Taryn Summerfield

Nuclear Factor-Kappa B Regulates Inducible Prostaglandin E Synthase Expression in Human Amnion Mesenchymal Cells

Abstract The human amnion is a major intrauterine source of prostaglandin (PG) E2, a potent mediator of uterine contractions and cervical ripening. During parturition, inflammatory cytokines promote PGE2 production through increased prostaglandin-endoperoxide synthase-2 (PTGS2, also known as cyclooxygenase-2) expression. This is mediated, in part, through activation of the transcription factor nuclear factor kappa B (NFkappaB). Prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1) acts downstream of PTGS2 and is inducibly expressed in most systems. We hypothesized that NFkappaB might regulate cytokine-induced PTGES expression in amnion cells. With amnion mesenchymal cells, we found that proinflammatory cytokines coordinately upregulated PTGS2 and PTGES mRNA expression. In parallel, increased expression of the PTGS2 and PTGES proteins was observed. In comparison, the expression of two other PGE synthases (PTGES2 and PTGES3) was unmodified. PTGES induction was blocked both in the presence of pharmacological NFkappaB inhibitors and following adenovirus-mediated overexpression of a dominant-negative NFkappaB pathway protein. In cells transiently transfected with a luciferase reporter bearing a portion (−597/+33) of the human PTGES gene promoter, interleukin-1beta (IL1B) produced a moderate increase in luciferase activity; this effect was abrogated in the presence of an indirect NFkappaB inhibitor (MG-132). Finally, a kappaB-like regulatory element was identified that, when mutated, markedly attenuated IL1B-responsive PTGES promoter activity. In conclusion, our results support a role for NFkappaB in cytokine-induced PTGES expression in amnion mesenchymal cells in vitro. By coordinately regulating PTGS2 and PTGES, NFkappaB may contribute to an inducible PGE2 biosynthesis pathway during human parturition.

Distinct cellular pools of perilipin 5 point to roles in lipid trafficking

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~1.15 g/ml) and size (~575 kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~15 nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.

Loss of Myoferlin Redirects Breast Cancer Cell Motility towards Collective Migration

Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231MYOF-KD) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA- 231LTVC) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA- 231MYOF-KD cells migrated directionally and collectively, while MDA-231LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231MYOF-KD cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA- 231MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA- 231MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA- 231LTVC-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes.

BackgroundThe objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor.MethodsPaired full-thickness FM and myometrial samples were collected from women in the following cohorts: preterm no labor (PNL, N = 22), spontaneous preterm labor (PTL, N = 21), term no labor (TNL, N = 23), and spontaneous term labor (STL, N = 21). NF-kappaB p65 localization was assessed by immunohistochemistry, and DNA binding activity was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based method.ResultsNuclear p65 labeling was rare in amnion and chorion, irrespective of clinical context. In decidua, nuclear p65 labeling was greater in the STL group relative to the TNL cohort, but there were no differences among the TNL, PTL, and PNL cohorts. In myometrium, diffuse p65 nuclear labeling was significantly associated with both term and preterm labor. There were no significant differences in ELISA-based p65 binding activity in amnion, choriodecidual, and myometrial specimens in the absence or presence of term labor. However, parallel experiments using cultured term fetal membranes demonstrated high levels of p65-like binding even the absence of cytokine stimulation, suggesting that this assay may be of limited value when applied to tissue specimens.ConclusionsThese results suggest that the decidua is an important site of NF-kappaB regulation in fetal membranes, and that mechanisms other than cytoplasmic sequestration may limit NF-kappaB activation prior to term.

Inflammatory Gene Regulatory Networks in Amnion Cells Following Cytokine Stimulation: Translational Systems Approach to Modeling Human Parturition

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1β, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.

Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition

BACKGROUND Inflammation is a proximate mediator of preterm birth and fetal injury. During inflammation several microRNAs (22 nucleotide noncoding ribonucleic acid (RNA) molecules) are up-regulated in response to cytokines such as interleukin-1β. MicroRNAs, in most cases, fine-tune gene expression, including both up-regulation and down-regulation of their target genes. However, the role of pro- and antiinflammatory microRNAs in this process is poorly understood. OBJECTIVE The principal goal of the work was to examine the inflammatory genomic profile of human decidual cells challenged with a proinflammatory cytokine known to be present in the setting of preterm parturition. We determined the coding (messenger RNA) and noncoding (microRNA) sequences to construct a network of interacting genes during inflammation using an in vitro model of decidual stromal cells. STUDY DESIGN The effects of interleukin-1β exposure on mature microRNA expression were tested in human decidual cell cultures using the multiplexed NanoString platform, whereas the global inflammatory transcriptional response was measured using oligonucleotide microarrays. Differential expression of select transcripts was confirmed by quantitative real time-polymerase chain reaction. Bioinformatics tools were used to infer transcription factor activation and regulatory interactions. RESULTS Interleukin-1β elicited up- and down-regulation of 350 and 78 nonredundant transcripts (false discovery rate < 0.1), respectively, including induction of numerous cytokines, chemokines, and other inflammatory mediators. Whereas this transcriptional response included marked changes in several microRNA gene loci, the pool of fully processed, mature microRNA was comparatively stable following a cytokine challenge. Of a total of 6 mature microRNAs identified as being differentially expressed by NanoString profiling, 2 (miR-146a and miR-155) were validated by quantitative real time-polymerase chain reaction. Using complementary bioinformatics approaches, activation of several inflammatory transcription factors could be inferred downstream of interleukin-1β based on the overall transcriptional response. Further analysis revealed that miR-146a and miR-155 both target genes involved in inflammatory signaling, including Toll-like receptor and mitogen-activated protein kinase pathways. CONCLUSION Stimulation of decidual cells with interleukin-1β alters the expression of microRNAs that function to temper proinflammatory signaling. In this setting, some microRNAs may be involved in tissue-level inflammation during the bulk of gestation and assist in pregnancy maintenance.

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research.

Agonist-Dependent Downregulation of Progesterone Receptors in Human Cervical Stromal Fibroblasts.

Progesterone (P4) maintains uterine quiescence during the majority of pregnancy, whereas diminished progesterone receptor (PR) expression and/or activity (ie, functional P4 withdrawal) promotes parturition. To investigate the regulation of PR expression in cervical stroma, fibroblasts from premenopausal hysterectomy specimens were prepared. Greater than 99% of the cultures were vimentin positive (mesenchymal cell marker) with only occasional cytokeratin-8 positivity (epithelial cell marker) and no evidence of CD31-positive (endothelial cell marker) cells. Cells were immunolabeled with antibodies directed against PRs (PR-A and PR-B), estrogen receptor α (ER-α), and glucocorticoid receptor-α/β (GR-α/β). All cells were uniformly immunopositive for ER-α and GR-α/β but did not express PRs. Incubation of cells with 10−8 mol/L 17β-estradiol induced a time-dependent increase in PR-A and PR-B messenger RNAs (mRNAs) by quantitative real-time polymerase chain reactions and proteins by immunoblotting and immunofluorescence. Incubation of cervical fibroblasts with PR ligands (medroxyprogesterone acetate or Org-2058) downregulated PR-A and PR-B levels. Coincubation of cells with PR ligands plus RU-486, a PR antagonist, partially abrogated agonist-induced receptor downregulation. Dexamethasone, a pure glucocorticoid, had no inhibitory effect on PR expression. These results indicate that progestins and estrogens regulate PR expression in cervical fibroblasts. We postulate that hormonal regulation of PR expression in the cervical stroma may contribute to functional P4 withdrawal in preparation for parturition.

Long-acting progestin-only contraceptives impair endometrial vasculature by inhibiting uterine vascular smooth muscle cell survival

Significance Over a million unintended pregnancies occur in the United States each year because of either discontinuation or misuse of contraceptives. The major reason for discontinuation of long-acting progestin-only contraceptives (LAPCs) is the occurrence of abnormal uterine bleeding (AUB). Uncovering the mechanisms underlying LAPC-induced AUB is essential to prevent their discontinuation. We found that LAPCs reduce proliferation of human and guinea pig endometrial vascular smooth muscle cells (VSMCs), resulting in production of thin-walled hyperdilated fragile microvessels. In cultured VSMCs, chemokine (C-C motif) ligand 2 reverses LAPC-mediated inhibition of VSMC proliferation, suggesting that LAPCs impair endometrial vascular integrity and that chemokine ligand 2 administration may prevent LAPC-induced AUB. Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impair vascular smooth muscle cell (VSMC) and pericyte proliferation and migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (αSMA) double-immunostaining assessed VSMC differentiation and proliferation in endometria from women before and after DepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA), or E2+MPA. Whole-genome profiling, proliferation, and migration assays were performed on cultured VSMCs treated with MPA or etonogestrel (ETO). Endometrial vessels of Depo-administered women displayed reduced αSMA immunoreactivity and fewer PCNA (+) nuclei among αSMA (+) cells (P < 0.008). Microarray analysis of VSMCs identified several MPA- and ETO-altered transcripts regulated by STAT1 signaling (P < 2.22 × 10−6), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduce VSMC proliferation and migration (P < 0.001). Recombinant CCL2 reversed this progestin-mediated inhibition, whereas a STAT1 inhibitor abolished the CCL2 effect. Similarly, the endometria of MPA treated OVX-GPs displayed decreased αSMA staining and fewer PCNA (+) nuclei in VSMC (P < 0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration.

Skin Microbiota in Obese Women at Risk for Surgical Site Infection After Cesarean Delivery

The obesity pandemic in the obstetrical population plus increased frequency of Cesarean delivery (CD) has increased vulnerability to surgical site infection (SSI). Here we characterized the microbiome at the site of skin incision before and after CD. Skin and relevant surgical sites were sampled before and after surgical antisepsis from obese (n = 31) and non-obese (n = 27) pregnant women. We quantified bacterial biomass by qPCR, microbial community composition by 16sRNA sequencing, assigned operational taxonomic units, and stained skin biopsies from incision for bacteria and biofilms. In obese women, incision site harbors significantly higher bacterial biomass of lower diversity. Phylum Firmicutes predominated over Actinobacteria, with phylotypes Clostridales and Bacteroidales over commensal Staphylococcus and Propionbacterium spp. Skin dysbiosis increased post-surgical prep and at end of surgery. Biofilms were identified post-prep in the majority (73%) of skin biopsies. At end of surgery, incision had significant gains in bacterial DNA and diversity, and obese women shared more genera with vagina and surgeon’s glove in CD. Our findings suggest microbiota at incision differs between obese and non-obese pregnant women, and changes throughout CD. An interaction between vaginal and cutaneous dysbiosis at the incision site may explain the a priori increased risk for SSI among obese pregnant women.