Hannibal Agains Harris Stowe 2017 Online
Hypertension. Author manuscript; bachelor in PMC 2021 Feb 1.
Published in concluding edited grade every bit:
PMCID: PMC7027931
NIHMSID: NIHMS1549044
Reduced mRNA expression of Regulator of Yard Protein Signaling-2 (RGS2) in the placenta is associated with human preeclampsia and sufficient to cause features of the disorder in mice
Katherine J. Perschbacher
1Department of Pharmacology, Academy of Iowa, Iowa City, IA 52242 Usa
Guorui Deng
oneDepartment of Pharmacology, Academy of Iowa, Iowa Metropolis, IA 52242 USA
Jeremy A. Sandgren
1Department of Pharmacology, University of Iowa, Iowa City, IA 52242 Us
John W. Walsh
iSection of Pharmacology, University of Iowa, Iowa City, IA 52242 Usa
Phillip C. Witcher
iSection of Pharmacology, Academy of Iowa, Iowa City, IA 52242 Usa
Sarah A. Sapouckey
1Department of Pharmacology, University of Iowa, Iowa City, IA 52242 United states
Caitlyn Due east. Owens
iDepartment of Pharmacology, Academy of Iowa, Iowa City, IA 52242 USA
Shao Yang Zhang
1Section of Pharmacology, University of Iowa, Iowa City, IA 52242 U.s.a.
Sabrina M. Scroggins
2Department of Obstetrics & Gynecology, University of Iowa, Iowa City, IA 52242 USA
Nicole A. Pearson
aneDepartment of Pharmacology, University of Iowa, Iowa Metropolis, IA 52242 The states
Eric J. Devor
twoDepartment of Obstetrics & Gynecology, University of Iowa, Iowa Metropolis, IA 52242 Usa
Julien A. Sebag
3Section of Physiology, Academy of Iowa, Iowa Metropolis, IA 52242 U.s.
Gary L. Pierce
4Section of Wellness & Human Physiology, University of Iowa, Iowa City, IA 52242 USA
5Abboud Cardiovascular Research Centre, Academy of Iowa, Iowa Urban center, IA 52242 United states of america
Rory A. Fisher
1Department of Pharmacology, University of Iowa, Iowa City, IA 52242 USA
Anne E. Kwitek
6Department of Physiology, Medical Higher of Wisconsin, Milwaukee, WI 53226 U.s.
7Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 U.s.a.
eightDepartment of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226 United states
Donna A. Santillan
2Department of Obstetrics & Gynecology, University of Iowa, Iowa City, IA 52242 U.s.
5Abboud Cardiovascular Enquiry Eye, University of Iowa, Iowa City, IA 52242 USA
Katherine N. Gibson-Corley
9Department of Pathology, University of Iowa, Iowa City, IA 52242 The states
Curt D. Sigmund
6Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 USA
7Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 USA
Mark K. Santillan
twoDepartment of Obstetrics & Gynecology, University of Iowa, Iowa City, IA 52242 U.s.
5Abboud Cardiovascular Research Center, University of Iowa, Iowa Urban center, IA 52242 USA
Justin 50. Grobe
6Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 USA
viiCardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 USA
xDepartment of Biomedical Engineering science, Medical College of Wisconsin, Milwaukee, WI 53226 The states
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Abstract
Cascade-specific termination of G protein signaling is catalyzed by the Regulator of M protein Signaling (RGS) family members, including RGS2. Angiotensin, vasopressin, and endothelin are implicated in preeclampsia, and RGS2 is known to inhibit G protein cascades activated past these hormones. Mutations in RGS2 are associated with human hypertension and increased hazard of developing preeclampsia and its sequelae. RGS family unit members are known to influence maternal vascular function, but the role of RGS2 inside the placenta has not been explored. Here, we hypothesized that reduced expression of RGS2 within the placenta represents a risk factor for the evolution of preeclampsia. Although cAMP/CREB signaling was enriched in placentas from human pregnancies affected by preeclampsia compared to clinically-matched controls and RGS2 is known to be a CREB-responsive cistron, RGS2 mRNA was reduced in placentas from pregnancies affected past preeclampsia. Experimentally reducing Rgs2 expression inside the feto-placental unit was sufficient to induce preeclampsia-like phenotypes in meaning wildtype C57BL/6J mice. Stimulation of RGS2 transcription within immortalized homo HTR8/SVneo trophoblasts by camp/CREB signaling was discovered to be dependent upon the activity of histone deacetylase activeness, and more specifically, histone deacetylase-9 (HDAC9), and HDAC9 expression was reduced in placentas from man pregnancies afflicted by preeclampsia. We conclude that reduced expression of RGS2 inside the placenta may mechanistically contribute to preeclampsia. More than generally, this work identifies RGS2 every bit an HDAC9-dependent CREB-responsive gene, which may contribute to reduced RGS2 expression in placenta during preeclampsia.
Keywords: Pre-Eclampsia, Pregnancy, Hypertension, Placenta, G protein signaling
Graphical Abstract
Summary
Reduced expression of RGS2 within the placenta is a risk factor for the development of PreE. Agreement the causes and consequences of reduced placental expression of RGS2 may place novel diagnostic and therapeutic approaches to PreE.
Introduction
The pregnancy-specific hypertensive disorder, preeclampsia (PreE), remains a dominant worldwide crusade of maternal and infant mortality, and a wealth of evidence supports a primary role for the placenta in PreE development and maintenance.1 Increased G Protein coupled receptor (GPCR) signaling in response to various hormones including angiotensin, endothelin, and vasopressin is implicated in human PreE and fauna models of the status.2–vi
Termination of K protein signaling after activation by GPCRs involves hydrolysis of a phosphate from the guanosine triphosphate (GTP) bound to the Gα subunit, and this is accomplished via enzymatic activity of the Gα subunit. Regulator of G protein Signaling (RGS) family unit members accelerate this GTPase activity. The B/R4 family of RGS proteins is increasingly recognized equally a potential contributor to cardiovascular control during pregnancy.seven Holobotovskyy demonstrated that loss of RGS5 in maternal myometrium is associated with hypertensive disorders of pregnancy, including PreE.eight The Staff group has also demonstrated that a specific single nucleotide polymorphism (SNP) in the 3' untranslated region of the RGS2 cistron (rs4606) in mothers is associated with PreE.9–eleven Mutations in RGS2 are associated with human hypertension,12, thirteen and reduced RGS2 expression has been demonstrated in hypertensive populations.14 Deletion of Rgs2 has as well been shown to increase resistance and reduce menstruum in uterine arteries of non-significant mice.15
Given the role of the placenta in the pathogenesis and maintenance of PreE, it follows that B/R4 family members such equally RGS2 expressed within the placenta may contribute to this disorder. Here we demonstrate that RGS2 expression is suppressed in man PreE placenta, and that feto-placental disruption of Rgs2 is sufficient to initiate selected characteristics of PreE in meaning C57BL/6J mice. Finally, we document that RGS2 transcription in immortalized HTR8/SVneo human trophoblasts is dependent upon histone deacetylase (HDAC9) activity. This discovery supports the concept that reduced HDAC9 activeness may account for the observed reduction in RGS2 expression in PreE placenta.
Methods
Please see associated Supplemental Methods and Data file for details; data and materials are available from the corresponding authors upon reasonable request.
De-identified placental tissue samples and clinical data were obtained from the University of Iowa Maternal Fetal Tissue Bank. Mice harboring a null allele for Rgs2 were obtained from the MMRRC (B6.129P2-Rgs2 tm1Dgen /Mmnc, 011639-UNC). All studies were approved by the University of Iowa Institutional Animal Care & Use Committee. HTR8/SVneo cells were purchased from American Blazon Culture Collection (CRL-3271).
Transcriptomes of embryonic/gestational mean solar day (GD) 12.5 placentas were assessed by RNA-sequencing; data are deposited in the NCBI Cistron Expression Omnibus (GSE122503).
Analytical comparisons were performed using t-exam, Isle of man-Whitney U, or ANOVA followed by Tukey or Bonferroni multiple-comparing procedures, as indicated. Differences were considered meaning at p<0.05 using ii-tailed tests.
Results
RGS2 is expressed in placenta, and this expression is reduced during PreE
We previously reported that RGS B/R4 family unit members are expressed in multiple layers of human placenta throughout gestation, and that RGS2 is among the most strongly expressed members.vii Reanalysis of unmarried-prison cell RNAseq data from human placentas at vi–fourteen weeks of gestation16 demonstrates that in RGS2 is expressed in many cell types (Figure 1A). Similar results are reported at viii weeks of gestation17 and in third-trimester (Figures S1A–B).18 Single-cell sequencing similarly demonstrates that Rgs2 is expressed in essentially all cells of mouse placenta at GD14.5 (Figure S1C).19 Fluorescent in situ hybridization (RNAscope) besides confirms Rgs2 expression beyond various layers of placenta in wildtype C57BL/6J mice (Figure S2).
RGS2 mRNA is reduced in placenta during PreE in humans.
(A) RGS2 expression in individual cell types of human placenta at 6–14 weeks of gestation, assessed by droplet single-cell sequencing (data from Vento-Tormo et al.16). Horizonal black lines stand for median expression level inside each prison cell type. (B) RGS2 expression in preterm homo placenta during PreE (command, Vaginal n = thirteen ; command, C-department n = 22, PreE, C-section n = 47; information from {"blazon":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE75010). *p<0.05 by t-test. (C) RGS2 expression in human placental tissue from clinically various normal pregnancies and PreE pregnancies (Unproblematic due north = half-dozen, Complicated n = 6, History of PreE n = 6, All controls northward = 18, PreE n =xviii). *p<0.05 past Mann-Whitney U. (B-C) Summary data presented as mean±SEM.
Comparison of gestational-age matched human placentas ({"type":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE7501020) revealed RGS2 expression is significantly reduced in preterm placentas from PreE pregnancies compared to preterm physiological pregnancies (Figures 1B, S3). RGS2 was also sensitive to delivery method, equally expression is lower in placentas delivered by Cesarean department than vaginal birth. Confirming these in silico studies, the expression of RGS2 was suppressed in placentas from pregnancies complicated by PreE obtained via the University of Iowa Maternal Fetal Tissue Banking concern (MFTB21) (Figures 1C. Reanalysis of {"type":"entrez-geo","attrs":{"text":"GSE93839","term_id":"93839"}}GSE9383918 and qPCR assay of MFTB samples (Figure S3C) likewise support differential expression of RGS2 across layers of the placenta.
Commercially-bachelor antibodies against man RGS2 failed quality-control experiments for Western blotting applications (Figure S4), and therefore rigorous quantification of RGS2 poly peptide is not possible at this time. In dissimilarity, analysis of mouse RGS2 protein in placentas from mice confirms both the quality of antibodies against mouse RGS2, and cytoplasmic localization of RGS2 in the cytoplasm of cells in specific layers of the placenta (Figure S5). These findings bolster the concept that RGS2 protein is expressed by trophoblasts, and its subcellular localization within the cytoplasm is expected to let RGS2 to perform its canonical second-messenger modulating function.
Disruption of Rgs2 in the feto-placental unit is sufficient to model some clinical and many molecular features of PreE in mice
To dissociate the effects of disrupting Rgs2 in the dam as well equally in the placenta, we used a selective convenance strategy in which wildtype C57BL/6J dams were mated with Rgs2-deficient (Rgs2-KO) sires or with wildtype littermates of these Rgs2-KO sires. These matings resulted in pregnant wildtype C57BL/6J dams conveying feto-placental units that were either heterozygous or wildtype for Rgs2, respectively (Effigy 2A). This convenance approach was selected instead of attempting to generate homozygous-null feto-placental units, as generating such feto-placental units would accept required a convenance strategy in which the dam was either homozygous-cypher (ie - Rgs2-KO) or heterozygous-nix for Rgs2, and it has been previously demonstrated that adult mice with either heterozygous or homozygous disruption of Rgs2 exhibit hypertension in the non-pregnant state.22 Time to come studies examining the issue of homozygous Rgs2 disruption in the placenta, peradventure as a model of preeclampsia superimposed upon chronic hypertension, are warranted merely beyond the objectives of the current written report. Heterozygous disruption of Rgs2 resulted in an judge l% reduction in Rgs2 mRNA in the placenta (Figure 2A), confirming that Rgs2 expression is sensitive to gene copy number, and that utilize of this breeding scheme results in suppression of Rgs2 mRNA within the feto-placental unit during pregnancy in an otherwise wildtype C57BL/6J dam.
Selective breeding paradigm to reduce Rgs2 in the feto-placental unit of pregnant C57BL/6J mice.
(A) Schematic illustrating breeding scheme, and Rgs2 mRNA in GD17.5 placentas from dams mated with wildtype littermate (Littermate) or Rgs2-scarce (Rgs2-KO) males (Littermate n=vii, Rgs2-KO n=12, each from an independent pregnancy). (B) Furnishings of reduced feto-placental Rgs2 on maternal systolic (SBP) and diastolic (DBP) claret pressure, and center rate (60 minutes) (Littermate n=v, Rgs2-KO n=5). (C) Maternal urine protein excretion (Littermate due north=vii, Rgs2-KO due north=ten). (D) Spiral artery number and diameter (Littermate northward=vii, Rgs2-KO n=14). (E) Thicknesses of placental layers, and per centum of area in placental layers staining positive for CD31 every bit an index of total vascularization (Littermate n=7, Rgs2-KO due north=15). (F) Hif-1α localization in nuclear and chromatin-precipitated fractions of placentas at GD17.5 (Littermate northward=6, Rgs2-KO due north=12). Summary data presented as mean±SEM. *p<0.05 by 2-tailed t-test (A, C), or Bonferroni correction for multiple comparisons (B, Eastward).
Blood pressure was assessed in dams in the final week of gestation using previously-implanted radiotelemetric probes. Systolic claret pressure (SBP) was not modified by sire genotype, simply diastolic blood pressure (DBP) and middle rate (Hour) were significantly increased throughout the light cycle in dams mated with an Rgs2-KO sire (Figures 2B, S6). Protein loss in urine was significantly increased for dams mated with an Rgs2-KO sire (Figure 2C) and tissue harm was suggested by increased plasma alanine transferase concentrations, though no major structural abnormalities (such every bit glomerular endotheliosis) were observed in kidneys from either group by electron microscopy (Figures S7, S8A).
At GD12.five and at GD17.5 the number of total feto-placental units and rate of spontaneous resorption were indistinguishable betwixt groups (Table S1). Placental masses and fetal masses were increased at GD12.5 but normalized by GD17.five in dams mated with Rgs2-KO sires (Figures S8B–C). No pregnant differences in number of spiral arteries or diameter of screw arteries were observed between groups at GD12.5 (Effigy 2D). No differences were observed in maximum invasion depth by individual cytokeratin-viii (CK8)-positive trophoblasts into the decidua layer, nor the maximum depth of CK8-positive, remodeled screw arteries (Effigy S8D–E). No differences in angiogenic factors were observed in placenta at GD17.5, including placental growth gene (PGF) or Fms-related tyrosine kinase i (FLT1) (Figure S9A–B). The labyrinth and spongiotrophoblast layers were thickened at GD12.5, but no departure in decidual thickness was noted (Figure 2E). Immunohistochemical detection of platelet endothelial cell adhesion molecule (CD31) was besides used to assess total vascularization of the placental layers at GD12.5. In contrast to assessments of trophoblast invasion, the fraction of CD31-positive cells was significantly reduced in each layer of the placenta from dams mated with Rgs2-KO sires (Effigy 2E), consistent with a reduction in full vascularization of the placenta despite normal morphology of the screw arteries.
Hypoxia is frequently documented in human PreE placenta, and commonly-used animal models of PreE such every bit the reduced uterine perfusion pressure (RUPP) model specifically rely upon mid-gestational placental hypoxia/ischemia as a master etiology,23 but no show for hypoxia was observed in placentas from dams mated with Rgs2-KO sires. Subcellular localization of HIF1α to the nucleus or to precipitated chromatin were both unchanged (Figure 2F), and expression levels of hypoxia-responsive genes were unchanged in placenta (Figure S9C–D). Further, transcriptomes of GD12.five placentas (including decidua) were assessed using RNA sequencing, and Gene Set Enrichment Assay (GSEA) failed to provide evidence for enrichment of canonical hypoxia target genes (GRD gene set: FDR q-value=0.22, normalized enrichment score (NES)=1.25; and Harris gene gear up: MSigDB: {"blazon":"entrez-nucleotide","attrs":{"text":"M10508","term_id":"210484","term_text":"M10508"}}M10508, FDR q-value=0.16, NES=1.30).
Differential gene expression analyses identified increased expression of 479 genes and decreased expression of 247 genes in the GD12.5 placentas from dams mated with Rgs2-KO sires (Figure 3A, S10). Many transcriptomic changes observed in these placentas were like to expression changes previously reported in homo PreE placentas (Figure 3B). Ingenuity Pathway Analysis (IPA) was then used to identify enriched pathways and networks, and many cistron expression patterns previously identified as contradistinct in placenta from humans with PreE were identified as significantly enriched in GD12.v placentas from dams mated with Rgs2-KO sires, including mitochondrial dysfunction, unfolded protein responses, prison cell death & survival, cell motion, jail cell growth & proliferation, oxidative stress, increased levels of ALT, and red blood cell product (Tables S2–S5).
Reduced feto-placental expression of Rgs2 induces changes in GD12.v placental gene expression consistent with PreE.
(A) Heat map illustrating relative expression of differentially upregulated (479) and downregulated (247) genes identified by DeSeq2 (FDR < 0.1) (Littermate northward=6, Rgs2-KO n=6 placentas, each from an contained pregnancy). (B) Venn diagrams demonstrating comparisons of the up- and downregulated genes in Rgs2-heterozygous placentas to genes similarly changed in human placentas from pregnancies affected past PreE.xx, 36–38 Numbers indicate the total number of genes significantly changed in the individual datasets, or shared between datasets.
Transcriptional control of RGS2 by CREB is altered in placenta during PreE
Previously, the rs4606 SNP "1000" allele for RGS2 in mothers was associated with PreE, and the same allele is suspected to reduce RGS2 transcript stability.nine–11, xiv In the electric current cohort, we adamant that expression of RGS2 in placenta did not correlate with rs4606 genotype (Figure S11). This suggests that other mechanisms also contribute to reduced RGS2 in the placenta during PreE.
Methylation of the RGS2 promoter has also been implicated in command of RGS2 expression,24 but little methylation was observed in whatsoever placental sample by the bisulfite conversion method, and no alter in methylation of the RGS2 promoter was observed during PreE (Figure S12).
Expression of Rgs2 is stimulated in vascular smooth muscle by camp/CREB through serine-133 phosphorylated CREB (pCREB) bounden to a specific cAMP response chemical element (CRE) sequence inside the Rgs2 promoter.25 We examined whether this pathway was functional in immortalized HTR-8/SVneo (HTR8) starting time-trimester human being trophoblasts. Increasing cAMP via forskolin (FSK) resulted in dose- and time-dependent increases in expression of RGS2, and chromatin immunoprecipitation (ChIP) confirmed FSK-stimulated binding of pCREB to the RGS2 promoter in these cells (Figure 4A).
Transcriptional regulation of RGS2 by the cAMP/CREB signaling pathway is impaired in human PreE.
(A) Dose- and fourth dimension-dependent responses of RGS2 mRNA in HTR8/SVneo cells treated with Forskolin (n=3 each dose / time), and serine-133 phosphorylated CREB (pCREB) presence at the RGS2 promoter in HTR8 cells after treatment with FSK (n=7 each). (B) cAMP concentrations (Control north=8, PreE n=eleven), ratio of serine-133 phosphorylated CREB to full CREB (Control n=4, PreE due north=five), and serine-133 phosphorylated CREB presence at the RGS2 promoter (Control n=7, PreE northward=6) in placenta from humans with or without PreE. (C) Correlation of RGS2 mRNA content to pCREB occupancy at the RGS2 promoter in human being placental tissue (Command n=v, PreE n=half dozen); comparison of curves past extra sum-of-squares: F=56.65 (ii,7), p<0.0001. (D) NR4A1, NR4A3, CREM, and RGS2 mRNA in HTR8/SVneo cells later stimulation past forskolin in the presence or absence of the not-selective HDAC inhibitor, SAHA (Vehicle n=eleven, SAHA n=14). (E) Serine-133 phosphorylated CREB occupancy at the RGS2 and FOS promoters of HTR8/SVneo cells (n=iv each). (F) Approved CREB target cistron expression in human placentas past in silico reanalysis of {"type":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE75010 (Control n=22, PreE n=47). Summary data presented every bit mean±SEM. *p<0.05 by t-test (A, B, F) or Tukey multiple-comparison procedure (D, E).
Nosotros examined the condition of the cAMP/CREB/RGS2 machinery in PreE placenta, as others have previously documented increased military camp/CREB activities in contained cohorts of PreE placenta.26, 27 In the present accomplice, the concentration of cAMP in placental tissue exhibited a non-pregnant increase during PreE, and CREB phosphorylation at serine balance 133 was significantly increased during PreE (Figures 4B, S13). Boosted in silico reanalysis of {"type":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE75010xx by GSEA also uncovered significant enrichment of CREB target gene expression during PreE (FDR q-value=0.037, NES=1.54). Despite these multiple lines of bear witness supporting normal or increased cAMP/CREB signaling in the placenta during PreE, pCREB bounden to the RGS2 promoter was significantly reduced in PreE placenta, consistent with an RGS2 locus-specific upshot (Figure 4B). Correlational analyses of the expression of RGS2 versus pCREB binding to the RGS2 promoter in individual placenta samples illustrates a complex alteration in the human relationship between pCREB binding and RGS2 expression during PreE, every bit both the gradient and intercept of the lines of regression are changed during PreE (Effigy 4C).
Previously, Fass demonstrated that inhibition of histone deacetylases (HDACs) increases expression of multiple CREB target genes in PC12 cells with FSK application, including FOS and NR4A1.28 In contrast, HDAC inhibition antagonized FSK-induced expression of a subset of CREB targets including NR4A3 and CREM (ICER). Consistent with the findings of Fass, we determined that FSK application increased expression of NR4A1, NR4A3, and CREM in cultured HTR8 cells, and the non-specific HDAC inhibitor, suberanilohydroxamic acid (SAHA) interfered with consecration of NR4A3 and CREM (Effigy 4D). Nosotros adjacent discovered that the consecration of RGS2 expression by FSK is dependent upon HDAC activity (Figure 4D). SAHA co-treatment likewise attenuated pCREB bounden to the RGS2 promoter (Figures 4E, S14). Chiefly, pCREB bounden to another (HDAC-contained) campsite responsive gene, FOS, was unaffected by SAHA treatment. Consistent with the concept that HDAC activity may be impaired in PreE placenta, mRNA levels of canonical CREB responsive genes such as FLT1, DUSP1, RAB25, LDHA and CRKL were increased in placenta during PreE, while the HDAC-dependent CREB responsive genes NR4A3 and CREM were unchanged (Figure 4F, S15).
Reductions in HDAC3, HDAC8, and HDAC9, and increased HDAC10 mRNA were identified in PreE placenta via in silico reanalysis of {"type":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE75010 (Figure 5A). These findings are consistent with the recent written report that HDAC9 expression is suppressed in PreE placenta, and that HDAC9 knockdown inhibits proliferation, migration and invasion of HTR8/svneo cells.29 Activities of HDAC3, HDAC8, and HDAC10 are inhibited by benzoylhydrazide scaffold compound UF010, but this compound has no upshot on HDAC9.30 UF010 had no inhibitory upshot upon FSK-mediated stimulation of RGS2 in HTR8 cells (Figure 5B), consistent with a role for HDAC9 in RGS2 control. Further, siRNA-mediated selective knockdown of HDAC9 but not HDAC8 in HTR8 cells resulted in reduced FSK-mediated stimulation of RGS2 merely not the SAHA-insensitive NR4A1 (Effigy 5C). Interestingly, HDAC9 knockdown had no inhibitory effect upon FSK-mediated stimulation of other SAHA-sensitive genes NR4A3 and CREM, indicating that subsets of HDAC-dependent CREB-responsive genes are regulated by distinct HDAC enzymes. These results lead us to the working model that reduced HDAC9 action in results in reduced CREB-mediated consecration of RGS2 in the trophoblast, which would exist expected to disinhibit GPCR signaling and promote placental dysfunction and PreE (Figure 5D).
Transcriptional control of RGS2 by CREB is dependent upon HDAC9 activity.
(A) HDAC cistron mRNA in human placentas by in silico reanalysis of {"type":"entrez-geo","attrs":{"text":"GSE75010","term_id":"75010"}}GSE75010 (Command n=22, PreE north=47). (B) RGS2 mRNA in HTR8/SVneo cells after stimulation with forskolin (FSK) in the presence or absenteeism of the selective HDAC inhibitor, UF010, which inhibits HDAC3, HDAC8, and HDAC10 but non HDAC9 (Vehicle n=19, UF010 n=18, FSK northward=18, FSK+UF010 n=12). (C) HDAC9, RGS2, NR4A1, NR4A3, and CREM mRNA in HTR8/SVneo cells after stimulation with FSK following selective knockdown of HDAC9 or HDAC8 by siRNA (scrambled siRNA control: n=seven vs n=eight; siRNA against HDAC9: north=4 vs n=5; siRNA against HDAC8: due north=3 vs n=3). Summary data presented as mean±SEM. *p<0.05 by Tukey multiple-comparison process. (D) Working model.
Word
A major role for the placenta in the development and maintenance of PreE is widely accepted, and the current written report demonstrates an association betwixt PreE with reduced expression of RGS2 in the man placenta, and the sufficiency of reduced feto-placental expression of Rgs2 to elicit several physiological and molecular PreE-similar phenotypes in wildtype C57BL/6J dams.
Previously we reported the sufficiency of chronic low-dose infusion of arginine vasopressin to induce all of the clinical features of PreE in C57BL/6J dams without inducing placental hypoxia.6 Similarly, here nosotros written report that reduced expression of Rgs2 in the feto-placental unit is sufficient to induce PreE-like clinical features without inducing placental hypoxia. Interestingly, both models induced other molecular signatures within the placenta that parallel PreE, including mitochondrial dysfunction, oxidative stress and unfolded poly peptide responses. Others have demonstrated that surgical consecration of placental hypoxia/ischemia (eg – the RUPP model) is sufficient to induce PreE-like phenotypes without modifying AVP secretion.31 These findings lead to the concept that abnormal GPCR signaling and hypoxia – which are both documented in human PreE – exist as independent, and possibly synergistic, contributors to the pathogenesis of PreE.six The variable contributions of multiple independent mechanisms to the pathogenesis of PreE may help explain the heterogeneity of clinical presentations of this disorder (early/belatedly, balmy/astringent, etc.) and the growing appreciation for multiple independent 'molecular subclasses' of PreE.20
RGS2 has previously been associated with hypertensive disorders of pregnancy such every bit PreE, though not specifically within the placenta. Mothers carrying the G allele for the rs4606 SNP in RGS2 exhibit increased risk of PreE.9–11 This association is modified by relevant co-variates such equally BMI,9 which may reflect obesity-associated increases in circulating hormones that activate RGS2-sensitive G protein signaling, such as angiotensin Two32 or arginine vasopressin33. Although HUNT2 studies demonstrated that the maternal genotype for rs4606 was associated with increased risk of PreE, information technology is notable that RGS2 mRNA expression levels were non assessed, and that the placental rs4606 genotypes were as well non assessed. The rs4606 genotype has been studied exterior the context of PreE, notwithstanding, as Semplicini et al. demonstrated that rs4606 is associated with homo hypertension and is associated with decreased RGS2 mRNA and protein in peripheral blood mononuclear cells.14 Our data that placental rs4606 genotype did not correlate with RGS2 mRNA marshal with findings from Mendelova, in which no clan between maternal rs4606 genotype and PreE status was observed.34 We conclude that rs4606 may confer moderate, maybe tissue-specific, changes in control of RGS2 just that this machinery is unlikely to account for the large reductions in RGS2 observed in placental tissue during PreE – and may betoken that RGS2 plays multiple independent roles in mother and placenta that each independently contribute to PreE.
Impaired transcriptional regulation of RGS2 could also occur due to mutations within the promoter, or deficiencies in critical transcriptional regulators. More than than 30 polymorphisms have been identified within the RGS2 gene in hypertensive humans, and three of these SNP's flank the functional CRE chemical element within the RGS2 promoter.25 It is possible that mutations in these locations could interfere with CREB bounden to the CRE sequence,25 however their low prevalence is unlikely to account for the reductions in RGS2 expression observed in PreE at the population level. The mechanism by which HDAC9 modulates CREB-mediated induction of transcription remains unclear merely probable involves a circuitous of interacting factors at the RGS2 promoter and upstream enhancer (Tables S8, S9), warranting future studies.
One major limitation facing the electric current study is the lack of tools available to assess RGS2 poly peptide levels or to assess its activity in human being tissues. Another major limitation facing the current study is the lack of data addressing cell- or layer-specific changes in factor expression in PreE. Ongoing piece of work by our group and others to explore cell-type specific changes in transcriptomes of PreE versus control placentas using single-cell RNAsequencing techniques promises to provide this type of assay. A third major limitation stems from the lack of an animal harboring a conditional Rgs2 allele (eg – Rgs2 flox ) to permit layer-specific disruptions of the cistron in mouse placenta. Our squad is currently working to regenerate such a model, as the only previously-reported brute of this blazon was lost in 2012.35 The current study provides articulate rationale for the pursuit of such tools and studies.
Supplementary Material
Supplemental Cloth
Long In Vivo Checklist
Acknowledgments
The authors gratefully acknowledge the participation of mothers in the University of Iowa MFTB. Multiple cadre facilities supported the project, including the University of Iowa Office of Animal Resources, Genome Editing Facility, and DNA Cadre Facility.
Sources of Funding
This work was supported by the American Heart Association (15SFRN23730000, 16PRE30980043, 16POST30960016, 17PRE33660633, 18PRE33960377, 18EIA33890055, 19POST34380239), National Institutes of Health (HL134850, HL084207, HD089940, UL1TR002537, P30CA086862), Roy Carver Trust, American Physiological Club, and University of Iowa Medical Student and Summer Undergraduate MSTP Research programs.
Footnotes
Disclosures
JLG, DAS, and MKS concur patents related to AVP for the prediction and treatment of PreE: US #9,937,182 (April 10, 2018), EU #2,954,324 (July 31, 2019), and PCT/US2018/027152.
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