PageArticles 1 of 42 in PresS. Am J Physiol Regul Integr Comp Physiol (April 11, 2007). doi:10.1152/ajpregu.00265.2005
Uteroplacental Insufficiency Decreases p53 Serine-15 Phosphorylation in Term IUGR Rat Lungs
O’Brien E.A1., Barnes V.2, Zhao L1., McKnight R.A.1, Yu X.1, Callaway C.W.1, Wang L.1, Sun J.C.1, Dahl M.J.1, Wint A.1, Wang Z.1, McIntyre T.M.2, Albertine K.H.1, Lane R.H.1
1
University of Utah School of Medicine, Department of Pediatrics, Division of
Neonatology, Salt Lake City, Utah 84158, 2Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195
Running head: IUGR Affects p53 in Neonatal Rat Lung
Contact Information Elizabeth A O’Brien University of Utah Division of Neonatology Williams Building P. O. Box 581289 Salt Lake City, UT 84158
[email protected] Phone (801) 581-4178 Fax (801) 585-7395
Copyright © 2007 by the American Physiological Society.
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2 Abstract 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 (P) 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 airspace mesenchyme. We also found that IUGR significantly decreased mRNA for targets downstream of p53, specifically, pro-apoptotic Bax and Apaf, Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis. Furthermore, we found that IUGR significantly increased mRNA for Bcl-2, an anti-apoptotic gene down regulated by p53. We conclude that in IUGR rats, uteroplacental insufficiency induces decreased lung mesenchymal p53 serine-15 P 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. Keywords: Chronic Lung Disease, Alveolar Simplification, Apoptosis
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3
Introduction 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 airspace walls, suggesting alterations in the balance between apoptosis and cell proliferation (18). Determining 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 weeks gestation (11, 13, 87).
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4 Although timing of alveolar formation varies among species, the histologically defined stages of lung development are conserved (4, 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, affect 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 up-regulates pro-apoptotic 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 down regulates Bcl-2, an anti-apoptotic 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 affects the p53-mediated stress response, we hypothesized that p53 mediates IUGRinduced acute lung injury making the IUGR lung more susceptible to subsequent injury. We used histologic 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 telangectasia mutated (ATM) and ataxia-telangectasia and Rad3-related kinase (ATR), enzymes known to
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5 phosphorylate serine-15 p53(19, 36, 49, 79). We evaluated the apoptosis cascade, cell cycle regulation, and angiogenesis to determine if downstream targets of p53 are altered in IUGR-induced lung injury. We looked for molecular evidence of pulmonary hypoplasia by measuring lung: body weight, DNA: tissue weight and DNA: 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, which includes dipalmitoylphosphatidyl-choline (DPPC), a surface tension lowering phospholipid in mature surfactant , in IUGR and control lungs (7, 15, 23, 28, 29, 60, 81).
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6 Materials and Methods 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 (1). 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 hour 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, approximately 48 hours after the initial operation, the dam was again anesthetized and pups were delivered by caesarian section. The pups were weighed, litter size noted, and tissue 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 -80ºC: n= 8 litters, 2-4 pups/litter, n=21 pups for histology/morphometry; n=3 litters, 2 pups/litter for 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 paraffinembedded 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
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7 analysis (9). We took calibrated digital images of fifteen random fields of view of lung parenchyma (9), excluding airways. Five linear measurements per field were made at the thinnest points in the distal airspace 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 (RT): cDNA was synthesized, using random hexamers and SUPERSCRIPT™ reverse transcriptase (GIBCO BRL, Gaithersberg, MD) from 1.0 µg of rat lung. Real Time PCR: Primers and probes (p53, Bax, Apaf, Bcl-2, Gadd45, and Tsp1) were designed, using Primer Express Software™ (Applied Biosystems, Foster CA) (Table 1); target probes were labeled with the fluorescent reporter dye FAM. Prior to the performance of real time PCR, all primer pairs were tested with serial Mg+2 and primer concentrations to determine the optimal reaction conditions and to demonstrate the specificity of each primer pair. Reporter dye emission was detected by an automated sequence detector combined with ABI Prism 7700 Sequence Detection System® software (Applied Biosystem, Foster CA). 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 threshold CT. CT has a linear relation with the logarithm of the initial template copy number (41). Real time PCR
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8 quantification was then performed using the Taqman® glyceraldehydes-3-phosphate dehydrogenase control. Prior to 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 then based upon value differences between the target and GAPDH control using comparative CT method (65). Cycle parameters were 55ºC x 5 min, 95ºC x 10 min, and then 40 cycles of 95ºC x 15 sec -> 58º x 60 sec. 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. Western blotting: 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) (Cell Signaling, Beverly, MA) polyclonal rabbit serine-15 phosphorylated p53 (1:2000) (Cell Signaling, Beverly, MA) for 1 hour at room temperature or with: monoclonal mouse DNA-PK (1:100) (Abcam, Cambridge, MA), monoclonal mouse ATM (1:100) (Abcam, Cambridge, MA), or polyclonal goat ATR primary antibody (1:100) (Santa Cruz, Santa Cruz, CA) for 2 hours at room temperature. The membranes were washed before incubation with the secondary anti-rabbit antibody (1:1000 dilution); (Cell Signaling, Beverly, MA) for p53 and serine-15 P p53, secondary anti-mouse antibody (1:2000) for DNA-PK and ATM (Cell Signaling, Beverly, MA), and secondary anti-goat antibody (1:3000) for ATR (Santa Cruz, Santa Cruz, CA). Enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) products were
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9 quantified by densitometry after standardization for loading. Each blot was replicated three times. Immunohistochemistry analysis for p53 Serine-15 P localization: Immunohistochemical staining methods were used that are standard in our laboratories (2, 3), including antigen retrieval (10 x Citra solution; Cat # 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 for10 min (Avidin/Biotin Blocking Kit; Cat # SP-2001, Vector Laboratories, Inc. 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 (Ser15) anti-mouse monoclonal antibody (Abcam Inc.; Cat # ab10803-50, Cambridge, MA) at 1:100 dilution in blocking buffer overnight at 4oC in a humidified chamber. The next day, the tissue sections were incubated in biotinylated horse anti-mouse IgG (Vector Laboratories, Inc.; Cat # BA-2001, Burlingame, CA) at room temperature for 30 min. Following incubation in TSATM Biotin System (PerkinElmer; Cat # NEL700, Boston, MA), according to the manufacturer’s instructions, the tissue sections were washed, stained with DAB (Sigma; St Louis, MO), counterstained with hematoxylin (Sigma; St. Louis, MO), 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
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10 the secondary antibody. All lung tissue sections were immunostained as a group. Digital images were taken with a Zeiss Axiophot microscope. Terminal deoxynucleotidyl transferase uridine nick end-label assay (TUNEL): Rat lung tissue sections were used to identify apoptotic nuclei, which were labeled using the terminal deoxynucleotidyl transferase uridine nick end-label technique (TUNEL) via the In Situ Death Detection Kit, POD (Roche, Nutley, NJ). Lung: Body Weight, DNA:Protein, DNA:Tissue weight ratios: Pups were weighed immediately following delivery. Lungs were isolated, weighed and snap-frozen in liquid nitrogen. Lung: 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 method and the DNA:tissue weight ratio was calculated (16, 17). Lung protein was isolated and quantified by the BCA method of Pierce (31). The DNA: 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 50mM. The volume of the solution was recorded and used as the portion of methanol/chloroform needed to form the mono-phase for the Bligh and Dyer extraction (8). The tissue was homogenized for two 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 x 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
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11 1 ml chloroform and applied to a pre-washed Aminopropyl SPE column to separate the lipid classes (45). The columns were prepared for use by first washing with Hexanes (2 x 1ml washes, Sigma; St. Louis, MO). The 1 ml samples were loaded onto the SPE columns. After the chloroform was eluted from the columns, the columns were washed using 1 ml of the following solvents to separate the lipid classes. Solvent A (2:1 {CHCl3: IPA (v/v)}) which elutes the free fatty acids, solvent B (2% Acetic Acid in Diethyl Ether) to elute the non-polar 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 60ºC for 45 min. The samples were extracted, using 2:1 (v/v) 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 GC and 5983 MS. The instrument parameters were as follows: EV 69.9, injection port 280ºC, Oven: 100-2 min -> 270ºC @ 8ºC/min; MS=230; Quad=150; Flow=1 ml/min; Column-DB5. (n=6 litters, 2 pups/litter, n=12 pups) Statistics: All data are expressed as mean percent of control ± SEM. 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 p0.05). Data are expressed as IUGR percent of control ± SE. n=6 rat pups grouped from 3 litters. *p
