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Maternal protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. We tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for observed gene expression changes.
We fed pregnant FVB/NJ mice from day post coitum 10.5 (DPC10.5) to DPC17.5, an isocaloric diet containing 50% less protein than normal chow. We used the Affymetrix Mouse 430A_2.0 array to measure gene expression changes in the placenta. We functionally annotated the regulated genes, and examined over-represented functional categories and performed pathway analysis. For selected genes, we confirmed the microarray results by use of qPCR.
We observed 244 probe sets, corresponding to 235 genes, regulated by protein restriction (p < 0.001), with ninety-one genes being up-regulated, and 153 down-regulated. Up-regulated genes included those involved in the p53 pathway, apoptosis, negative regulators of cell growth, negative regulators of cell metabolism and genes related to epigenetic control. Down-regulated genes included those involved in nucleotide metabolism.
Microarray analysis has allowed us to describe the genetic response to maternal protein deprivation in the mouse placenta. We observed that negative regulators of cell growth and metabolism in conjunction with genes involved in epigenesis were up-regulated, suggesting that protein deprivation may contribute to growth restriction and long-term epigenetic changes in stressed tissues and organs. The challenge will be to understand the cellular and molecular mechanisms of these gene expression responses.
Successful placental development is crucial for optimal growth, maturation, and survival of the embryo/fetus. The placenta, a fetomaternal organ joining mother and offspring during pregnancy in mammals, serves as an endocrine organ in the “maternal-placental-fetal” complex, in addition to its role in the exchange of respiratory gases, exchange of nutrients, an immunologic barrier, and other functions. As has been recognized for many years, deviation in the normal gene expression pattern may lead to altered placental phenotype, as well as a modified phenotype of the conceptus. Previously, we have examined developmental gene expression patterns in the developing murine placenta, and reported numerous placental genes are up- or down-regulated to a significant degree, and that specific functional groups of genes are regulated at the different developmental ages  and with maternal hypoxia . However, a number of stressors during gestation can lead to altered placental and fetal growth and development. One of the important stressors is maternal malnutrition, which during pregnancy may have deleterious consequences for the progeny. Historical data point to these effects in human populations. For instance, during WWII, the people of both Holland and Russia were subjected to severe dietary restrictions due to interdiction of food supplies by the German army . The children born under these conditions not only were small for gestational age, but they also developed significant health problems later in life [4, 5]. Several major sequelae have been described including those of the cardiovascular system, type II diabetes, and mood and personality disorders .
Nutritional deprivation influences not only placental growth and morphology, but also alters the hormonal milieu of the developing fetus, and causes subsequent cardiovascular, hormonal and behavioral consequences in the adult [7, 8]. These epidemiologic observations have led to speculation regarding the mechanism of changes in the placenta, and their effects on the developing fetus. The observations made in human subjects have been confirmed in several animal models. An important question, is the extent to which these observed effects result from an overall caloric restriction, as opposed to a qualitative component in the diet that triggers the responses. Evidence from several animal models points to protein deprivation as a major factor in these defects . For example, in the rat the growth reducing effects of a low calorie diet can only be reversed by a dietary increase in protein levels; vitamin supplements, and caloric increases, while carbohydrates failed to reverse the observed effects . Other studies have revealed that dietary amino acid balance is a key mediator of some of the cardiovascular and metabolic effects observed in response to protein deprivation . However, no studies have examined the global changes in the placental gene expression with maternal protein restriction. We thus tested the hypothesis that, by the use of microarray technology, we could identify specific genes and cellular pathways in the developing placenta that are responsive to maternal protein deprivation, and propose a potential mechanism for phenotypic changes that have been observed.
Eight-week old FVB/NJ male and female mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and housed at the Animal Research Facility, Loma Linda University, Loma Linda, CA under conditions of 14 h light, 10 h darkness, ambient temperature of 20°C, and relative humidity of 30-60%. All experimental protocols were in compliance with the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Animals, and were approved by the Institutional Animal Care and Use Committee of Loma Linda University.
Mice were bred by overnight monogamous pairing of virgin females with a male, the male was removed in the morning, and that day was considered 0.5 day post coitum (0.5 dpc). Mice were weighed daily and pregnancy was confirmed by examining vaginal plugs on day 0.5 and weight gain by 10.5 dpc. At 17.5 dpc the pregnant females were euthanized. The uterus was removed rapidly and placed in a petri dish containing RNA Later solution (Ambion, Austin, TX). Entire placentae were isolated under a dissection microscope and maternal deciduas and endometrial tissues were removed. The isolated and cleaned placentae were snap frozen in liquid nitrogen, and stored at -80 °C for later analysis. RNA was isolated from the entire placentae using the TRIZOL reagent kit (Life Technologies, Rockville, MD), and was stored at -80 °C until further analysis. We confirmed the developmental stages of the embryos by visual inspection according to a modified Theiler staging system . Details of the staging system are available online at http://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.html.
The mice were initially fed a normal mouse chow (20% protein content by weight, diet # TD91352). At 0.5 dpc the pregnant mice were divided into two groups, one group (n=3) were continued on normal mouse chow (control) and another group (n=3) were switched to a custom protein diet (10% protein by weight, diet # TD92208) (Teklad, Indianapolis, IA). The 50% protein deprivation was continued from 10.5 dpc to 17.5 dpc (total 7 days.). Studies in several species suggest that severe protein reduction leads to fetal programming of adulthood diseases in the offspring such as hypertension, schizophrenia, behavioral abnormalities etc [12-14]. Studies also indicate that maternal protein deprivation causes altered gene expression in different organs during different time points in the offspring lifespan and lead to these disorders. However, changes in the placental gene expression with this degree of protein deprivation are unknown, and were the focus of present study. The timing of the protein restriction was chosen in order to avoid interfering with fertilization and implantation of the embryo. We also sought to focus on the mature placenta, as in the mouse the allantoic fusion does not occur until 8 dpc and the placenta is not fully formed until 10.5 dpc. The diets were designed to ensure that mice would receive the same amount of calories and nutrients, but a reduced amount of protein. Maternal food intake and maternal weights were measured daily in order to assure isocaloric food intake.
The RNA was processed for use on the Affymetrix Mouse 430A_2.0 array (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Briefly, 5 μg of total RNA was reverse transcribed to double stranded cDNA (Superscript II kit, Life Technologies). The double stranded cDNA was used in an in-vitro transcription reaction to generate biotynilated cRNA probes. The cRNA probes were purified, fragmented, and hybridized to the Affymetrix chip. Washes and staining were performed in an Affymetrix Gene Chip Fluidics station 400. The Affymetrix arrays were scanned using a Gene Array Scanner (Hewlett Packard, Austin, TX), and processed at the Microarray Facility, University of California Irvine, (Irvine, CA). The hybridizations were performed in triplicate for control and protein restricted conditions. All the placentas obtained from one mouse were pooled, and the total RNA isolated was considered as one RNA sample. Six such RNA samples, three each from protein restricted and control mice dams were used for microarray hybridization. Analyses were performed using BRB ArrayTools developed by Dr. Richard Simon and Amy Peng Lam (http://linus.nci.nih.gov/BRBArrayTools.html). We analyzed the data using the random variance method at a significance of p < 0.001 . The genes were assigned to functional classes based on the GO database (http://www.geneontology.org/GO.annotation.html ), and significantly over-represented GO categories in the gene sets were analyzed using the Gene Ontology Tree Machine (http://genereg.ornl.gov/gotm/). We also manually functionally annotated genes using Pubmed searches.
In an effort to validate the results of the microarray analysis, we chose several genes that were shown to be regulated by gestational protein restriction for analysis using real time PCR. RNA was isolated from mice different than the ones used for the microarray (n=5). Exon spanning primers were designed using the Universal Probe Library Assay Design Center (Roche, Indianapolis, IN). The primers were synthesized by Integrated DNA technologies (Coralville, CA). The primer sequences selected are shown in Table 1. Total RNA (1 μg per reaction) was reverse transcribed using random hexamers and the SuperScript II reverse transcriptase kit (Invitrogen, Carlsbad, CA). Relative expression was normalized to 18S RNA and fold changes were calculated using the ΔΔCt method. Samples were analyzed on the Roche LightCycler 1.5 (Roche, Indianapolis, IN).
In response to protein deprivation the placental weights remained unchanged while pup weights were significantly reduced (p< 0.05) as shown in Figure 1.
To evaluate the genetic response to protein deprivation we used the Affymetrix Mouse 430A_2.0 oligonucleotide array to compare gene expression levels between normal placentae at 17.5 dpc, and those from pregnancies in which the mothers were exposed to seven days of protein deprivation. Of 22,690 genes examined by on the microarray, using the random variance model , we observed 244 probe sets, corresponding to 235 genes, that were influenced by protein restriction (p < 0.001; some probe sets hybridize to different areas of the same gene. This is a design of the Affymetrix chip which serves as an internal control). As a consequence of maternal protein deprivation, 91 of these probe sets were up-regulated, while 153 were down-regulated. As noted in Table 2, among the gene ontology classes most over-represented in the up-regulated group, were regulators of apoptosis (Bcl2-like 2, p53, endophilin, Fas-activated serine/threonine kinase), negative regulators of cell growth (farnesyltransferase CAAX box beta, cadherin 5, CCAAT/enhancer binding protein (C/EBP) alpha, inositol polyphosphate-5-phosphatase D, p53), and negative regulators of cellular metabolism (nuclear receptor co-repressor 2, histone deacetylase 7A, SPEN homolog, transcriptional regulator). A number of genes involved in the p53 pathway were up-regulated. The genes rai17 and hipk2 were up-regulated, both of which are activators of p53. Rai17 induces the expression of p53 and is a cofactor of p53-mediated gene regulation . Hipk2 is a kinase that phosphorylates Serine 46 on the p53 protein and activates its pro-apoptotic effects . We also noted up-regulation of the gene jmy, a co-factor of p53. Jmy is up-regulated in response to DNA damage and binds to p53 in a protein complex that enhances its activity . In addition we noted the up-regulation of two genes, Cebpa and Inpp5d, which are induced by p53. Cebpa is a leucine zipper transcription factor involved in the terminal differentiation of several cell types. It is up-regulated in response to UV radiation, and serves as a DNA damage induced G1 checkpoint in the cell . Inpp5d is a phosphatase involved in inositol-mediated signaling, and has a potential anti-survival effect on the cell. It has been identified as a p53 transcriptional target .
Overall, the present study shows that the major pathways up-regulated with maternal protein deprivation are the p53 pathway, regulators of apoptosis, negative regulators of cell growth and metabolism and certain epigenetic regulators such as histone deacetylases, methionine adenosyl-transferase II alpha. In contrast, as noted in Table 3, among down-regulated genes, particularly striking were those genes related to nucleotide metabolism, and certain epigenetic regulators such as histone 2, Mcm6 and telomeric repeat binding factor 1. The major placental gene pathways up- or down- regulated by maternal protein deprivation. We verified the expression of Cebpa, p53, Rai17, Jmy, Hipk2 and Inpp5d by the use of real-time qPCR (Table 4). The expression of these genes were altered to similar extent as observed during our Microarray analysis.
Microarray analysis is an invaluable tool to examine genetic mechanisms of cancer growth, development, responses to stress and, other processes. Numerous studies have been conducted utilizing this powerful tool of cDNA and oligo microarray, to elucidate the gene expression patterns in various physiological and pathological conditions. Previously, by the use of microarray analysis we have reported the changes in placental gene expression with fetal development  and acute maternal hypoxia . In the present study we report the alterations in the placental gene expression with the maternal protein deprivation. Maternal protein restriction may play an important role in several disorders. Epidemiologic data in humans and studies in laboratory animals provide useful lessons on the effects of caloric restriction/malnutrition on fetal development and disease prevalence in adulthood [4, 5, 12, 21-26]. Those fetuses exposed to maternal caloric restriction in mid-gestation had a much greater incidence of bronchitis and other pulmonary disease  and renal disease as evidenced by microalbuminuria . Females conceived during the famine also had a much higher prevalence of obesity as adults . The cellular/molecular mechanisms of these in utero “programming” effects are unknown.
Studies in ruminants also have demonstrated that under-nutrition can have profound consequences for the fetus. In sheep, restricted maternal nutrition in early to mid-gestation was associated with an increase in placental weight, an increase in crown-rump length, and lower fetal to placental weight ratios . Maternal under-nutrition also caused an alteration of cardiovascular homeostatic regulation by the renin-angiotensin system, and exposed the lambs to higher levels of glucocorticoids . These hormonal effects also were associated with hypertension in the lambs . Protein restriction in bovines also caused an increase in placental weight and an altered placental morphology .
Studies in rats have shown similar effects. Maternal protein restriction in rats triggers hypertension in the pups in adulthood. These effects appear to be mediated through a suppression of the renin-angiotensin system in the pups . An alteration of placental glucocorticoid metabolism also was observed in placentae of rats fed a protein restricted diet. The activity of 11β-hydoxysteroid dehydrogenase, an enzyme present in the placenta, which normally protects the pups from maternal glucocorticoid excess, was reduced in protein restricted rats . Another hormonal alteration in nutritionally deprived rats was an increase in somatostatin expression in the periventricular nucleus of the pups. This led to much lower levels of growth hormone, and had deleterious effects on the growth of the pups post-partum . Fetal undernourishment also led to neuronal sequelae. The facial motor nucleus in pups was under-developed, and led to a functional decrease in the ability of pups to suckle and chew.
A study somewhat similar to ours was conducted in the rat, revealing an increase in genes involved in apoptosis, and p53 . A direct comparison of the results between the studies is difficult, however, because of intra-species differences and the different timing of the protein restriction. Nonetheless, it is of interest to note similar themes emerging. The earliest large-scale studies on caloric restriction were related to the slowdown of aging process in mice skeletal muscles . In the present study, maternal protein restriction showed up-regulation of the genes responsible for the negative regulation of cell growth and metabolism in the placenta. It is of interest to observe that caloric or protein restriction in different tissues and at different ages effect similar groups of gene responsible for the decrease in cellular growth and metabolism. However, further studies are needed to examine the biologic mechanisms by which protein/caloric restriction produces up-regulation of this particular pathway.
In a previous study, we used the Affymetrix oligonucleotide array to define developmental changes in gene expression from 10.5 dpc to 17.5 dpc in the mouse placenta . In addition, we have reported on significant changes in mouse placental gene expression in response to maternal hypoxia for 48 hours, from 15.5 dpc to 17.5 dpc . In the present study of placental gene regulation in response to maternal dietary protein restriction, we demonstrate a profound down-regulation of cell growth and proliferation and an up-regulation of genes coding for apoptotic proteins (Tables (Tables22 and and3).3). Of particular interest, p53 along with rai17, Hipk2, jmy, Cebpa and Inpp5d (proteins that either activate, or are cofactors of, or are induced by p53), an important regulator of cell growth and proliferation were up-regulated. This pathway serves as a G1 checkpoint, and arrests growth and/or induces apoptosis in response to cellular damage. Mutations in the p53 gene have been implicated in a number of cancers and other pathological processes . Hipk2, an upstream regulator of p53, activates its transcriptional activity and pro-apoptotic activities through phosporylation at Ser 46 . Cebpa, a transcription factor induced by p53, mediates some of the downstream effects of p53 activation . Several studies on the effects of nutritional deprivation have demonstrated that the p53 pathway is a crucial mediator of the observed biological effects. Mice deficient in p53 (p53 -/-) are more susceptible to cancer, but caloric restriction partially reversed that effect .
The present study has demonstrated a significant up-regulation of genes responsible for apoptosis regulation such as Bcl2-like 2, p53, endophilin, Fas-activated serine/threonine kinase. Apoptosis and its associated regulatory mechanisms are physiological events crucial to the maintenance of homeostasis in the placenta and other organs. Imbalance of these processes may cause various pathological conditions, may compromise placental function and, consequently, pregnancy success. Increased apoptosis occurs in the placentas of pregnant women with several developmental abnormalities, while increased Bcl-2 expression is generally associated with pregnancy-associated tumors and decreased expression is associated with placentas of the diabetic women . Another important finding of the study was upregulation of Fas-activated serine/threonine phosphoprotein (FAST). FAST is a survival protein, bound to the outer mitochondrial membrane and mediates alternative and constitutive splicing, which may affect the expression of several other genes .
A potentially important finding of the present study, is that protein deprivation altered the expression of several genes involved in DNA methylation and histone acetylation, which are involved in epigenetic regulation of gene expression. The expression levels of histone deacetylase 7A and methionine adenosyltransferase II alpha, were significantly elevated. Histone actetylation triggers changes in chromatin structure, and regulates transcriptional availability of genes. In turn, histone deacetylation increases histone affinity for DNA, thereby repressing transcription . Methionine adenosyltransferase II alpha synthesizes AdoMet, the direct precursor used for DNA methylation by methyltransferases. Histone 2 (h3c2) is down-regulated, along with Mcm6 and telomeric repeat binding factor 1. These proteins contribute to DNA replication, stability, and structure [42, 43]. Recent studies in the human, have demonstrated in small for gestational age pregnancies an altered DNA methylation pattern in imprinted regions of the genome, and that imprinted genes are expressed in an unbalanced manner in pregnancies affected by intrauterine growth retardation [44, 45].
In several animal models, in addition to potential deleterious effects, a positive aspect of nutritional deprivation in the adult is that of prolonged lifespan and reduced cancer rates. A proposed mechanism for these benefits is that nutritional restriction in the absence of malnutrition inhibits cellular proliferation and induces apoptosis. This effect has been shown in mice lacking p53, in which -/- and +/- mutants have lowered spontaneous cancer rates when fed a complete, but calorically reduced diet . In the adult and aging animal, nutritional restriction has been shown to have beneficial effects that increased life span . In contrast, a different picture has emerged in the fetus. As discussed above, caloric and protein deprivation have been shown to trigger fetal programming of adult disease, and lead to an increased prevalence of metabolic disorders in adulthood. In the developing fetus, numerous animal studies have shown the negative long-term effects of caloric and protein deprivation on the cardiovascular, renal, nervous system and metabolism (for review see ). Both fetal and placental growths are essential for the long-term well-being of the individual. Thus, one would anticipate that profound inhibition of cellular growth at key time points during development would have grave long-term consequences for the embryo/fetus. This suggests that the timing of the treatment is a key determinant in the effect on the organism.
The present data support the hypothesis that maternal protein restriction triggers an up-regulation of apoptosis-related genes, an increase in the p53 pathway, a change in epigenetic modulators, and an overall down-regulation of cellular proliferation and growth associated genes. The upregulation of inhibitory transcription factors, and other key negative cellular regulators altered by protein deprivation, offers a picture of profound and global down-regulation of the entire cellular proliferative machinery. These results suggest numerous avenues for future research, and raise a number of fundamental questions regarding energy/protein balance and cellular growth. A critical challenge will be to understand the cellular and molecular mechanisms of these epigenetic responses.
We thank Brenda Kreutzer for her assistance in the preparation of this manuscript and JD Heck of the DNA Array Core, University of California Irvine, Irvine, CA for technical assistance. This work was supported, in part by USPHS grant HD-03807 to LDL.