Mol. Hum. Reprod. Advance Access originally published online on September 2, 2006
Molecular Human Reproduction 2006 12(11):677-685; doi:10.1093/molehr/gal071
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Nitration of p38 MAPK in the placenta: association of nitration with reduced catalytic activity of p38 MAPK in pre-eclampsia
1Department of Obstetrics and Gynecology and 2Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, PO Box 670526, Cincinnati, OH 45267-0526, USA. E-mail: rose.webster{at}uc.edu
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Abstract |
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Peroxynitrite, a potent pro-oxidant formed from the interaction of superoxide and nitric oxide, has been widely reported to be nitrating tyrosine residues in proteins resulting in the formation of nitrotyrosine. Biological nitration of tyrosine, a footprint of oxidative injury, has been found to occur in various pathological states including pre-eclampsia, a leading cause of maternal mortality and increased perinatal mortality. Oxidative stress is a major contributor to endothelial dysfunction in pre-eclampsia. Previously, we have demonstrated increased nitrotyrosine immunostaining in placental villous vascular endothelium, surrounding vascular smooth muscle and villous stroma from pre-eclamptic or diabetic pregnancies. Immunoprecipitation (IP) with antinitrotyrosine antibodies followed by immunoblot analysis identified increased nitration of phospho-p38 mitogen-activated protein kinase (MAPK) in the pre-eclamptic placenta. The catalytic activity of p38 MAPK and concentration of phospho-p38 MAPK was also found to be reduced in placentae from pre-eclamptic pregnancies. Comparison of peptide masses of a 42-kDa protein obtained by mass spectrometry with masses of a theoretical tryptic digest of p38 MAPK that was modified by phosphorylation and nitration identified the protein to be p38 MAPK.
Key words: nitration/nitrotyrosine/p38 MAPK/peroxynitrite/pre-eclampsia
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Introduction |
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Pre-eclampsia complicates about 5–7% of all pregnancies and is associated with fetal growth restriction, premature delivery and maternal death. It is diagnosed primarily by the onset of hypertension and proteinuria in the latter half of gestation. Vascular and endothelial dysfunction are well-documented characteristics of pre-eclampsia (Duley, 2003
). Reduced endovascular trophoblast invasion and uteroplacental artery remodelling, the key pathologic features of pre-eclampsia, lead to relative hypoxia and increased oxidative stress in trophoblast tissue (Goldman-Wohl and Yagel, 2002). This results in the putative release of placental factors that may act upon the maternal systemic vasculature resulting in widespread oxidative stress and vascular dysfunction. Despite active research for many decades, the aetiology of this disorder, which is unique to human pregnancy, remains an enigma. It is obvious that a single mechanism responsible for the pre-eclampsia syndrome does not exist although several compelling studies demonstrate that hypertension may develop because of oxidative stress (Roberts et al., 2000; Vaziri et al., 2000, 2002; Barton et al., 2001; Makino et al., 2002; Zhou et al., 2002). Nitric oxide, as a vasodilator, appears to play a crucial role in the gradual decrease in the vascular resistance of the placenta throughout pregnancy enabling it to meet the growing needs of the fetus (Myatt et al., 1991; Bisseling et al., 2003). Nitric oxide produced by syncytiotrophoblasts also appears to prevent platelet adhesion to the trophoblast surface thereby maintaining blood flow in the intervillous space (Myatt et al., 1991). Increased expression of the type III endothelial nitric oxide synthase (eNOS) isoform has been reported in the placentae of pregnancies complicated by pre-eclampsia (Myatt et al., 1997). The combination of increased oxidative stress and nitric oxide in the placentae of pregnancies complicated by pre-eclampsia potentiates the formation of the potent pro-oxidant peroxynitrite (Ischiropoulos and al-Mehdi, 1995; Buttery et al., 1996). Peroxynitrite has the ability to nitrate tyrosine residues in proteins resulting in the formation of nitrotyrosine residues, which are used as fingerprints of peroxynitrite formation and action (Radi, 2004). We have found nitrotyrosine residues, which are markers of both oxidative stress and nitrative stress, in the villous vessels and stroma of placentae from pregnancies complicated by either pre-eclampsia or pregestational diabetes (Myatt et al., 1996, 2000; Lyall et al., 1998; Stanek et al., 2001). In addition, we have shown that peroxynitrite treatment of the normal placental vasculature in vitro leads to the formation of nitrotyrosine residues and alters vascular reactivity of the placenta to resemble that observed in placentae from pregnancies complicated by either pre-eclampsia or pregestational diabetes (Kossenjans et al., 2000).
p38 mitogen-activated protein kinase (MAPK), a member of the highly conserved MAPK superfamily, regulates diverse cellular processes in response to a plethora of extracellular stimuli (Ichijo, 1999). p38 MAPK is essential for placental organogenesis and for trophoblast growth and invasion (Adams et al., 2000; Li et al., 2003). p38 MAPK also has been reported to regulate placental lactogen gene in trophoblasts (Peters et al., 2000). Also, p38 MAPK has been implicated to regulate implantation and differentiation in mouse models (Scherle et al., 2000; Natale et al., 2004). It is well established that dual phosphorylation of p38 MAPK at threonine-180 and tyrosine-182 is a critical requirement for its catalytic activity (Brancho et al., 2003) This particular tyrosine residue or any other tyrosine residues in the catalytic domain of the kinase may also be modified by nitrative stress resulting from the oxidative environment prevalent in pre-eclampsia, and this could potentially down-regulate the activity of p38 MAPK. Tyr-nitration of p38 MAPK has been both previously suggested (Kiroycheva et al., 2000) and tentatively identified (Bruckdorfer, 2001) in other systems.
In this study, we hypothesized that nitrated proteins would be increased in the placentae from pregnancies complicated by pre-eclampsia where oxidative stress and nitrative stress are observed. We have used a proteomic approach of immunoprecipitation (IP) combined with mass spectrometry to begin to selectively isolate and identify the nitrated proteins in the placenta.
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Products
Antinitrotyrosine antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Protein A sepharose was purchased from Amersham Biosciences (Piscataway, NJ, USA). Non-radioactive p38 MAPK assay kit, phospho-p38 and p38 MAPK antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Polyclonal antibodies to phospho-p38 were raised by immunizing rabbits with a synthetic phospho-Thr180/Tyr183 peptide by the manufacturer. Antibodies used against p38 MAPK and phospho-p38 MAPK were against the alpha form of p38 MAPK. Western Blot Chemiluminescence reagent was purchased from Perkin Elmer (Boston, MA, USA). Precast 8–16% gradient gels were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Molecular weight markers used were broad range for silver staining from Amersham Biosciences Corp., kaleidoscopic marker from Invitrogen Life Technologies and molecular weight standards from Santa Cruz Biotechnology Inc. (SantaCruz, CA, USA) for westerns. The BCA kit for protein estimation was obtained from Pierce (Rockford, IL, USA).
Tissue preparation
Tissues were collected under a protocol approved by the Institutional Review Board at the University of Cincinnati Medical Center. Placentae were collected immediately following delivery from normal pregnancies (mean gestational age for this group was 39.4, Table I) and those complicated by severe pre-eclampsia (mean gestational age was 30.1, Table I). These two groups are not matched by gestational age. In this study, for the control group, only those placentae obtained from pregnancies without any other known complications were used. Each group comprised nine patients for most experiments except where indicated otherwise. Severe pre-eclampsia was defined as occurrence of hypertension (sustained blood pressure >160 mmHg systolic and 110 mmHg diastolic), edema and proteinuria (>3+ protein on dip stick) after 20 weeks of gestation in a previously normotensive woman. Critical clinical characteristics of the patient groups are shown in Table I. Villous tissue was dissected out from beneath the chorionic plate, avoiding the basal plate, and stored at –80°C. Tissue was subsequently thawed and lysed at 4°C using a tissue tearor. Lysates (10% w/v) were prepared in lysis buffer 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) 2%, 20 mM Tris, pH 7.5, 1 mM ethylene diammine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 2 µM leupeptin, 5.8 µM pepstatin, 20 µM 4-(2-aminoethyl)-bezenesulphonyl fluoride (AEBSF) and 5 µM N-tosyl-L-lysine chloromethyl ketone (TLCK). Lysates were centrifuged at 20000 x g at 4°C for 5 min to remove debris, and supernatants were used for all experiments.
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IP
IP was carried out essentially according to Rane et al. (2001) using supernatants containing 1 mg of protein diluted in IP buffer containing 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 200 µM AEBSF, 200 µM sodium orthovanadate, 0.5% Nonidet P-40, 0.5% Triton X-100, 20 mM sodium fluoride, 0.15 M sodium chloride, 2 µM leupeptin, 5.8 µM pepstatin and 5 µM TLCK. The lysates were precleared by incubation with 20 µl of protein A sepharose for 1 h at 4°C. Following centrifugation at 10000 x g for 1 min, 10 µl of antinitrotyrosine antibody or p38 MAPK antibody or phospho-p38 MAPK antibody was incubated with the supernatants overnight at 4°C on a rotator. Protein A sepharose was then added followed by incubation for an additional 1 h at 4°C on the rotator. Beads were washed three times with lysis buffer, resuspended in 50 µl of 1x Laemmli buffer and boiled at 95°C for 5 min. Immunoprecipitates were separated by sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE) and silver stained or Coomassie stained or western blotted as appropriate. To aid nitrotyrosine detection on western blots, a modification of the Laemmli buffer was used, replacing dithiotrietol (DTT) with 10 mM iodoacetamide (IAA). This prevents reduction of nitrotyrosine to aminotyrosine, which enables detection of nitrated proteins (Balabanli et al., 1999).
Western blotting
Following lysate preparation, proteins were separated on 8–16% gradient precast gels, transferred onto nitrocellulose membranes and blocked with 5% milk in 0.1% Tween, 20 mM Tris (pH7.5)-buffered saline (TTBS) (w/v) for 1 h. Blots were probed with antinitrotyrosine (1:2000), anti-p38 (1:500) or anti-phospho-p38 (1:500) antiserum in 5% bovine serum albumin/TTBS overnight and were detected using peroxidase-conjugated secondary antibody (1:10000) in 5% milk/TTBS for 1 h. In the case of antinitrotyrosine, blots were blocked with 1% BSA, and both primary and secondary antibodies were made up in the same. Products were visualized by chemiluminescence (Perkin Elmer). Band intensity was measured using AlphaImager software from Alpha Innotech. Equal protein loading was confirmed by Ponceau S staining.
p38 MAPK activity assays
The assay was carried out essentially according to the manufacturer’s protocol. Total activity of p38 MAPK was measured in placental lysates. Activity was also measured in phospho-p38 immunoprecipitates or nitrotyrosine immunoprecipitates as required. Reactions were carried out with kinase buffer containing 25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, 10 mM MgCl2, 200 µM adenosine triphosphate (ATP) and 2 µg of activating transcription factor-2 (ATF-2) at 30°C for 30 min. Reactions were stopped with 25 µl of 3x Laemmli buffer, and reaction mixtures were boiled at 95°C for 5 min. Reaction mixtures were separated on 12% SDS–PAGE gels and western blotted for phospho-ATF-2. The blots were then quantified. Higher levels of phospho-p38 activity were reflected in higher amounts of phospho-ATF-2. This is an assay widely used in measurement of p38 MAPK activity (Samet et al., 1998; He et al., 2003). It has also been demonstrated that inhibitors of p38 MAPK can inhibit phosphorylation of ATF-2 (Lewis et al., 2005).
Statistical analysis
The significance of difference between the data from the two independent groups of samples (normotensive and pre-eclamptic) was determined by the non-directional two-tailed Student’s t-test in preliminary experiments. Preliminary experiments were carried out with three samples each. Power analysis was carried out on the preliminary data obtained, and this indicated that seven samples were necessary to obtain 90% power at a significance level of P < 0.05. Experiments carried out with nine samples had a power of 96.7% at the same significance level. In those experiments that were repeated with more samples (>3), the unidirectional one-tailed Student’s t-test was used. Each experiment included at least triplicate experiments for each variable tested. All results are expressed as mean ± SEM of three independent experiments. Differences were considered to be significant when the P value < 0.05. Statistical analyses were carried out on densitometric data obtained from western blots.
Trypsin digestion
To obtain peptides for mass spectrometric analysis, antinitrotyrosine immunoprecipitated proteins from one representative pre-eclamptic placenta were separated by SDS–PAGE and Coomassie stained. The same sample was also separated by SDS–PAGE and western blotted for phospho-p38 MAPK. The phospho-p38-immunoreactive band on the film obtained after western blot was aligned to the Coomassie-stained gel, and the corresponding band was excised and digested with trypsin. Before digestion, the excised gel pieces were destained for 10 min, washing three times with 200 µl of 25 mM NH4HCO3/50% acetonitrile. For the protein reduction, a freshly prepared 10 mM DTT was added to the gels, enough to cover them, vortexed briefly and incubated at 56°C for 45 min. The samples were cooled to room temperature and DTT solution was removed followed by the addition of 55 mM solution of IAA in 25 mM NH4HCO3 enough to cover the gel pieces; this addition was done for alkylation. The IAA solution was removed and discarded; samples were cleaned with 25 mM NH4HCO3 for 10 min and then twice with 50% acetonitrile/25 mM NH4HCO3. Samples were then dried for 20 min in speed vac, and then trypsin solution was added. The gel pieces were rehydrated in 0.01 µg/µl of trypsin solution and then incubated at 4°C for 45 min. The trypsin solution was made in 25 mM NH4HCO3. All the trypsin solution was removed and a small amount of 25 mM NH4HCO3 solution added to keep the gels hydrated during digestion. The digestion was performed overnight at 37°C in a water bath. The peptides were extracted from the gels by using 5% formic acid and 50% acetonitrile/25 mM NH4HCO3 solution. Extraction of the peptides was done twice, solutions were combined, volumes were reduced to 
20 µl and 10 µl of the eluted peptide was loaded on a capillary column ready for (LC-MS) run. Two microlitres was applied by a thin film-spotting procedure for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) analysis using 
-cyanohydroxycinnamic acid as the matrix on stainless steel targets.
Mass spectrometry
Mass spectral data were obtained at the WM Keck Foundation Biotechnology Laboratory, Yale University, using a Micromass TofSpec SE and a 337-nm N2 laser at an accelerating voltage of 25 kV in the positive linear ion mode. Peptide masses obtained by MALDI-MS analysis were searched against databases using the programs ProFound and Pepsea. These programs rely on the National Center for Biotechnology Information (NCBI) and European Molecular Biology Laboratory (EMBL) non-redundant databases, respectively, to identify the intact proteins. We then compared actual peptide masses obtained from our protein in this way with peptide masses obtained from a theoretical (Nicholson et al., 2005) tryptic digest of p38 MAPK (Database: Swiss Prot, accession number Q16539
[GenBank]
) modified by phosphorylation and nitration. Theoretical tryptic digest masses were generated using the MS-Digest program of Protein Prospector (http://prospector.ucsf.edu). Peptide mass matching was carried out with a mass tolerance of upto 1 Da. Peptide mass matching has been reported by others (Thongboonkerd et al., 2002; Singh et al., 2003; Emanuelsson et al., 2005) with over a dalton difference.
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Nitrated proteins in the placenta
Our earlier observations of increased nitrotyrosine immunostaining in the pre-eclamptic placenta led us to our current hypothesis that there would be correspondingly higher levels of nitrated proteins in the pre-eclamptic placenta and initiated the search for nitrated proteins. To selectively isolate and identify the nitrated proteins, we first separated the antinitrotyrosine immunoprecipitated proteins by SDS–PAGE and silver stained the gels. Figure 1 (panel A) shows at least six or more proteins of sizes ranging from 20 to 90 kDa, identified by silver staining that are nitrated in both normal and pre-eclamptic placentae (n = 3, each group) examined. This was confirmed by western blot analysis of total lysates with antinitrotyrosine antibody, which also showed nitration in both normal and pre-eclamptic placentae (Figure 1, panel B, n = 3, each group) with similar molecular weight ranges of proteins being identified. Preincubation of the antinitrotyrosine antibody with 10 mM nitrotyrosine was used to check the specificity of the antibody, and this blocked the nitrotyrosine immunoreactivity in the lysates on western blot (Figure 1, panel B).
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Identification of phospho-p38 as a nitrated protein and effect on catalytic activity of phospho-p38
Major differences in the levels of nitrated proteins between normal and pre-eclamptic placentae were found around 42 kDa range (Figure 1, panel A), suggesting that it might correspond to p38 MAPK, among other MAPKs. We explored the possibility that p38 MAPK was a target of increased nitration in the pre-eclamptic placenta. To achieve this aim, we immunoprecipitated nitrotyrosine-containing proteins with the antinitrotyrosine antibody and cross-blotted them with phospho-p38 MAPK antibody, which gave recognition of a band at 42 kDa (Figure 2A). The apparent intensity of this band was significantly greater (n = 9, P < 0.0001) in antinitrotyrosine immunoprecipitates from pre-eclamptic placentae than that from normal placenta (Figure 2B). Negative control of IP with protein A sepharose beads alone gave no bands on cross-blotting, confirming the specificity of the IP protocol (data not shown).
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To directly examine whether Tyr-nitration affected functionality of p38 MAPK in the placenta, we tested antinitrotyrosine immunoprecipitates for their ability to phosphorylate ATF-2, a measure of the catalytic activity of p38 MAPK. There was no significant difference in overall p38 MAPK activity shown by antinitrotyrosine immunoprecipitates from pre-eclamptic placentae (Figure 2C and D) when compared with normotensive pregnancies. When activity was normalized to the amount of phospho-p38 MAPK present in the antinitrotyrosine immunoprecipitates, however, the specific catalytic activity obtained from pregnancies complicated by pre-eclampsia was 65% less (P < 0.03, Figure 2E) than that from normotensive pregnancies.
The catalytic activity was drastically reduced in phospho-p38 MAPK immunoprecipitates from pre-eclamptic pregnancies, and they exhibited 81% lower activity (Figure 3, panel A, P < 0.05) than in immunoprecipitates from normal placentae. It is significant that phospho-p38 immunoprecipitates from pre-eclamptic placentae also showed a higher extent of nitration (Figure 3, panel B) compared with normal. This demonstrates a correlation between higher level of nitration of phospho-p38 and lower catalytic activity. For additional evidence of nitration, we have also, in separate experiments, immunoprecipitated with antibody that recognizes total p38 MAPK (non-phosphorylated plus phosphorylated, Figure 3c) and cross-blotted with antinitrotyrosine antibody. This blot was also stripped and probed for total p38 (Figure 3d).
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Western blotting of total placental lysates also revealed that the amount of phospho-p38 MAPK was significantly less in placentae of pregnancies complicated by pre-eclampsia compared with normotensive pregnancies (P < 0.001, Figure 4). Not surprisingly, the overall catalytic activity of phospho-p38 MAPK was 32% less (P < 0.05) in placental lysates from pregnancies complicated by pre-eclampsia compared with normotensive pregnancies (Figure 5).
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Confirmation of nitration of p38 MAPK by MALDI-MS
To obtain unequivocal identification by MALDI-MS, we excised the protein band corresponding to phospho-p38 MAPK from an SDS–PAGE gel on which the antinitrotyrosine immunoprecipitate from a pre-eclamptic placenta had been fractionated and digested with trypsin (Table II). Because of the extensive nitration combined with phosphorylation that presumably prevented a definitive identification of the protein, masses obtained by MALDI-MS did not match any peptide sequences in any available database. Accordingly, the sequences were subsequently compared with those obtained by theoretical tryptic digest of p38 MAPK (Database: Swiss Prot., accession number Q16539
[GenBank]
). This theoretical digest was carried out after modification (enabled by the software) of its serine, threonine and tyrosine residues by phosphorylation and nitration of tyrosine. Interestingly, eight of the peptide masses from the excised phospho-p38 MAPK band matched those of the theoretical digest masses of p38 MAPK modified by phosphorylation and nitration in all the possible residues of serine, threonine and tyrosine (Table I). In a database search using MOWSE, three masses are considered enough for a positive identification. The peptide 166–186 (Table I) contains the sites of dual phosphorylation, T(180) and Y(182), that is critical for p38 MAPK catalytic activity. The mass spectrometry data thus indicate that phospho-p38 MAPK is nitrated in the pre-eclamptic placentae.
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Discussion |
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The data presented here demonstrate that p38 MAPK is a nitrated protein in the placentae, with the amount of nitrated protein being significantly higher (P < 0.0001) in the placentae of pregnancies complicated by pre-eclampsia than in normal pregnancies. It needs to be borne in mind that these data were generated using two distinct groups of completely normal and severe pre-eclamptic women. The two patient groups used were significantly (P < 0.0001) different in their gestational ages. The primary reason for not using age-matched normals was due to the fact that no pregnancies which deliver at 28–32 weeks can be considered as true normals. Most, if not all, of them have some underlying pathology (Barrett, 2002
; Badr et al., 2005; Crider et al., 2005). Although risk factors for preterm birth include among others preterm premature rupture of membranes (pPROM), cervical insufficiency, pathologic uterine distension, uterine anomalies, intrauterine infection/inflammation and social factors, the final common pathway appears to be activation of the inflammatory cascade (Engel et al., 2005), which was yet another reason for not using age-matched normals. Moreover, because oxidative stress and, therefore by association, nitrative stress have been reported (Wang and Walsh, 1998; Casanueva and Viteri, 2003; Jauniaux et al., 2003) to increase with increasing gestational age, a true normal preterm group would have potentially exhibited even lower levels of nitrated protein and thus increased the intensity of the differences that we have obtained. This study specifically looked at early onset severe pre-eclampsia so as to ensure maximal phenotypic differences.
The combined tools of IP and mass spectrometry confirm the nitration of phospho-p38 MAPK from human placental lysates. We speculated that the extensive nitration combined with the phosphorylation prevents a definitive identification of the protein from conventional database searches. However, eight peptide masses of the unidentified protein match the theoretical tryptic digest of p38 MAPK, when it was modified by nitration and phosphorylation in its serine, threonine and tyrosine residues. Although such an extensive modification may not take place in an in vivo situation, this assumption was necessary so as not to miss any peptides that could be potentially modified. Peptide matches obtained cover 33% of the entire p38 MAPK sequence and essentially confirm the identification of nitrated p38 MAPK. It is highly likely that nitration of p38 MAPK interferes with its interaction with other proteins. Several reports (Choi, 2000; Hagemann and Blank, 2001; Taylor and Starnes, 2003) do exist concerning interaction of active p38 MAPK with other proteins and also the ability of p38 MAPK to regulate various gene functions (Ono and Han, 2000). Nitration of p38 MAPK demonstrated here would therefore adversely impair several critical functions at both cellular and genetic levels.
The overall reduced concentration of phospho-p38 MAPK that we observed in the pre-eclamptic placentae may be because of several reasons. It may be hypothesized that gestational age could influence activity and expression of p38 MAPK in the pre-eclamptic group. However, to our knowledge there have been no reports on the change in p38 MAPK activity or expression in the placenta with gestational age. Yet, because our data presented here (Figure 2A) demonstrate increased nitration of phospho-p38 in the pre-eclamptic placentae, we propose that the reduction in the amount of phospho-p38 MAPK is due to nitration. It is possible that the nitration of phospho-p38 MAPK reduces its affinity for the specific antibody as previously suggested by Kiroycheva et al. (2000). We have also estimated the catalytic activity of p38 MAPK in total placental lysates where the assay was not compromised by the use of immunoprecipitating antibodies. Here, again the catalytic activity of p38 MAPK was significantly (P < 0.05, Figure 5) less in pre-eclampsia. We have established the decreased activity of p38 MAPK in pre-eclampsia in three different ways, namely by measuring activity in antinitrotyrosine, phospho-p38 immunoprecipitates and also in total placental lysates. On the basis of data presented here and our in vitro data (Webster et al., 2006) that demonstrated total inhibition of catalytic activity on nitration of p38 MAPK, we suggest that the reduced catalytic activity of p38 MAPK observed in the pre-eclamptic placenta is due to nitration.
Many proteins including manganese superoxide dismutase (MnSOD) (Yamakura et al., 1998), p53 (Yamakura et al., 1998; Chazotte-Aubert et al., 2000; Cobbs et al., 2001), cytochrome P450 (Roberts et al., 1998), Ca-ATPase of the sarcoplasmic reticulum (Viner et al., 1996), mitochondrial creatine kinase (Stachowiak et al., 1998), tyrosine hydroxylase (Ara et al., 1998) and prostacyclin synthase (Zou et al., 1997, 1999a,b) have been reported to be nitrated. Protein nitration has been most commonly shown to inhibit protein function, e.g. MnSOD (MacMillan-Crow et al., 1996), p53 (Chazotte-Aubert et al., 2000), NF
B (Park et al., 2005) and tyrosine hydoxylase (Ara et al., 1998). However, in some cases, nitration either has no effect, e.g. protein kinase epsilon (Balafanova et al., 2002), or activates protein function as is the case with cyclooxygenase-2 (Salvemini, 1997), poly ADP ribose polymerase (Szabados et al., 1999) and fibrinogen (Gole et al., 2000). In addition, cytoskeletal proteins, such as actin and tubulin, have also been shown to be nitrated (Ischiropoulos, 1998), which has been postulated to have a physiological role in vivo.
Proteins that are tyrosine nitrated have been reported to be poor substrates for tyrosine kinases (Gow et al., 1996; Kong et al., 1996). This would result in decreased phosphorylation of p38 MAPK when it is nitrated and may explain the decreased activity and amounts of phospho-p38 MAPK seen in our experiments as well as in those of Kiroycheva et al. (2000). Proteolytic degradation that has been seen to occur for other nitrated proteins (Grune et al., 1998) could also reduce the intact amount of a protein. Another possibility is that nitration on tyrosine leads to displacement of phosphotyrosine which has been reported to occur in lyn kinase (Mallozzi et al., 2001; Minetti et al., 2002). In the case of lyn kinase, phosphotyrosine displacement leads to activation of the kinase. However, the reduced catalytic activity of p38 MAPK that we observed seems to rule out such a possibility. Nitration has also been reported to be mimicking phosphorylation in the case of src kinase (Minetti et al., 2002).
The detection of nitration in both normal and pre-eclamptic placenta suggests that nitration may be a normal negative regulator for phosphorylation. It has been shown that both peroxynitrite (Jope et al., 2000) and nitric oxide (Li et al., 2001) have the ability to induce phosphorylation of p38 MAPK protein. Yet, there are reports (Kiroycheva et al., 2000) of peroxynitrite causing reduced expression of phospho-p38 MAPK protein. Our results show an association between the reduced amounts of phospho-p38 MAPK and increasing levels of nitration in the placenta. This increased nitration is also associated with lowered p38 MAPK catalytic activity.
Potentially, the nitration of p38 MAPK observed in term placenta could be because of a biphasic effect of peroxynitrite. Initially, peroxynitrite induces phosphorylation and then the increasing concentrations of peroxynitrite bring about nitration of the kinase. Such a biphasic effect of peroxynitrite has been reported in other situations (Jope et al., 2000). Higher levels of peroxynitrite caused by increased oxidative stress in pre-eclampsia may lead to increased nitration of p38 MAPK. Increased nitration of p38 MAPK correlates with the higher levels of nitrotyrosine staining, detected in the pre-eclamptic placenta (Myatt et al., 1996).
Pre-eclampsia is associated with abnormal trophoblast invasion and development of the placenta (Roberts and Cooper, 2001; Roberts and Lain, 2002; McMaster et al., 2004). Targeted inactivation of the p38 MAPK is accompanied by early embryonic lethality in mice and is associated with defects in placental development because of loss of labyrinth and reduced spongiotrophoblast layers in the placenta, reduced vascularization of labyrinth and increased apoptosis (Adams et al., 2000). Nitric oxide and progesterone may synergistically activate p38 MAPK to induce apoptosis in endometrial epithelial cells (EECs), a process that may facilitate implantation (Li et al., 2001). If this is so, then inactivation of p38 MAPK following nitration may be one more potential cause for poor trophoblast invasion and consequently, implantation observed in pre-eclampsia (Waite et al., 2002). If this is the case though, substantial nitration of p38 MAPK may have to occur before implantation. Our data presented here provide another possible cause for inadequate implantation observed in pre-eclampsia. These data along with earlier reports by Miller et al. (1996) of elevated nitrative response in animal models of fetal growth restriction clearly suggest a link between nitrative stress and fetal growth restriction which accompanies pre-eclampsia.
Our data clearly demonstrate a coexistence of loss of function and nitration of p38 MAPK. We have identified a critical signalling molecule p38 MAPK to be nitrated. Our data demonstrate that the increased nitration of p38 MAPK is associated with a decrease in p38 MAPK catalytic activity which could be one of the causes for both poor implantation and growth restriction observed in pre-eclampsia. It is necessary to use both in vitro studies and in vivo cell culture to demonstrate the direct effect of nitration on functionality of p38 MAPK. Such studies are currently in progress in our laboratory.
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Acknowledgements |
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This work was supported by NIH RO1 grant HL07529. Mass spectrometric services were obtained from W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.
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