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Am J Physiol Regul Integr Comp Physiol 293: R314-R322, 2007. First published April 11, 2007; doi:10.1152/ajpregu.00265.2005
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Uteroplacental insufficiency decreases p53 serine-15 phosphorylation in term IUGR rat lungs

E. A. O'Brien,1 V. Barnes,2 L. Zhao,1 R. A. McKnight,1 X. Yu,1 C. W. Callaway,1 L. Wang,1 J. C. Sun,1 M. J. Dahl,1 A. Wint,1 Z. Wang,1 T. M. McIntyre,2 K. H. Albertine,1 and R. H. Lane1

1Division of Neonatology, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah; and 2Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio

Submitted 13 April 2005 ; accepted in final form 9 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intrauterine growth restriction (IUGR) increases the incidence of chronic lung disease (CLD). The molecular mechanisms responsible for IUGR-induced acute lung injury that predispose the IUGR infant to CLD are unknown. p53, a transcription factor, plays a pivotal role in determining cellular response to stress by affecting apoptosis, cell cycle regulation, and angiogenesis, processes required for thinning of lung mesenchyme. Because thickened lung mesenchyme is characteristic of CLD, we hypothesized that IUGR-induced changes in lung growth are associated with alterations in p53 expression and/or modification. We induced IUGR through bilateral uterine artery ligation of pregnant rats. Uteroplacental insufficiency significantly decreased serine-15-phosphorylated (serine-15P) p53, an active form of p53, in IUGR rat lung. Moreover, we found that decreased phosphorylation of lung p53 serine-15 localized to thickened distal air space mesenchyme. We also found that IUGR significantly decreased mRNA for targets downstream of p53, specifically, proapoptotic Bax and Apaf, as well as Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis. Furthermore, we found that IUGR significantly increased mRNA for Bcl-2, an antiapoptotic gene downregulated by p53. We conclude that in IUGR rats, uteroplacental insufficiency induces decreased lung mesenchymal p53 serine-15P in association with distal lung mesenchymal thickening. We speculate that decreased p53 serine-15P in IUGR rat lungs alters lung phenotype, making the IUGR lung more susceptible to subsequent injury.

intrauterine growth restriction; chronic lung disease; alveolar simplification; apoptosis

UTEROPLACENTAL INSUFFICIENCY is a leading cause of intrauterine growth restriction (IUGR) in western countries (30). IUGR is associated with increased incidence of multiple morbidities such as respiratory distress syndrome (28). Furthermore, infants who are IUGR also have increased incidence of chronic lung disease (CLD) (5, 30, 50, 73, 88). In addition, IUGR infants have increased risk for impaired lung function (6, 32, 77). The molecular mechanisms responsible for IUGR-induced lung injury are unknown.

Apoptosis of lung fibroblasts, cellular proliferation, and angiogenesis may contribute to thinning of lung mesenchyme, a process necessary for alveolar formation (10, 47, 72, 76). CLD, which is frequent in IUGR infants, is characterized histologically by decreased alveolar secondary septation and thickened distal air space walls, suggesting alterations in the balance between apoptosis and cell proliferation (18). Determining the factors that direct alveolar formation has proven elusive because formation of alveoli occurs late in development and spans the perinatal and postnatal periods.

For this reason, we induced IUGR using an established rat model of uteroplacental insufficiency (51–53, 56, 57). IUGR pups are 20–25% lighter than the sham-operated control animals (controls), birth weights are normally distributed within and among litters, and litter size does not differ significantly between control and IUGR groups (51–57). Notably, at term birth, rat lungs are in the saccular stage of lung development, equivalent to a human fetus ranging from 24 to 36 wk of gestation (11, 13, 87).

Although timing of alveolar formation varies among species, the histologically defined stages of lung development are conserved (3, 12, 20, 21). Alveolar formation is manifest by the development of alveolar secondary septae, or crests, which divide the terminal air sacs into anatomic alveoli (85). Attenuation of the lung mesenchyme is then required to create the thin air/blood barrier needed for optimal diffusion of gas.

Uteroplacental insufficiency in both human and rat alters the intrauterine environment, exposing the fetus to stressors such as hypoglycemia, hypoinsulinemia, acidosis, hypoxia, and decreased branched chain amino acids and results in IUGR (22). Hypoxia, as well as hypoxia plus nutrient deprivation and acidosis, affects activation of p53 (33, 70). p53 is a transcription factor that plays a pivotal role in determining cellular response to stress by affecting the apoptosis cascade as well as cell cycle regulation and angiogenesis (39, 42, 83). Specifically, p53 upregulates proapoptotic downstream targets Bax and Apaf, as well as Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis (25, 27, 42, 43, 59, 69, 74, 83, 84). Furthermore, p53 downregulates Bcl-2, an antiapoptotic downstream target (61). Notably, p53 is critical for the stress response in the lung and plays a role in lung development (68, 78). Because hypoxia, acidosis, and nutrient deprivation characterize the intrauterine milieu of the IUGR fetus and affect the p53-mediated stress response, we hypothesized that p53 mediates IUGR-induced acute lung injury, making the IUGR lung more susceptible to subsequent injury.

We used histological and morphometric analysis to describe the lung phenotype observed in IUGR rat pups. We determined p53 status in IUGR rat lung. We determined protein levels of DNA-dependent protein kinase (DNA-PK), ataxia-telangiectasia mutated (ATM), and ataxia-telangiectasia and Rad3-related kinase (ATR), enzymes known to phosphorylate serine-15 p53 (19, 36, 49, 79). We evaluated the apoptosis cascade, cell cycle regulation, and angiogenesis to determine whether downstream targets of p53 are altered in IUGR-induced lung injury. We looked for molecular evidence of pulmonary hypoplasia by measuring lung-to-body weight, DNA-to-tissue weight, and DNA-to-protein ratios in IUGR rat lungs as well as controls (14, 67). Finally, because IUGR is associated with an increased incidence of respiratory distress syndrome, which is characterized by surfactant deficiency, and because other animal models of IUGR involving starvation and hypoxia decrease surfactant phospholipid synthesis, as well as alveolar surface area, we measured total phospholipids and dipalmitoyl phospholipids, including dipalmitoyl phosphatidylcholine (DPPC), a surface tension-lowering phospholipid in mature surfactant, in IUGR and control lungs (7, 15, 23, 28, 29, 60, 81).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All animal procedures were approved by the University of Utah Animal Research Committee and are in accordance with the American Physiological Society's guiding principles (4). These surgical procedures have been described previously (48, 51, 55, 80). Time-dated Sprague-Dawley pregnant rats were individually housed under standard conditions with 12:12-h light-dark cycles and allowed free access to standard rat chow and water. On day 19 of gestation (term is 21.5 days), maternal rats were anesthetized with intraperitoneal xylazine (8 mg/kg) and ketamine (40 mg/kg), and both uterine arteries were ligated (IUGR group). Control rats underwent identical anesthetic and surgical procedures, except for arterial ligation (control group). After surgery, if there was evidence of infection or distress, euthanasia was performed. At term gestation, ~48 h after the initial operation, the dam was again anesthetized, and pups were delivered by caesarian section. The pups were weighed, litter size was noted, and tissue was collected. Lungs were either 1) insufflated with 10% formalin through the trachea at 20 cmH2O or 2) snap-frozen in liquid nitrogen and stored at –80°C (n = 8 litters, 2–4 pups/litter, n = 21 pups for histology/morphometry; n = 3 litters, 2 pups/litter, n = 6 pups for molecular analysis).

Histology. IUGR and control lungs were isolated and insufflated with 10% formalin through the trachea as described above. Heart-lung blocks were paraffin-embedded and serially sectioned at 5 µm. Slides were stained with standard hematoxylin and eosin stain.

Morphometric analysis. Hematoxylin and eosin-stained slides were used for morphometric analysis. We followed the principles of unbiased sampling and blinded analysis (9). We took calibrated digital images of 15 random fields of view of lung parenchyma (9), excluding airways. Five linear measurements per field were made at the thinnest points in the distal air space walls. This sampling procedure was repeated on a total of three adjacent tissue sections per rat (225 measurements per rat). Linear measurements were averaged per pup, and results were divided by pup weight (24) (n = 8 litters, 2–4 pups/litter, n = 21 pups).

RNA isolation. The method of Chomczynski and Sacchi was used to isolate RNA, as previously described, including treatment with DNase (40, 42, 58, 71, 75). Gel electrophoresis confirmed the integrity of the samples.

Reverse transcription. cDNA was synthesized, using random hexamers and SuperScript reverse transcriptase (GIBCO BRL, Gaithersburg, MD) from 1.0 µg of rat lung.

Real-time PCR. Primers and probes (p53, Bax, Apaf, Bcl-2, Gadd45, and Tsp-1) were designed, using Primer Express software (Applied Biosystems, Foster City, CA) (Table 1); target probes were labeled with the fluorescent reporter dye FAM. Before the performance of real-time PCR, all primer pairs were tested with serial Mg2+ and primer concentrations to determine the optimal reaction conditions and to demonstrate the specificity of each primer pair. Reporter dye emission was detected using an automated sequence detector combined with ABI Prism 7700 Sequence Detection System software (Applied Biosystems). An algorithm normalized the reporter signal (Rn) to a passive reference and multiplied the standard deviation of the background Rn in the first cycles by a default factor of 10 to determine the cycle threshold (CT). CT has a linear relation with the logarithm of the initial template copy number (41). Real-time PCR quantification was then performed using the TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. Before the use of GAPDH as a control, serial dilutions of cDNA were quantified to prove the validity of using GAPDH as an internal control. Relative quantification of PCR products are therefore based on value differences between the target and GAPDH control using the comparative CT method (65). Cycle parameters were 55°C for 5 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s followed by 58°C for 60 s. For every sample, each PCR reaction was performed on three separate occasions; in each set of reactions every sample is present in triplicate. All studies were done on term (D0) IUGR and control rat lungs.


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  		Table 1. PCR primers for real-time PCR

 
Western blot analysis. Protein was isolated by centrifugation following homogenization in Laemmeli lysis buffer. Protein concentration was determined using Bradford's dye-binding assay. Standard Western blotting technique was used (46). The membrane was incubated with Blotto solution and then with polycolonal rabbit p53 primary antibody (1:250 dilution; Cell Signaling, Beverly, MA) or polyclonal rabbit serine-15-phosphorylated (serine-15P) p53 (1:2,000 dilution; Cell Signaling) for 1 h at room temperature or with monoclonal mouse DNA-PK (1:100 dilution; Abcam, Cambridge, MA), monoclonal mouse ATM (1:100 dilution; Abcam), or polyclonal goat ATR primary antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. The membranes were washed before incubation with the secondary anti-rabbit antibody (1:1,000 dilution; Cell Signaling) for p53 and serine-15P p53, secondary anti-mouse antibody (1:2,000 dilution) for DNA-PK and ATM (Cell Signaling), and secondary anti-goat antibody (1:3,000 dilution) for ATR (Santa Cruz Biotechnology). Enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) products were quantified by densitometry after standardization for loading. Each blot was replicated three times.

Immunohistochemistry analysis for p53 serine-15P localization. Immunohistochemical staining methods were used that are standard in our laboratories (1, 2), including antigen retrieval (10x Citra solution, catalog no. HK-086-9K; BioGenex, San Ramon, CA) according to the manufacturer's instructions. Sections were then incubated in a 3% H2O2 solution for 30 min at room temperature to quench endogenous peroxidase activity. The tissue sections were incubated with avidin solution for 10 min, followed by incubation with biotin solution for 10 min (avidin/biotin blocking kit, catalog no. SP-2001; Vector Laboratories, Burlingame, CA). The tissue sections subsequently were treated with blocking buffer that contained 10% normal horse serum (room temperature for 30 min). Tissue sections were then incubated in phospho-p53 (serine-15) anti-mouse monoclonal antibody (catalog no. ab10803-50; Abcam) at 1:100 dilution in blocking buffer overnight at 4°C in a humidified chamber. The next day, the tissue sections were incubated in biotinylated horse anti-mouse IgG (catalog no. BA-2001; Vector Laboratories) at room temperature for 30 min. Following incubation in TSA biotin system (tyramide signal amplification, catalog no. NEL700; PerkinElmer, Boston, MA) according to the manufacturer's instructions, the tissue sections were washed, stained with diaminobenzidine (Sigma, St. Louis, MO), counterstained with hematoxylin (Sigma), dehydrated, and coverslipped with Cytoseal 60 (Stephens Scientific, Kalamazoo, MI). Negative staining controls included substitution of the primary antibody with species-matched, isotype-matched anti-insulin antibody, omission of the primary antibody, or omission of the secondary antibody. All lung tissue sections were immunostained as a group. Digital images were taken with a Zeiss Axiophot microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end label assay. Rat lung tissue sections were used to identify apoptotic nuclei, which were labeled using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique via the In Situ Death Detection kit, POD (peroxidase; Roche, Nutley, NJ).

Ratios of lung to body weight, DNA to protein, and DNA to tissue weight. Pups were weighed immediately following delivery. Lungs were isolated, weighed, and snap-frozen in liquid nitrogen. Lung-to-body weight ratios were calculated (n = 8–12). Tissue homogenization and DNA isolation were performed on 50 mg of frozen lung tissue/rat pup. DNA isolation was performed using standard methods, and the DNA-to-tissue weight ratio was calculated (16, 17).

Lung protein was isolated and quantified using the bicinchoninic acid method of Pierce (31). The DNA-to-protein ratio was calculated.

Phospholipid analysis. Lung samples were weighed and added to a solution containing 2:1 acidic methanol-chloroform. The acidic methanol was made by adding glacial acetic acid to a final concentration of 50 mM. The volume of the solution was recorded and used as the portion of methanol-chloroform needed to form the monophase for the Bligh and Dyer extraction (8). The tissue was homogenized for 2 min with a Polytron PT-10–35 homogenizer, and the second step of the Bligh and Dryer extraction was performed (8). Phases were separated by centrifugation at 547 g for 15 min. The aqueous phase was washed once again with chloroform. The two chloroform fractions were combined and dried under a steady stream of nitrogen. The lipid was suspended in 1 ml of chloroform and applied to a prewashed aminopropyl solid-phase extraction (SPE) column to separate the lipid classes (45). The columns were prepared for use by first washing with hexanes (2 x 1-ml washes; Sigma). The 1-ml samples were loaded onto the SPE columns. After the chloroform was eluted from the columns, the columns were washed with 1 ml of the following solvents to separate the lipid classes: solvent A [2:1 (vol/vol) CHCl3-isopropyl alcohol], which elutes the free fatty acids, solvent B (2% acetic acid in diethyl ether) to elute the nonpolar lipids, and finally, solvent C (methanol) was used to elute the polar phospholipids. The methanol fraction was collected and dried under a stream of N2 (45). The dried lipid residue was suspended in 500 µl of a 5% solution of hydrofluoric acid and incubated at 60°C for 45 min. The samples were extracted using 2:1 (vol/vol) pentane and water. The pentane (top layer) was dried under nitrogen stream and immediately resuspended in dichloromethane (Acros Organics). The lipid analysis was performed in EI mode, using an Agilent 6890 gas chromatograph and 5983 mass spectrometer. The instrument parameters were as follows: EV, 69.9; injection port, 280°C; oven, 100°C at 2 min -> 270°C at 8°C/min; MS source, 230°C; MS quad, 150°C; flow, 1 ml/min; column DB5 (n = 6 litters, 2 pups/litter, n = 12 pups).

Statistics. All data are expressed as means ± SE. For real-time RT-PCR and Western blotting, statistical analysis was performed using ANOVA (Fisher's protected least significance difference) and Student's unpaired t-test. For morphometric analysis, statistical analysis was performed using the Wilcoxon signed-rank test and two-sample t-test. We accepted P < 0.05 as indicating statistical significance.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Histology. Lung histology appeared different between the IUGR and control rat pups (Fig. 1). The difference was in the architecture of the mesenchymal core of the developing distal air space walls, which appeared thicker in the IUGR rat pups compared with control rat pups. Histological changes were quantified by morphometric analysis. IUGR increased distal air space wall thickness relative to body weight (IUGR distal air space thickness 2.05 ± 0.12 µm/g body weight compared with controls, 1.22 ± 0.06 µm/g body wt; P < 0.05) (Fig. 2).


Figure 1
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  		Fig. 1. Terminal deoxynucleotidyl transferase-mediated dUTP nick end (TUNEL) labeling (A and B) and serine-15-phosphorylated (serine-15P) p53-positive cells (C and D) in the lung parenchyma of neonatal rat pups on day 0 (D0) of life. Images in A and C show lung tissue from control rats; images in B and D show lung tissue from intrauterine growth restriction (IUGR) rats. Fewer TUNEL-positive and serine-15P p53-positive cells (arrow in A and C) are visible in the IUGR group compared with the control group. Also, the distal air space walls appear thicker in the IUGR group. Inset in C shows an immunostained control (omission of the primary antibody). Images in A–D are of the same magnification.

 

Figure 2
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  		Fig. 2. Distal air space wall thickness in the lung parenchyma of neonatal rat pups on D0 of life. IUGR significantly increased distal air space wall thickness per gram of body weight in term rat lungs in the saccular stage of lung development. Data are means ± SE; n = 8 litters, 2–4 pups/litter, n = 21 pups. *P < 0.05.

 
p53 mRNA and protein. Although there was a tendency toward increased p53 mRNA, uteroplacental insufficiency did not significantly change p53 mRNA (Fig. 3A) or total protein (Fig. 3B) in D0 IUGR rat lungs compared with control rat lungs.


Figure 3
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  		Fig. 3. A: quantification of p53 mRNA using real-time RT-PCR in D0 IUGR and control (C) rat lungs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. IUGR did not significantly alter p53 mRNA (p53 mRNA: IUGR, 130 ± 37% of control, P > 0.05). B: quantification of p53 total protein and serine-15P p53 using Western blotting and densitometry in D0 IUGR and control rat lungs. GAPDH was used as internal control. Uteroplacental insufficiency decreased serine-15P p53 to ~70% of controls, whereas p53 total protein was not decreased (p53 total protein: IUGR, 101.5 ± 14% of control, P > 0.05). Data are means ± SE expressed as IUGR percentages of control; n = 6 rat pups grouped from 3 litters. *P < 0.01.

 
Serine-15P p53 protein and immunohistochemistry. Because neither p53 mRNA nor total protein was different between the two groups of rat pups, we determined whether an activated form of p53 was affected by IUGR. Therefore, Western blotting was performed to measure serine-15P p53. IUGR decreased serine-15P p53 to 65 ± 13% of controls (P < 0.05) (Fig. 3B). Furthermore, using immunohistochemistry, serine-15P p53 protein expression was decreased in mesenchymal cells located in the distal air space walls of IUGR rats compared with controls (Fig. 1). Corresponding with decreased p53 serine-15 phosphorylation, IUGR decreased DNA-PK and ATR protein, whereas IUGR increased ATM (IUGR: DNA-PK, 76 ± 3.2% of controls; ATR, 86 ± 4% of controls; ATM, 311 ± 22.9% of controls; P < 0.05).

RNA levels for downstream targets of p53 and TUNEL staining. A decrease in activated p53 protein implied that downstream targets of p53 also were decreased. To test this possibility, we assessed a number of molecular and histological markers of apoptosis. First, we measured mRNA expression for proapoptotic Bax and Apaf. Expression of both Bax and Apaf mRNA was significantly decreased to 68 ± 8.1 and 76.5 ± 4.7% of controls, respectively (P < 0.01) (Fig. 4). mRNA expression of other genes involved in organ development was also downregulated. For example, Gadd45 mRNA, the product of a gene involved in growth arrest, and Tsp-1 mRNA, another downstream target of p53 that is involved in angiogenesis, were significantly lower in the IUGR rat lung tissue (Gadd45, 78.8 ± 5.9%; Tsp-1, 88.2 ± 2.2%) compared with controls (P < 0.05) (Fig. 4). Conversely, IUGR significantly increased antiapoptotic Bcl-2 mRNA expression to 185 ± 24% of controls (P < 0.05) (Fig. 4). Consistent with decreased mRNA expression of proapoptotic downstream targets of p53, fewer TUNEL-positive cells were visible in the distal air space mesenchyme of the IUGR rat lungs compared with controls (Fig. 1).


Figure 4
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  		Fig. 4. Quantification of downstream targets of p53 proapoptotic Bax and Apaf, antiapoptotic Bcl-2, Gadd45 (involved in growth arrest), and Tsp-1 (involved in angiogenesis) using real-time RT-PCR in D0 IUGR and control rat lungs. GAPDH was used as internal control. Data are means ± SE expressed as IUGR percentages of control; n = 6 rat pups grouped from 3 litters. *P < 0.01; **P < 0.05.

 
Molecular markers of pulmonary hypoplasia. To determine whether the IUGR rat lungs were hypoplastic, we measured lung-to-body weight ratios. However, this analysis showed no significant difference between IUGR and control rats (lung/body weight: IUGR, 0.038 ± 0.002 vs. control, 0.037 ± 0.002; P > 0.05). At the molecular level, DNA-to-tissue weight and DNA-to-protein ratios were measured. IUGR significantly decreased DNA/tissue weight and DNA/protein compared with controls (Fig. 5).


Figure 5
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  		Fig. 5. A: quantification of the DNA/tissue weight in D0 IUGR and control rat lungs. IUGR decreased the ratio of DNA to tissue weight to 5.7 ± 1.9 µg DNA/mg tissue compared with 7.2 ± 1.7 µg DNA/mg tissue in the control rat lungs. Data are means ± SE expressed as µg DNA/mg tissue; n = 6 rat pups grouped from 3 litters. *P < 0.05. B: quantification of DNA/protein in D0 IUGR and control rat lungs. IUGR decreased the ratio of DNA to protein to 0.30 ± 0.1 µg DNA/µg protein compared with 0.36 ± 0.01 µg DNA/µg protein in the control rat lungs. Data are means ± SE expressed as µg DNA/µg protein; n = 6 rat pups grouped from 3 litters. *P < 0.05. Uteroplacental insufficiency significantly decreased both DNA/tissue weight and DNA/protein in the D0 rat lung.

 
Phospholipid analysis. To determine whether uteroplacental insufficiency-induced IUGR altered surfactant phospholipid content, we measured dipalmitoyl phospholipids, because DPPC is one of the tensoactive components in surfactant being preferentially released into the air spaces of most mammals, as well as total phospholipid content. Similar to starvation-induced IUGR, uteroplacental insufficiency-induced IUGR significantly decreased total dipalmitoyl phospholipids and dipalmitoyl phospholipids per milligram of lung weight compared with controls [dipalmitoyl phospholipids: IUGR, 0.61 ± 0.05 ng compared with control, 1.17 ± 0.21 ng; P < 0.05, n = 12 pups; dipalmitoyl phospholipids (ng/mg lung wt): IUGR, 5.17 x 10–3 ± 0.00039 ng/mg compared with control, 9.84 x 10–3 ± 0.00189 ng/mg; P < 0.05, n = 12 pups] (Fig. 6). Total phospholipid content was not significantly altered.


Figure 6
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  		Fig. 6. Quantification of dipalmitoyl phospholipids and dipalmitoyl phospholipids per mg of lung weight in D0 IUGR and control lungs. A: IUGR significantly decreased total dipalmitoyl phospholipid: IUGR, 0.61 ± 0.05 ng compared with control, 1.17 ± 0.21 ng dipalmitoyl phospholipid. B: controlling for lung weight, IUGR also significantly decreased dipalmitoyl phospholipid: IUGR, 0.00517 ± 0.00039 ng/mg lung weight compared with control, 0.00984 ± 0.00189 ng/mg lung weight. Data are means ± SE; n = 12 pups. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of our study show that uteroplacental insufficiency and subsequent IUGR decreased serine-15 phosphorylation/activation of p53, reduced expression of proapoptotic molecules that are downstream of activated p53, and led to the appearance of thickened lung mesenchyme. Despite the well-established link between IUGR and altered lung phenotype, a specific molecular mechanism responsible for this phenotypic change has not been identified. Our novel finding of decreased active p53 in thickened mesenchyme of the IUGR rat lung identifies a role for p53 in IUGR-induced lung morbidity, thus suggesting a molecular mechanism for the lung's dysmorphogenesis.

Our results show that whereas IUGR decreased p53 phosphorylation and expression of its proapoptotic downstream targets, it increased expression of Bcl-2, an antiapoptotic downstream target negatively regulated by p53 (25, 27, 42, 61, 74, 83). p53 is a transcription factor that plays a pivotal role in determining cellular response to stress by regulating the apoptosis cascade, cell cycle, and angiogenesis (38, 83). p53 is also important for stress responses in the lung. For example, in response to hyperoxic stress, p53 protein accumulates in airway and alveolar epithelial cells in adult mice in association with increased apoptosis (68). Furthermore, neonatal mice exposed to hyperoxia had increased apoptosis in peripheral lung cells in association with increased Bax expression (64). Thus IUGR-induced decreased active p53 in neonatal lung mesenchyme may alter the lung's ability to respond to stress, such as hyperoxia, making it more susceptible to injury. Although not statistically different, we speculate that our findings of increased p53 mRNA, in the context of decreased serine-15P p53, represents a feedback response attempting to correct p53 protein levels.

p53 is kept at low levels in the cell under normal conditions, and its activity is regulated at many levels (42, 83). In response to cellular stress, p53 undergoes many different posttranslational modifications. Serine-15 phosphorylation causes p53 to dissociate from MDM2, a ubiquitin ligase, allowing p53 to accumulate in the cell and activate downstream targets (66, 82, 83). Serine-15 p53 is phosphorylated by a family of kinases called phosphatidylinositol 3-kinases, which include DNA-PK, ATM, and ATR (19, 36, 49, 79). RKO cells, a human colon cancer cell line, exposed to hypoxia had an ATR-dependent increase in serine-15P p53, with subsequent accumulation of p53 and growth arrest (36). Furthermore, hypoxia-induced ATR-dependent p53 serine-15 phosphorylation occurs in solid tumors (37). Acidosis also increases p53 protein and apoptosis in cultured colonic epithelial cells derived from colorectal adenomas (86). Although we know that hypoxia affects phosphorylation of serine-15 p53 in colorectal cells, little is known about serine-15P p53 in lungs exposed to hypoxia. We showed decreased DNA-PK and ATR in association with decreased serine-15 phosphorylation in response to uteroplacental insufficiency and IUGR, an insult characterized by the combination of hypoxia, acidosis, hypoglycemia, and hypoinsulinemia. In this regard, our results are in contrast to the aforementioned studies, which demonstrated increased serine-15P p53 in response to hypoxia and acidosis. Supporting our results, hypoxia in fetal alveolar type II epithelial cells decreased the ratio Bax/Bcl-2 (34, 35). Our findings are novel because we looked at p53 activation in the developing lung in vivo, and our translational in vivo model is characterized by a dynamic and multifactorial insult.

The underlying change in phenotype seen in the saccular stage lungs of IUGR rat pups born at term suggests that IUGR makes the lung more susceptible to injury from volume and oxygen trauma, stressors frequently experienced by IUGR neonates who are often born at this same stage of lung development. This suggestion is supported by animal studies linking intrauterine stress and IUGR to pulmonary hypoplasia, an altered surfactant phospholipid profile, and CLD. Specifically, uteroplacental insufficiency, induced by umbilico-placental embolization in pregnant ewes, resulted in IUGR lambs that were hypoxemic and had increased minute ventilation and oxygen consumption at 8 wk of age. Conversely, the lambs had decreased diffusing capacity, functional residual capacity, and total lung capacity, as well as pulmonary DNA and protein concentrations (26, 44). Other studies have shown that IUGR lamb lungs had fewer alveoli per respiratory unit and thicker interalveolar septae at 8 wk of postnatal life, changes that persisted into adulthood (62, 63). Our findings are consistent with the results of those studies and extend them by demonstrating an underlying dysregulation of molecular signaling downstream from p53.

Thickened lung mesenchyme compared with the decreased ratios DNA/protein and DNA/tissue weight may seem incongruous. However, the morphometric analysis of distal air space wall thickness focused on the mesenchymal compartment in the lungs, whereas the DNA/protein and DNA/tissue weight measurements were made on whole lungs. We speculate that distal air space mesenchymal thickening occurs at the expense of the highly active epithelial compartment, which is suggested by decreased dipalmitoyl phospholipids, and may lead to the decreased DNA/protein and DNA/tissue weight.

Some limitations of our study are important to consider. Caution must be used when attempting to apply data obtained from a rat model to human pathophysiology. In humans, the impact and timing of uteroplacental insufficiency ranges across a continuum and is impacted by environmental and genetic variables. In contrast, the laboratory rat used in this study is inbred and experiences a homogeneous environment and diet. In this model of uteroplacental insufficiency, the fetal rat is exposed to a severe insult that occurs relatively late in gestation. However, several investigators have demonstrated that IUGR infants, despite their variable environments and genetic backgrounds, have significantly higher incidence of CLD (5, 30, 50, 73, 88). Furthermore, Greenough et al.'s (32) recent data show that this IUGR induced alteration in pulmonary function persists at follow up. In addition, the methods employed for phospholipid analysis quantified the dipalmitoylated components of rat lung surfactant, which may include dipalmitoyl phosphatidylglycerol, making the DPPC assessments an approximation. However, the data do demonstrate a significant difference in the phospholipid profile of IUGR rat lung surfactant.

In summary, uteroplacental insufficiency and subsequent IUGR lead to decreased p53 serine-15 phosphorylation in lung mesenchyme in association with thickened distal air space walls, decreased DNA content, and an altered surfactant phospholipid profile in the newborn rat. Decreased activation of lung mesenchymal cell p53, via decreased p53 serine-15 phosphorylation, results in altered expression of key downstream targets of p53 that are involved in apoptosis and cell cycle regulation, as well as angiogenesis. Alterations in p53 activation may represent a mechanism through which uteroplacental insufficiency and IUGR alter the balance between apoptosis and cell proliferation in developing lung, thereby predisposing the affected individual to lung injury and altered lung phenotype.


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This work was supported by National Institutes of Health (NIH) Grants HD01410 (to E. A. O'Brien), HD41075 (to R. H. Lane), and HL62875 (to K. H. Albertine) and the Primary Children's Medical Center Foundation. This work was also supported in part by NIH Grant MO1 RR-018390. We also acknowledge the University of Utah Children's Health Research Center.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. A. O'Brien, Univ. of Utah, Division of Neonatology, Williams Bldg., PO Box 581289, Salt Lake City, UT 84158 (e-mail: elizabeth.obrien{at}hsc.utah.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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