Preeclampsia‐associated stresses activate Gadd45a signaling and sFlt‐1 in placental explants

Accumulating evidence suggests that placental stresses during pregnancy can play an important role in the pathogenesis of preeclampsia. A common signal pathway that senses and converts placental stresses into intracellular stress response may be contributing to this pathology. Based on our previous findings, we extended our investigation to establish that Gadd45a stress signaling regulates sFlt‐1 levels, particularly in placenta, when exposed to various preeclampsia‐associated stresses including AT‐1 receptor agonist (Angiotensin II), hypoxia, and inflammatory cytokines. Using a placental explant model, we found that Gadd45a was induced in response to all the preeclampsia stresses stated above. Although stress induced Gadd45a was associated with the activation of its downstream effectors phospho‐p38 and phospho‐JNK, the subsequent regulation of sFlt‐1 levels occurred through either one of these effectors, but not both. These observations indicate that Gadd45a signaling may work as a hub connecting placental stresses and the pathogenesis of preeclampsia. It also provides evidence to justify testing the role of Gadd45 in the etiology of preeclampsia using in vivo mouse (i.e., Gadd45a null mice) models. J. Cell. Physiol. 228: 362–370, 2013. © 2012 Wiley Periodicals, Inc.     

Gadd45α as an upstream signaling molecule of p38 MAPK triggers oxidative stress-induced sFlt-1 and sEng upregulation in preeclampsia

Preeclampsia (PE) is known to be associated with increased circulating levels of anti-angiogenic factors, such as soluble fms-related tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). However, the way that placental oxidative stress results in the elevation of these two factors remains enigmatic. We have observed the overexpression of growth arrest and DNA damage-inducible 45 alpha (Gadd45α) and excessive activation of p38 mitogen-activated protein kinase (MAPK) in preeclamptic placentas compared with normotensive controls, together with increased levels of sFlt-1 and sEng in maternal sera in patients with PE. Moreover, Gadd45α knockdown or p38 inhibition provides protective effects in hypoxia/reoxygenation (H/R)-exposed human umbilical vein endothelial cells (HUVECs) by suppressing oxidative stress, inhibiting apoptosis, and promoting their potential for in vitro angiogenesis. A regulatory signaling pathway in which H/R intervention causes the induction of Gadd45α leading to p38 activation and ultimately an increase in sFlt-1 and sEng secretion in HUVECs has concurrently been established. Our study opens up a promising new avenue of investigation for increasing the understanding of the origin of sFlt-1 and sEng in PE and provides novel therapeutic targets for pregnancy complications arising from placental endothelial dysfunction.     

Soluble endoglin contributes to the pathogenesis of preeclampsia

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-beta coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-beta1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-beta signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.     

MiR-141–3p promotes hypoxia-induced autophagy in human placental trophoblast cells

Preeclampsia (PE) is a pregnancy-specific disorder and a significant contributor to maternal, fetal and neonatal morbidity and mortality worldwide. Its pathogenesis is generally accepted as insufficient trophoblast invasion of the maternal endometrium and inadequate remodeling of the maternal spiral arteries. These impairments lead to elevated levels of hypoxia and oxidative stress. Autophagy has become a highly researched field in obstetrics, and this process may be essential for preimplantation development beyond the four- and eight-cell stages, and for blastocyst survival, extra-villous trophoblast functions, invasion and vascular remodeling. Several studies have shown that autophagy activation, shown by an increase in autophagy vacuoles or microtubule-associated protein 1 A/1B-light chain 3 (LC3) dots, was more common in PE than in normal pregnancy. Thus, changes in autophagic status are seen in preeclamptic placentas. MicroRNA-141–3p (miR-141–3p), a multifunctional miRNA, is involved in a variety of physiological and pathological processes, including PE and autophagy. However, the influence of miR-141–3p on autophagy regulation in trophoblast cells has yet to be described. Therefore, the objective of our study was to investigate the role of miR-141–3p in autophagy induced by hypoxia in human placental trophoblast cells. Our results found that hypoxia induced autophagy in trophoblast cells and dramatically elevated the expression of miR-141–3p. Overexpression of miR-141–3p improved autophagic activity, whereas low expression of miR-141–3p inhibited autophagic activity. Therefore, our data demonstrated that miR-141–3p promoted hypoxia-induced autophagy in placental trophoblast cells, which may be related to the development of preeclampsia.     

Comparative N-Glycoproteomic and Phosphoproteomic Profiling of Human Placental Plasma Membrane between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

Preeclampsia is a serious complication of pregnancy, which affects 2–8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane.     

Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans

Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.     

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n = 8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n = 9); normal pregnant rats injected with MBG (NPM, n = 8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n = 8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1 nM. Treatment with MBG (≥ 1 nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.     

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra-peritoneal cavity following intra-peritoneal administration of the bacterial cell wall pathogen-associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine and IL-8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages.     

Reduced placental docosahexaenoic acid levels associated with increased levels of sFlt-1 in preeclampsia

Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p<0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p<0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p<0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.     

Angiotensin II and angiotensin-(1-7) decrease sFlt1 release in normal but not preeclamptic chorionic villi: an <Emphasis Type="Italic">in vitro</Emphasis> study

Background  

During preeclampsia, placental angiogenesis is impaired. Factors released from the placenta including vascular endothelial growth factor (VEGF), placental growth factor (PLGF), soluble VEGF receptor 1 (sFlt1), and soluble endoglin (sEng) are regulatory molecules of placental development and function. While the renin angiotensin system has been shown to regulate angiogenic factors in other research fields, these mechanisms have not been extensively studied during pregnancy.     

Casein kinase 2- and protein kinase A-regulated adenomatous polyposis coli and beta-catenin cellular localization is dependent on p38 MAPK

Skin cancer is the most common form of malignancy in the world with epidemic proportions. Identifying the biochemical and molecular mechanisms underlying the events leading to tumors is paramount to designing new and effective treatments that may aid in treating and/or preventing skin cancers. Herein we identify p38 MAPK, along with its positive modulator, Gadd45a, as important regulators of nucleocytoplasmic shuttling of the adenomatous polyposis coli (APC) tumor suppressor. APC normally functions to block beta-catenin from promoting cell proliferation and migration/invasion. Keratinocytes lacking proper p38 MAPK activation, either due to lack of Gadd45a or through the use of p38 MAPK-specific inhibitors, are unable to effectively transport APC into the nucleus. We also show that p38 MAPK is able to directly associate with and modulate both casein kinase 2 (CK2) and protein kinase A (PKA), which promote and block APC nuclear import, respectively. We demonstrate that p38 MAPK is able to not only enhance CK2 kinase activity but also suppress PKA kinase activity. Moreover, lack of normal p38 MAPK activity in either Gadd45a-null keratinocytes or in p38 MAPK inhibitor treated keratinocytes leads to decreased CK2 activity and increased PKA activity. In either case, disruption of APC nuclear import results in elevated levels of free cellular, and potentially oncogenic, beta-catenin. Numerous tumors, including skin cancers, are associated with high levels of beta-catenin, and our data indicate that p38 MAPK signaling, along with Gadd45a, may provide tumor suppressor-like functions in part by promoting APC nuclear localization and effective beta-catenin regulation.     

Serum iron and copper status and oxidative stress in severe and mild preeclampsia

Our aim was to investigate parameters of iron and copper status and oxidative stress and antioxidant function in women with healthy pregnancy, mild and severe preeclampsia with a view to exploring the possible contribution of these parameters to the aetiology. Thirty healthy, 30 mild preeclamptic and 30 severe preeclamptic pregnant women were included. Serum and placental lipid peroxides, and serum vitamin E and total carotene levels were measured by colorimetric assay. Cholesterol, copper, iron, total iron binding capacity (TIBC), ceruloplasmin and transferrin concentrations were measured by commercially available procedures. Data were analysed statistically using one-way analysis of variance and Pearson correlation test. Logistic regression procedures were used to calculate odds ratios. Lipid peroxides in serum and placental tissue, and iron, copper and ceruloplasmin levels in serum were significantly increased, and transferrin, TIBC, vitamin E/total cholesterol and total carotene/total cholesterol ratios in serum were significantly decreased especially in women with severe preeclampsia. Significant correlations were detected between serum iron and lipid peroxides in serum and placental tissue and between serum iron and vitamin E/total cholesterol in severe preeclamptic pregnancy. Furthermore, there were significant correlations between serum malondialdehyde and ceruloplasmin and vitamin E/total cholesterol in women with severe preeclampsia, and changes in serum and placental lipid peroxides and serum iron concentrations were significantly associated with preeclampsia. In conclusion, ischaemic placental tissue may be a primary source of potentially toxic iron in preeclampsia and the released iron species may contribute to the aetiology and would exacerbate lipid peroxidation and endothelial cell injury, which may be abated by antioxidant supplementation.     

Preeclamptic women are deficient of interleukin-10 as assessed by cytokine release of trophoblast cells in vitro

BACKGROUND: It is well known that the acceptance of the fetoplacental unit in human pregnancy requires maternal immune tolerance, which is thought to be regulated locally by the placenta. Therefore an anti-inflammatory cytokine such as IL-10 plays a critical role in different pregnancy disorders including preeclampsia. In the present study, we examined the expression of both proinflammatory (TNF-alpha, IL-1beta, IL-2) and immunoregulatory (IL-6, IL-10) cytokines from normal term and preeclamptic patients in human trophoblast cultures. METHODS: Eleven patients with preeclampsia and 11 patients with a normal pregnancy at term were included in the study. Trophoblast cells isolated from placentas were cultured up to 48 h under standard tissue culture conditions and cytokine release was determined by ELISA. IL-10 synthesis was significantly decreased in the third trimester in preeclamptic patients in comparison with the control group. RESULTS: There were no significant differences in IL-1beta, IL-2, IL-6 or TNF-alpha expression but a significant alteration in IL-10 release in trophoblast cultures in vitro in term placentas from preeclamptic patients compared with normal pregnancy. CONCLUSIONS: Because IL-10 is a potent regulator of anti-inflammatory immune response these abnormalities may be associated with the inadequate placental development in preeclampsia.     

miR-34a expression,epigenetic regulation,and function in human placental diseases

Preeclampsia (PE) is the major pregnancy-induced hypertensive disorder responsible for maternal and fetal morbidity and mortality that can be associated with intrauterine growth restriction (IUGR). PE and IUGR are thought to be due to a placental defect, occurring early during pregnancy. Several placental microRNAs (miRNAs) have been shown to be deregulated in the context of placental diseases and could thus play a role in the pathophysiology of PE. Here, we show that pri-miR-34a is overexpressed in preeclamptic placentas and that its placental expression is much higher during the first trimester of pregnancy than at term, suggesting a possible developmental role. We explored pri-miR-34a regulation and showed that P53, a known activator of miR-34a, is reduced in all pathological placentas and that hypoxia can induce pri-miR-34a expression in JEG-3 cells. We also studied the methylation status of the miR-34a promoter and revealed hypomethylation in all preeclamptic placentas (associated or not with IUGR), whereas hypoxia induced a hypermethylation in JEG-3 cells at 72 h. Despite the overexpression of pri-miR-34a in preeclampsia, there was a striking decrease of the mature miR-34a in this condition, suggesting preeclampsia-driven alteration of pri-miR-34a maturation. SERPINA3, a protease inhibitor involved in placental diseases, is elevated in IUGR and PE. We show here that miR-34a overexpression in JEG-3 downregulates SERPINA3. The low level of mature miR-34a could thus be an important mechanism contributing to SERPINA3 upregulation in placental diseases. Overall, our results support a role for miR-34a in the pathophysiology of preeclampsia, through deregulation of the pri-miRNA expression and its altered maturation.     

gadd45 is not required for activation of c-Jun N-terminal kinase or p38 during acute stress.

Cells respond to environmental stress with activation of c-Jun N-terminal kinase (JNK) and p38. Recent studies have implicated Gadd45 and two related proteins, MyD118/Gadd45beta and CR6/Gadd45gamma, as initiators of JNK/p38 signaling via their interaction with an upstream kinase MTK1. It was proposed that stress-induced expression of the Gadd45-related proteins leads to MTK1 activation and subsequent JNK/p38 activation. Using embryo fibroblasts from gadd45-null mice, we have addressed the requirement for Gadd45 in mediating JNK/p38 activation during acute stress. Comparison of JNK/p38 activities in response to methyl methanesulfonate, hydrogen peroxide, UVC irradiation, sorbitol, and anisomycin treatment of gadd45(+/+) and gadd45(-/-) fibroblasts revealed no deficiency in JNK/p38 activation in gadd45(-/-) fibroblasts. In addition, in wild type cells, JNK and p38 activation significantly preceded gadd45 induction with all stresses. Examination of myd118/gadd45beta and cr6/gadd45gamma expression in gadd45(+/+) and gadd45(-/-) fibroblasts revealed similar induction patterns in the two cell types, which, like gadd45 expression, was delayed relative to JNK/p38 activation. We conclude that gadd45 expression is not required for activation of JNK/p38 by environmental stresses, nor are stress-induced increases in myd118/gadd45beta and cr6/gadd45gamma expression necessary for kinase activation in response to such insults.     

Gadd45alpha regulates p38-dependent dendritic cell cytokine production and Th1 differentiation

Gadd45alpha inhibits the activation of p38 by the T cell alternative pathway involving phosphorylation of p38 Tyr(323). Given that T cell p38 may play a role in Th1 development, the response to Th-skewing Ags was analyzed in Gadd45alpha(-/-) mice. Despite constitutively increased p38 activity in Gadd45alpha(-/-) T cells, the Th1 immune response to Toxoplasma gondii Ag (STAg), was diminished. In contrast to T cells, dendritic cells (DC) lacked the alternative p38 activation pathway. Gadd45alpha(-/-) DCs responded to STAg with low levels of MAP kinase cascade-dependent p38 activation, IL-12 production, and CD40 expression. Wild-type T cells transferred into Gadd45alpha(-/-) recipients had a diminished Th1 response to STAg, whereas Gadd45alpha(-/-) T cells transferred into wild-type hosts behaved normally. Therefore, Gadd45alpha has tissue-specific and opposing functions on p38 activity, and Gadd45alpha-regulated p38 activation in DCs is a critical event in Th1 polarization in vivo.