Adaptations to changes in oxygen are critical to ensure proper placental development, and impairments in oxygen sensing mechanisms characterize placental pathologies such as preeclampsia. In this study, we examined the involvement of SUMOylation, a reversible posttranslational modification, in the regulation of the asparaginyl hydroxylase Factor Inhibiting Hypoxia Inducible Factor 1 (FIH1) in the human placenta in development and in disease status. FIH1 protein abundance and spatial distribution in the developing placenta directly correlated with oxygen tension in vivo. Immunofluorescence analysis showed that early on FIH1 primarily localized to nuclei of cytotrophoblast cells, while after 10 weeks of gestation it was present in nuclei and cytoplasm of both cytotrophoblast and syncytiotrophoblast cells. Exposure of choriocarcinoma JEG-3 cells to hypoxia induced FIH1 SUMOylation by promoting its association to SUMO2/3. Transfection of JEG-3 cells with FIH1 constructs containing SUMO-mutated sites revealed that SUMOylation of FIH1 by SUMO2/3 targeted it for proteasomal degradation, particularly in hypoxia. SUMOylation of FIH1 directly impacted on HIF1A activity as determined by HIF-responsive luciferase assay. Co-immunoprecipitation analyses revealed enhanced FIH1-SUMO2/3 associations early in development, when FIH1 levels are low, while deSUMOylation of FIH1 by SENP3 increased later in gestation, when FIH1 levels are rising. In preeclampsia, decreased FIH1 protein expression associated with impaired deSUMOylation by SENP3 and increased association with the ubiquitin ligase RNF4. We propose a novel mode of regulation of FIH1 stability by dynamic SUMOylation and deSUMOylation in the human placenta in response to changing oxygen tension, thereby mediating HIF1A transcriptional activity in physiological and pathological conditions.

Preeclampsia (PE) is a major obstetric complication that is challenging to predict. Currently, there are limited tools to assess placental health/function in crucial gestational periods for diagnosis and early prediction. The glycoprotein fibronectin (FN) is augmented in PE placentae, and associated with reduced activity of JMJD6, an oxygen sensor that regulates placental FN processing. Evidence implicates placenta-derived small extracellular vesicles (sEVs) in the pathogenesis of pregnancy-associated disorders. Here, we examined the utility of FN and JMJD6 in placental sEVs as putative markers for early- and late-onset PE (E-PE and L-PE). Maternal plasma was obtained from venous blood collected longitudinally during pregnancy (10-14, 16-22, and 26-32 weeks of gestation and at delivery) in normotensive term control, preterm control, L-PE, E-PE, and gestational hypertensive individuals. Placenta-derived sEVs were isolated and their FN and JMJD6 content and JMJD6 activity were measured. In women that went on to develop preeclampsia, FN content of circulating placental sEVs was significantly elevated as early as 10 to 14 weeks of gestation and remained augmented until the time of delivery. This was accompanied by a depletion in JMJD6 content. Multivariate receiver operating characteristic analysis revealed high predictive power for FN and JMJD6 as early markers of E-PE and L-PE. In vitro, hypoxia or JMJD6 loss promoted FN accumulation in sEVs that was reverted on restoring cellular iron balance with the natural compound, Hinokitiol. Elevated FN, along with diminished JMJD6 in circulating placental sEVs, serves as an early molecular signature for the detection of different hypertensive disorders of pregnancy and their severity.

Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHDs), specifically PHD2, causes placental hypoxia-inducible factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia, yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/- cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new-onset hypertension at midgestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small molecule inhibitor of HIF1, to pregnant Phd2-/- cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension had been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.

Dynamic changes in physiologic oxygen are required for proper placenta development; yet, when low-oxygen levels persist, placental development is halted, culminating in preeclampsia (PE), a serious complication of pregnancy. Considering mitochondria's function is intimately linked to oxygen changes, we investigated the impact of oxygen on mitochondrial dynamics in placental mesenchymal stromal cells (pMSCs) that are vital for proper placental development. Transmission electron microscopy, proximity ligation assays for mitochondrial VDAC1 and endoplasmic reticulum IP3R, and immunoanalyses of p-DRP1 and OPA1, demonstrate that low-oxygen conditions in early 1st trimester and PE promote mitochondrial fission in pMSCs. Increased mitochondrial fission of mesenchymal cells was confirmed in whole PE placental tissue sections. Inhibition of DRP1 oligomerization with MDiVi-1 shows that low oxygen-induced mitochondrial fission is a direct consequence of DRP1 activation, likely via HIF1. Mitophagy, a downstream event prompted by mitochondrial fission, is a prominent outcome in PE, but not 1st trimester pMSCs. We also investigated whether mesenchymal-epithelial interactions affect mitochondrial dynamics of trophoblasts in PE placentae. Exposure of trophoblastic JEG3 cells to exosomes of preeclamptic pMSCs caused heightened mitochondrial fission in the cells via a sphingomyelin-dependent mechanism that was restored by MDiVi-1. Our data uncovered dichotomous regulation of mitochondrial fission and health in human placental mesenchymal cells under physiologic and pathologic hypoxic conditions and its impact on neighboring trophoblast cells.

Preeclampsia, a serious hypertensive disorder of pregnancy, is characterized by elevated ceramide (CER) content that is responsible for heightened trophoblast cell death rates via apoptosis and autophagy. Whether trophoblast cells undergo necroptosis, a newly characterized form of regulated necrosis, and the potential role of CER in this process remain to be established. Herein, we report that exposure of both JEG3 cells and primary isolated cytotrophoblasts to C16:0 CER in conjunction with a caspase-8 inhibitor (Q-VD-OPh) promoted necroptotic cell death, as evidenced by increased expression and association of receptor-interacting protein kinases RIP1 and RIP3, as well as phosphorylation of mixed lineage kinase domain-like (MLKL) protein. MLKL activation and oligomerization could be abrogated by pretreatment with the necroptosis inhibitor necrostatin-1 (Nec-1). CER+Q-VD-OPH-treated primary trophoblasts displayed striking necrotic morphology along with disrupted fusion processes as evidenced by maintenance of E-cadherin-stained membrane boundaries and reduced glial cell missing-1 expression, but these events were effectively reversed using Nec-1. Of clinical relevance, we established an increased susceptibility to necroptotic cell death in preeclamptic placentae relative to normotensive controls. In preeclampsia, increased necrosome (RIP1/RIP3) protein levels, as well as MLKL activation and oligomerization associated with necrotic cytotrophoblast morphology. In addition, caspase-8 activity was reduced in severe early-onset preeclampsia cases. This study is the first to report that trophoblast cells undergo CER-induced necroptotic cell death, thereby contributing to the increased placental dysfunction and cell death found in preeclampsia.

Persistent low oxygen is implicated in the pathogenesis of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Emerging evidence implicates a novel family of Jumonji C catalytic domain proteins as mediators of hypoxic gene expression. Here, we investigated the regulatory relationship between Jumonji C domain containing protein 6 (JMJD6) and hypoxia-inducible factor (HIF)1A in the human placenta at physiological and pathological conditions. JMJD6 expression inversely correlated with changes in oxygen tension during early placental development, ie, high at 7-9 weeks when-partial pressure of O2 is low and declining afterwards when-partial pressure of O2 increases. Moreover, JMJD6 protein was significantly elevated in early-onset preeclamptic placentae, localizing to the syncytiotrophoblast layer and syncytial knots. Exposure of primary isolated trophoblast cells, human villous explants, and JEG3 choriocarcinoma cells to low oxygen (3%) and sodium nitroprusside (inducer of oxidative stress) also resulted in elevated JMJD6 levels, which was abrogated by HIF1A knockdown. In normoxia, knockdown of JMJD6 in JEG3 cells stabilized HIF1A with a concomitant decrease in von Hippel-Lindau (VHL) tumor suppressor protein, a negative regulator of HIF1A stability. In contrast, overexpression of JMJD6 enhanced VHL expression and destabilized HIF1A. JMJD6 regulation of VHL stability did not involve the ubiquitin-proteasome system but likely occurred through lysyl hydroxylation and small ubiquitin-like modifier 1-dependent small ubiquitin-like modifierylation. In summary, our data signify a novel role for JMJD6 as an oxygen sensor in the human placenta, and alterations in the JMJD6-VHL-HIF1A feedback loop may indirectly contribute to elevated HIF1A found in preeclampsia.

Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol.

The von Hippel Lindau (VHL) protein is a key executor of the cellular hypoxic response that is compromised in preeclampsia, a serious disorder complicating 5% to 7% of pregnancies. To date, the mechanisms controlling VHL gene expression in the human placenta remain elusive.

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

Hypoxia-inducible factor 1A (HIF1A) stability is tightly regulated by hydroxylation and ubiquitination. Emerging evidence indicates that HIF1A is also controlled by the interplay between SUMO-specific ligases, which execute protein SUMOylation, and Sentrin/SUMO-specific proteases that de-SUMOylate target proteins. Given the significance of HIF1A in the human placenta, we investigated whether placental HIF1A is subject to SUMOylation in physiological and pathological conditions.

The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2'-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.

Gestational diabetes mellitus (GDM), a common pregnancy disorder, increases the risk of fetal overgrowth and later metabolic morbidity in the offspring. The placenta likely mediates these sequelae, but the exact mechanisms remain elusive. Mitochondrial dynamics refers to the joining and division of these organelles, in attempts to maintain cellular homeostasis in stress conditions or alterations in oxygen and fuel availability. These remodeling processes are critical to optimize mitochondrial function, and their disturbances characterize diabetes and obesity.

Aberrant ceramide build-up in preeclampsia, a serious disorder of pregnancy, causes exuberant autophagy-mediated trophoblast cell death. The significance of ceramide accumulation for lysosomal biogenesis in preeclampsia is unknown. Here we report that lysosome formation is markedly increased in trophoblast cells of early-onset preeclamptic placentae, in particular in syncytiotrophoblasts. This is accompanied by augmented levels of transcription factor EB (TFEB). In vitro and in vivo experiments demonstrate that ceramide increases TFEB expression and nuclear translocation and induces lysosomal formation and exocytosis. Further, we show that TFEB directly regulates the expression of lysosomal sphingomyelin phosphodiesterase (L-SMPD1) that degrades sphingomyelin to ceramide. In early-onset preeclampsia, ceramide-induced lysosomal exocytosis carries L-SMPD1 to the apical membrane of the syncytial epithelium, resulting in ceramide accumulation in lipid rafts and release of active L-SMPD1 via ceramide-enriched exosomes into the maternal circulation. The SMPD1-containing exosomes promote endothelial activation and impair endothelial tubule formation in vitro. Both exosome-induced processes are attenuated by SMPD1 inhibitors. These findings suggest that ceramide-induced lysosomal biogenesis and exocytosis in preeclamptic placentae contributes to maternal endothelial dysfunction, characteristic of this pathology.