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Fetal Protein Deprivation and Translational Responses in the Placenta
CB Duckvworth, DA Novak, MJ Beveridge, M Rahmani
The implications of altering fetal nutrition are thought to be profound, as changes that occur in the developing
fetus may lead to pathologic states later in life. Preliminary studies using pregnant rat dams fed a low protein
diet have indicated an increased amount of mRNA in the corresponding placentas. However, the amount of
amino acid transport was diminished, suggesting a block in translation. The present study sought to investigate
the mechanisms by which the initiation of translation is controlled. Significant to the proposed pathways are
the protein kinases GCN2 and mTOR; the translation initiation factors eIF-2a, 4EBP1 and eIF-4E; the
transcription factor ATF4; and 4F2HC, the heavy chain subunit of several amino acid transporters.
Immunoblot analyses were performed using the isolated trophoblasts and placental homogenates collected
from timed-pregnant rat dams fed 8% protein and 19% protein diets. Results demonstrated a 2.5-fold increase in
the amount of eIF-4E in placental homogenates from the low protein group as compared to the control group.
In terms of phosphorylation, the relative amount of p-eIF-4E as a fraction was decreased in the low protein group
of placental homogenates. Additionally, 4F2HC in placental homogenates and ATF4 in isolated trophoblasts were
both significantly higher in the low protein groups. These findings are consistent with the hypothesized roles
the factors were thought to play in the regulation of translation and protein synthesis. p-GCN2
unexpectedly decreased in the low protein group of the placental homogenates, which may be explained with
further investigation. This will include assaying total GCN2, a goal that is facilitated by the very recent availability of
a commercial antibody against total GCN2.
Previous research has shown that the offspring of mothers fed a low protein or low calorie diet is smaller for
than those fed normally. Suboptimal maternal nutrition is an important cause of intrauterine growth
retardation (IUGR), a condition that occurs in both humans and rodent models.1-3 In rodents this can be produced
via nutrient restriction, specifically by the ingestion of a low protein diet during pregnancy.4 IUGR is thought
to profoundly impact both the immediate and long-term health of affected individuals.5,6 During the final 75%
of gestation in humans and the final 50% of gestation in rats, the placenta is the primary channel of
nutrient exchange. The mechanism by which this vital organ reacts to starvation is not yet clear, although it has
been suggested that there is some manner in which the mother's body down-regulates the nutrients that are
passed through the placenta to the fetus. David Barker and his colleagues were the first to postulate the idea of
fetal "programming." They proposed that during critical periods of rapid fetal growth, insufficient nutrition can
cause permanent physiological changes. These changes, which attempt to counteract poor nutrition in the
short-term, are thought to lead to the development of disease in the long-term. Their data documented some
of these diseases as including cardiovascular disease, glucose intolerance, hypertension and type 2 diabetes
mellitus, among others.7
In preliminary studies, control and experimental groups of pregnant rats were established. They were pair-fed,
with the control group receiving a 19% protein diet and the experimental group receiving an isocaloric 8%
protein diet for 20 days. The placentas of each group were collected for RNA studies and western blot
analyses. Results indicated that there was an increased amount of steady-state mRNA for the sodium-coupled
neutral amino acid transporters SNAT1 and SNAT2 in the low protein group. However, the amount of amino
acid transport decreased, which suggested a block in translation. The present study sought to further
investigate poor fetal nutrition at the placental level, as well as examine the effects arising at the cellular
level. Trophoblast cells, which are present in the placenta, were examined in addition to freshly prepared
It was hypothesized that the aforementioned fetal alterations are primarily due to changes in the initiation
of translation within the placenta. Although many proteins are affected in this pathway (Figure 1), several of the
key factors are modified primarily by phosphorylation and dephosphorylation. In particular, it is known that
amino acid starvation increases the activity of the protein kinase GCN2. This, in turn, dephosphorylates the elF-
2a eukaryoticc initiation factor) complex, thus deactivating it. This deactivation proceeds down a chain of
reactions, eventually turning off translation. In another process, the protein kinase mTOR (mammalian target
of rapamycin) phosphorylates the translation initiation factor 4EBP1 (4E binding protein). This removes 4EBP1 from
a complex with translation initiation factors including eIF-4E. By removing the binding protein, translation is free
to proceed. These positive and negative influences on translation can help elucidate the mechanism by
which environmental conditions affect protein synthesis and growth.
Gr h factorsnitogens ents
4I IJ 1 4/
Figure 1. Translation initiation as affected by growth factors, nutrients and hormones
Another key component in this pathway is the activating transcription factor ATF4. As previously stated,
preliminary data showed an increased amount of RNA transcribed in placentas from the low protein group. It
is therefore probable that a concurrent increase in the translation of ATF4 occurs in this group. Also essential
for growth is 4F2HC, a subunit of several amino acid transporters. By controlling the expression of this
particular protein, the cell is able to mitigate the transport of several amino acid substrates simultaneously.
The availability of nutrients and growth factors controls translation in a complex way, often with multiple input
points and pathways. It is hypothesized that many of the changes investigated in this study can be generalized
to other important proteins and can provide valuable insight into the placenta's response to malnutrition.
Timed-pregnant Sprague-Dawley rats were obtained from Harlan on day 5 of gestation. They were weight
matched into low protein and control groups and pair-fed diets consisting of 8% and 19% protein, respectively.
The diets were obtained from Purina Inc, and made isocaloric through the addition of sucrose. On day 20, the
animals were anaesthetized with pentobarbital, and placentas were collected for either whole tissue
homogenization or trophoblast isolation.
Placental Homogenate Preparation
Placentas from 12 pregnant rat dams were used for the preparation of placental homogenates. The placentas
were collected over two experimental replications, with a total of 6 placentas from each the control and low
protein groups. The placentas were taken from consistent locations within the uterus, after which they
were homogenized and placed in a buffer containing protease inhibitors.
Isolation of Trophoblasts
Placentas from pregnant rat dams were used to obtain 4 parallel (pair-fed control/ low protein) trophoblast
isolations. 3 rats were used per preparation, using all of the placentas collected. Once harvested, the placentas
were placed in a sterile beaker with 50 ml of Hanks Buffered Saline Solution (HBSS) at 40C. They were then
minced with a sterile razor blade and placed in a sterile 125 ml flask with 50 ml of dissociation media.
The dissociation media consisted of: 10% HBSS, 0.1% collagenase, 0.1% hyaluronidase, 0.01% DNase, 1% FBS.
The mixture was loosely covered with foil and incubated in a 370C shaking water bath for 1 hour. The tissue was
next centrifuged for 5 minutes at 150 xg and 40C, and the resulting pellet was resuspended in 20 ml HBSS.
Using successive layers of sterile nylon mesh (250 pm, 100 pm, 50 pm), the tissue was filtered and rinsed
with HBSS. The cells were layered onto Percoll (1 ml cells / 5 ml Percoll) and centrifuged for 15 minutes at 700
xg and 40C. Trophoblasts were collected in the second layer of cells, washed twice with HBSS and resuspended
in appropriate media. Cell counts were performed to measure viability, which was between 85-90%.
Protein Extraction and Western Blotting
Protein content of the placental homogenates and isolated trophoblast preparations was measured by means of
a Lowry protein assay. Using standard techniques, protein aliquots (50 pg/ lane) were separated on either 7.5%
or 15% SDS-PAGE and electrotransferred to a 0.45 pm nitrocellulose membrane. Immunoblots were prepared
using antibodies which were specific to the phosphorylated and dephosphorylated forms of important components
in the translation initiation pathway shown in Figure 1. They included the following primary antibodies: eIF-4E
(Santa Cruz Biotech), phosphor(Ser 209)-eIF-4E (Cell Signaling), 4E-BP1 (Lab Vision Corp), phosphor(Ser 65,
Thr 70)-4E-BP1(Santa Cruz Biotech), eIF-2a (Cell Signaling), phosphor(Ser 51)-eIF-2a (Cell Signaling), phospho
(Thr 898)-GCN2 (Cell Signaling), mTOR (Santa Cruz Biotech), ATF4 (Santa Cruz Biotech), and 4F2HC (Santa
Cruz Biotech). As 4E-BP1, p-4E-BP1, eIF-2a, p-eIF-2a have low molecular weights, these antibodies were used
on blots prepared from 15% acrylamide gels. The remaining antibodies were used on blots prepared from
7.5% acrylamide gels. The secondary antibodies used were donkey anti-goat (DAG) for 4F2HC and goat anti-
rabbit (GARb) for all other primary antibodies. Conditions for detection of each were optimized in the
laboratory. Immunoreactive bands were detected using enhanced chemiluminescence and x-ray film.
Densitometry Measurement and Statistical Analyses
Blots were scanned and analyzed using Scion Image (2000 Scion Corporation) to measure respective
densitometries. Statistical analyses were performed using two-tailed, paired t-tests in the trophoblasts and
two-tailed, unpaired t-tests in the placental homogenates.
Immunoblot analyses revealed a 2.5-fold increase in the amount of eIF-4E in placental homogenates from the
low protein group as compared to the control group (Figures 2-3). Though the total amount of p-eIF-4E did
not significantly differ between low protein and control groups, the relative amount as a fraction was decreased in
the low protein group of placental homogenates. There were similar qualitative findings in the isolated
trophoblast preparations. 4F2HC in placental homogenates and ATF4 in isolated trophoblasts were both
significantly higher in the low protein groups. In the placental homogenates, p-GCN2 decreased in the low
protein group, which was unexpected. No significant differences were found between control and low protein
groups in trophoblasts and placental homogenates for the following: 4E-BP1, p-4E-BP1, eIF-2a, p-eIF-2a or mTOR.
19% protien 8% protein 19% protein 8% protein
Figure 2. Immunoblot analysis of placental homogenates (n = 12) from female pregnant rats fed
the described diets for 20 days
- Placental homogenates
r--- Isolated trophoblasts
pLP/pC 4F2HC ATF4 pGCN2
Figure 3. Immunoblot analysis of placental homogenates (n = 6) and isolated trophoblast preparations
(n = 4). Antibodies utilized are as defined in Table 1. Shown are arbitrary densitometric Units (+
SE) representing the low protein group normalized to the control group (= 1). Significance determined
by paired t-test (2-tail) in the isolated trophoblast groups; unpaired t-test (2 tailed) in the case
of placental homogenates. * signifies p < 0.05; ** signifies p < 0.005. pLP/pC signifies the ratio
of phosphorylated eIF4E in the low protein group to that in the control group; lower values signify
less relative phosphorylation in the low-protein group.
The data for eIF-4E showed the largest difference between relative amounts of phosphorylated and
dephosphorylated forms in placental homogenates. That the placentas from the low protein group contained
relatively more eIF-4E than p-eIF-4E, as a fraction of the total, intuitively suggests diminished translation.
This conclusion is further supported by the similar trend seen in the trophoblast preparations. Overall, this is
perhaps a result of a relative decrease in phosphorylation by the upstream protein kinases MNK1 and 2. This is
a possibility that can be further examined through the use of MNK knockout mice.
The results for 4F2HC and ATF4 confirm the roles these components were thought to play in the initiation
of translation and their interactions with each other. The increased expression of 4F2HC in the low protein
placental homogenates reflects an attempt to up-regulate the activities of several amino acid transporters.
The proteins in which 4F2HC is a subunit transport a significant proportion of amino acids moving into and out of
the cell. Consequently, the cell can rapidly alter the transport of amino acids in response to environmental
conditions via regulation of the expression of 4F2HC. Although in vivo the repercussions of protein deprivation
are unclear, in vitro it is associated with the up-regulation of 4F2HC mRNA, as has been seen in SNAT1
and SNAT29,10. In 4F2HC, transcriptional up-regulation occurs via the GCN2 pathway and requires the presence
of ATF 4, which is consistent with the observed increase of ATF4. In mammalian cells, the translation of ATF4 is
aided by GCN2 via EIF2a. This additionally leads to the induction of a variety of starvation induced
genes. Considering this, it is surprising that the amount of p-GCN2 was diminished in the low protein group
of placental homogenates, despite an increase in ATF4. An explanation will require further investigation,
including assaying total GCN2 and assessing GCN2 in isolated trophoblasts. Of great interest to this goal is the
very recent availability of a commercial antibody against total GCN2 (Cell Signaling Technologies). This, along
with immunoprecipitation western analysis, will facilitate future studies.
The research thus far, and that found in relevant literature, has focused on two main pathways of
translation initiation: those involving eIF-4E and its modifiers, and those pertaining to eIF-2a. The reported
changes in the placenta were found in the absence of alterations in the amount eIF-2a or in its level
of phosphorylation, suggesting that there may be another means by which amino acid deprivation enhances
the expression of ATF 4. It has recently been shown that, in the livers of fasting rats, there is a decrease in
the phosphorylation of eIF-2a11. This finding is counter-intuitive to expectations based on previous studies and
shows that much is still not understood in this pathway. Additionally surprising was that the levels of 4E-BP1
were not significantly different between the control and low-protein groups. It may be possible that some of the
other 4E-BP binding proteins were, in fact, present but were not detected by the antisera employed. The use
other antigens may be required to fully illuminate some of the complex processes that occur in response
to suboptimal fetal nutrition. No comparable work completed in the placenta is available to compare with the
present study. Although the amount of non-placental in vivo work is also limited, it is clear that the
intrinsic responses of organs to stress may be different from that observed in isolated cell models. Exploration
of these changes within the placenta can yield valuable insight through the use of mice carrying GCN2 null and elF-
2a "always on" mutations.
Future studies will seek to confirm the relationship of the alterations in translational control in vivo by using
the aforementioned knockout mouse models to determine the effects of a low-protein maternal diet during
gestation. Fetal and placental growth, mortality, protein synthesis and potential compensatory pathways will
be examined. This will further define the function of selected effectors, their pathways and the physiology of
the placental response in vivo to protein deprivation.
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