Domestic Animal Endocrinology 25 (2003) 61–67
Is poly(ADP-ribose) polymerase involved in
bovine placental retention?
M. Kankofer a,∗ , L. Guz b
a
Department of Biochemistry, Faculty of Veterinary Medicine, Agricultural University,
Lubartowska 58 a, Lublin 20-123, Poland
b Department of Fish Diseases and Biology, Faculty of Veterinary Medicine,
Agricultural University, Akademicka 12, Lublin 20-033, Poland
Abstract
Poly(ADP-ribose) polymerase (PARP) is the enzyme which utilises NAD to synthesise poly(ADPribose) polymers. This process appears in response to DNA lesions.
Oxidative stress, which might be involved in bovine placental retention, is the reason for oxidative
DNA injury.
In this mini-review, the relationship between PARP activity and bovine placental retention is
discussed. The results of our experiments on PARP activity in placental tissues showed that the
enzyme of 113 kDa and its cleavage products were present in retained as well as released fetal
membranes. Western blotting technique showed different intensities in the staining of bands which
might suggest different activities of the enzyme.
© 2003 Elsevier Science Inc. All rights reserved.
Keywords: Poly(ADP-ribose) polymerase (PARP); Cow; Retained placenta
1. Poly(ADP-ribosyl)ation and poly(ADP-ribose) polymerase
Poly(ADP-ribosyl)ation is the process which appears in response to DNA lesions (DNA
nicks and nitric base damage generating nicks), induced by different endo- or exogenous
agents such as oxidative injury [1]. The DNA-bound, catalytically activated poly(ADPribose) polymerase (PARP) utilizes NAD to synthesize poly(ADP-ribose) (pADPr) on a
variety of nuclear target proteins including histones. This transient, post-translational modification of nuclear proteins may cause the loss of their biological functions due to a massive
increase in the negative charge [2]. pADPr can modify the function of PARP itself via covalent or non-covalent interactions [3–5]. Automodified PARP loses its ability to bind DNA
strand breaks due to electrostatic repulsion between pADPr and DNA, exposing the DNA
∗
Corresponding author. Tel.: +48-81-747-4023; fax: +48-81-445-6006.
E-mail address: kankofer@agros.ar.lublin.pl (M. Kankofer).
0739-7240/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0739-7240(03)00045-6
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strand breaks to proteins involved in the repair process [2]. In this way the enzyme may
influence and regulate DNA repair processes [6–9].
The synthesis of pADPr is catalysed by PARP. PARP (EC 2.4.2.30) is a 113 kDa multifunctional enzymatic protein which seems to detect nick damage of DNA and fragmented
DNA. PARP activation leads to DNA repair and recovery after low levels of DNA damage
while cell death is observed at high levels of DNA damage [10,11]. PARP inhibitors prevent oxidant-induced necrotic death and force a switch from necrosis to apoptosis [12–14].
3-Aminobenzamide is a potent PARP inhibitor and is believed to interact with the catalytic
domain at the C-terminus of the enzyme [15].
The enzyme contains three following domains:
1. DNA binding domain (46 kDa) which contains two zinc fingers. This domain is responsible for the recognition and binding to both single and double strand breaks as well as
a bipartite nuclear localization signal.
2. Carboxyl terminal domain (55 kDa) that bears the NAD+ binding site and the catalytic
activity. It represents the most highly conserved part of the enzyme.
3. Automodification domain (16 kDa) is rich in glutamic acid residues which are sites
for the covalent binding of pADPr. This domain contains a breast cancer susceptibility
protein (BRCT), a putative site of interactions with other proteins.
The enzyme uses NAD+ as a substrate [11]. In case of PARP overactivation, it may
lead not only to depletion of nicotinamide coenzyme, indispensable for oxidoreductases
activity, but also, after release of a substantial amount of protons, to acidification [16] and
a decrease in the cellular ATP level [10]. Affar et al. [16] reported also that apart from
PARP activation, the inhibition of the ATP-dependent Na+ /H+ exchanger can be the cause
of acidification. He also showed that rapid acidification after high levels of DNA damage
can suppress apoptosis while permitting necrotic death.
pADPr is degraded by poly(ADP-ribose) glycohydrolase (PARG), a 110 kDa protein,
which is the principal enzyme responsible for the catabolism of pADPr [17]. There is
evidence, based on studies of kinetics of NAD+ consumption and polymer accumulation
following DNA damage, suggesting that PARP and PARG act sequentially and are closely
coordinated [18]. pADPr are quickly removed and degraded through internal and external
cleavage of ribose-ribosyl bonds via endo- and exoglicosidic modes of action, liberating
free (ADP-ribose) monomers and shorter polymers [19]. As in the case of PARP, PARG is
susceptible for cleavage by caspases during apoptosis [20].
PARP is efficiently cleaved and inactivated in programmed cell death into a 24 kDa
fragment containing the N-terminal DNA binding site and an 89 kDa peptide comprising
of the central automodification domain, the C-terminal NAD binding site and the catalytic
domain [21]. The site of PARP cleavage is located within the nuclear localization signal and
is highly conserved. Activation of cytosolic proteases and a fairly specific degradation of
proteins, including PARP, are important for apoptosis [10]. There is evidence that caspases
are responsible for PARP cleavage. The poly(ADP-ribosyl)ation of PARP may accelerate
its proteolysis [22]. During the execution phase of apoptosis, PARP appears to be among
the earliest death substrates to be cleaved [15].
A new surprising connection of PARP and the transcription factor NF-kappaB was described by Le Page et al. [23]. He demonstrated that the inhibition of PARP-1 impaired
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the ability of NF-kappaB to function as a transcriptional activator in the expression of the
inducible nitric oxide synthase (iNOS) gene. This transcription factor NF-kappaB, activated
by oxidative stress and chemical agents which damage DNA, is essential for many processes
including DNA damage and repair and in immune responses [24].
Experiments of Lautier et al. [25] showed a 20-fold stimulation of poly(ADP-ribose)
biosynthesis when induced by reactive oxygen species. The authors also stated that the
decrease in NAD levels, after exposure of cells to reactive oxygen species, was caused by
stimulation of poly(ADP-ribosyl)ation.
The observations in knock out mice showed no effects on cell cycle profile and the
ability to synthesize pADPr following treatment with genotoxic agents. It gave the idea that
another enzyme may be present. The enzyme designated sPARP-1 (short) is identical to
the catalytic domain of PARP-1 and shares most of the well documented features of the
carboxyl terminal part of PARP-1 [26]. It is localized in the nucleus and is a product of the
same gene. It is strand break-independent, but strongly stimulated by genotoxic treatments
such as alkylation, UV irradiation, suggesting the involvement of PARP-1 and sPARP-1 in
different types of DNA damage-inducible response pathways.
Worth mentioning is that PARP activity was not described in bovine placenta.
2. Retention of fetal membranes, oxidative stress and DNA damage
Retention of fetal membranes (RF), which is one of the most important bovine postpartum diseases, is supposed to be connected with alterations in prostaglandin [27,28] as
well as steroid hormone [29] metabolism. Some of the arachidonic acid cascade enzymes,
which are involved in the metabolism of PGs, are nicotinamide-dependent [30]. These coenzymes can be depleted when PARP is active and can cause the alterations in prostaglandin
metabolism. Therefore, it might be possible that poly(ADP-ribosyl)ation and prostaglandin
metabolism can be considered also in terms of the processes of proper and improper placental
release.
Previous experiments showed that although the level of PGF2␣ was lower in cases of
retained placenta, it was not connected with marked decrease in 15-hydroxyprostaglandin
dehydrogenase activity, which is responsible for PGF2␣ catabolism [31].
The activity of 9-keto prostaglandin reductase, which shifts reversibly PGE2 into PGF2␣
and uses NADPH as coenzyme, increased in retained placental tissues as compared to
properly released placenta [32].
Previous experiments on antioxidative/oxidative status that occurs in placental tissues
during releasing and retaining of bovine placenta have shown that not only activity of enzymatic but also the levels of non-enzymatic antioxidants were altered in retained placenta.
Although the mechanisms used by the enzymes for the neutralization of reactive oxygen
species are different, the efficiency of antioxidative system should be considered as joint
action of all four enzymes (glutathione peroxidase—GSH-Px, glutathione transferase—
GSH-Tr, catalase—CAT and superoxide dismutase—SOD). There is evidence that GSH-Px
activity was significantly higher in retained placenta. The opposite results were noticed for
GSH-Tr activity. The activity of CAT and SOD differed with respect to type of placental
tissues and mode of delivery between retained and released fetal membranes [33].
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The antioxidant biomarkers, both non-enzymatic and enzymatic, measure the capacity
to react to oxidant conditions but give only scant information on damage undergone by the
cell, tissue or organism. Such damage is actually reflected by the biomolecules and a series
of oxidative lesions that have been proposed as both invasive and non-invasive biomarkers
for lipids, proteins and nucleic acids.
Lipid peroxidation processes in placental tissues, measured by means of the level of
TBA-reactive substances, conjugated dienes and hydroperoxides, showed increased intensity in cases of retained placenta as compared to healthy animals [34]. Placental protein
peroxidation, reflected by the level of SH-groups as well as bityrosine and formylokinurenine, was also altered in cows affected by retained placenta [35]. 8-iso-Prostaglandin F2␣
(8-iso-PGF2␣ ) is considered as a marker of oxidative tissue damage. The concentrations
of free and total 8-iso-PGF2␣ in bovine placental tissues were higher in retained than in
released fetal membranes [36].
The concentration of 8-hydroxy-deoxy-guanosine (8-OH-dG), which is a marker of oxidative DNA damage, was changed in retained placenta cases in comparison to healthy animals [37]. This may suggest the imbalance between production and neutralization of reactive
oxygen species as well as any oxidative DNA damage resulting from oxidative/antioxidative
imbalance in retained placental tissues. The possible strand breaks requiring repair mechanisms activity and PARP involvement may occur in such cases. The results of the above
mentioned experiments gave additional evidence to support the search for the relationship
between PARP activity and the retention of bovine placenta.
Partial examination of bovine placental proteins by means of SDS–PAGE and zymography of metalloproteinases showed differences in the number of fractions as well as differences in metalloproteinases activities in cases of retained and released placenta [38,39].
This may suggest the alterations in proteolytic processes in bovine placenta during improper
placental release.
The importance of PARP for DNA repair process, the consequences for metabolism
and the possible indirect relationship with proper and improper placental release raise two
questions: is there any relationship between disturbances in prostaglandins metabolism and
poly(ADP-ribosyl)ation processes? and is PARP involved in bovine placental retention?
For answering this question we tried to detect the presence of PARP in bovine placenta by
use of bovine anti-PARP antibody and Western blotting technique. We expected to describe
eventually existing differences between retained and released placenta with respect to time
and mode of delivery.
For this study we collected placentomes immediately after spontaneous delivery of calves
at term (282–288 days of pregnancy) or after extraction of a calves during caesarian section before term (272–277 days of pregnancy) and at term from cows divided into the six
following groups:
A
B
C
D
E
F
Caesarian section before term with RF
Caesarian section before term without RF
Spontaneous delivery at term with RF
Spontaneous delivery at term without RF
Caesarian section at term with RF
Caesarian section at term without RF
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65
Human placental samples were included into analysis as the control of experimental
procedure.
3. Poly(ADP-ribose) polymerase in bovine placenta
Placental samples were analysed in comparison to bovine PARP standard. The bands
located at the same position as the enzyme standard were defined as PARP and were present
in all examined tissues. Human placenta showed only one band.
The cleavage products of the enzyme were present in all bovine samples. Their number
was different with respect to time and mode of delivery but three of bands were present
in all samples and referred to the molecular weights of approximately 97, 89 and 65 kDa,
respectively.
Although Western blotting is only a semi-quantitative method, it was possible to notice
differences in the intensity of the staining of bands suggesting indirectly the differences
in enzyme amount. Generally, the intensity was lower in fetal than in maternal parts of
placenta. The presence of non-specific staining was checked with second antibody—no
bands were detected.
4. Conclusions
The study confirmed that PARP molecules as well as their cleavage products are present
in bovine retained as well as properly released placenta but its patterns are different with
respect to kind of tissue, time and mode of delivery.
In conclusion, this preliminary study showed that PARP activity is detectable in bovine
as well as human placenta. Further experiments are necessary to describe in detail the kind
of relationship between PARP and the process of releasing and retaining bovine placenta.
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