibrotic lungs may therefore relate to ongoing proliferation within this compartment. Conversely, epithelial p66Shc expression after 2A11 infusion appears to be independent of any initial increase in proliferation. In this population, Shc isoform expression may represent a mechanism by which bombesin signaling regulates epithelial cell mitogenesis.
p66Shc also mediates oxidative stress signaling on serine phosphorylation, resulting in activation of the mitochondrial apoptosis pathway, culminating in caspase activation and cytochrome-c release (27, 28). In the current study, both cleaved PARP and p66Shc localized to the mesenchyme of 140-day baboons with BPD, and attenuation of BPD in the same model shifted both p66Shc and cleaved PARP expression to the epithelium. These findings are therefore broadly consistent with the putative role of p66Shc in mediating oxidative stress responses. However, the large increase in p66Shc expression associated with 2A11 treatment infers that its function in BPD is adaptive. In contrast, p66Shc has been characterized as playing a maladaptive role in oxidative lung disease. Mice deficient in p66Shc are resistant to oxidative injury from hydrogen peroxide and paraquat, and live 30% longer (7). Susceptibility to oxidative stress is restored by induced p66Shc expression. It is unclear how these findings may be reconciled; it is possible that p66Shc confers different functions in neonates and adults.
The expression and localization of p66Shc within the neonatal lung may therefore identify a population of cells that is both rapidly proliferating and at increased risk of oxidative stresseCinduced cell death. The shift of p66Shc expression from the epithelium to the mesenchyme in severely fibrotic BPD may directly regulate both cell populations. We note that many of the signaling pathways converging on Shc have been implicated in the regulation of lung cell proliferation and survival, including those of epidermal growth factor (29), insulin (4), and fibroblast growth factors (30). The elucidation of the mechanisms governing Shc isoform expression may provide additional insight into the determinants of pulmonary fibrosis after premature birth. Moreover, the signaling pathways converging on Shc may be manipulated to determine how mitogenic and oxidative stress signaling interact to regulate fibrogenic precursors.
Acknowledgments
Key R-10 support personnel include Vicki Winters and the pathology staff at University of Texas Health Sciences Center at San Antonio, and the Neonatal Intensive Care Unit and animal production staffs at the Southwest Foundation for Biomedical Research. Histology technical support was provided by Pablo Bringas Jr. and Valentino Santos of the University of Southern California Center for Craniofacial Molecular Biology.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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