Mono-(2-ethyl-5-hydroxyhexyl) phthalate promotes uterine leiomyoma cell survival through tryptophan-kynurenine-AHR pathway activation.

Uterine leiomyoma is the most common tumor in women and causes severe morbidity in 15 to 30% of reproductive-age women. Epidemiological studies consistently indicate a correlation between leiomyoma development and exposure to endocrine-disrupting chemical phthalates, especially di-(2-ethylhexyl) phthalate (DEHP); however, the underlying mechanisms are unknown. Here, among the most commonly encountered phthalate metabolites, we found the strongest association between the urine levels of mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), the principal DEHP metabolite, and the risk of uterine leiomyoma diagnosis (n = 712 patients). The treatment of primary leiomyoma and smooth muscle cells (n = 29) with various mixtures of phthalate metabolites, at concentrations equivalent to those detected in urine samples, significantly increased cell viability and decreased apoptosis. MEHHP had the strongest effects on both cell viability and apoptosis. MEHHP increased cellular tryptophan and kynurenine levels strikingly and induced the expression of the tryptophan transporters SLC7A5 and SLC7A8, as well as, tryptophan 2,3-dioxygenase (TDO2), the key enzyme catalyzing the conversion of tryptophan to kynurenine that is the endogenous ligand of aryl hydrocarbon receptor (AHR). MEHHP stimulated nuclear localization of AHR and up-regulated the expression of CYP1A1 and CYP1B1, two prototype targets of AHR. siRNA knockdown or pharmacological inhibition of SLC7A5/SLC7A8, TDO2, or AHR abolished MEHHP-mediated effects on leiomyoma cell survival. These findings indicate that MEHHP promotes leiomyoma cell survival by activating the tryptophan-kynurenine-AHR pathway. This study pinpoints MEHHP exposure as a high-risk factor for leiomyoma growth, uncovers a mechanism by which exposure to environmental phthalate impacts leiomyoma pathogenesis, and may lead to the development of novel druggable targets.

Lyophilization of human amniotic fluid is feasible without affecting biological activity.

BACKGROUND: Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs. METHODS: We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74. RESULTS: After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01). CONCLUSIONS: Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.

Transcriptional Profiling of Human Endocervical Tissues Reveals Distinct Gene Expression in the Follicular and Luteal Phases of the Menstrual Cycle.

The endocervix plays an important role in providing appropriate protective mechanisms of the upper female reproductive tract (FRT) while at the same time providing the appropriate milieu for sperm transport. Hormone fluctuations throughout the menstrual cycle contribute to changes in the mucosal environment that render the FRT vulnerable to infectious diseases. The objective of this study was to identify genes in human endocervix tissues that were differentially expressed in the follicular versus the luteal phases of the menstrual cycle using gene expression profiling. A microarray using the IIlumina platform was performed with eight endocervix tissues from follicular and four tissues from luteal phases of the menstrual cycle. Data analysis revealed significant differential expression of 110 genes between the two phases, with a P value <0.05 and a fold change cutoff of 1.5. Categorization of these genes, using Ingenuity Pathway Analysis, MetaCore from Thomson Reuters, and DAVID, revealed genes associated with extracellular matrix remodeling and cell-matrix interactions, amino acid metabolism, and lipid metabolism, as well as immune regulation in the follicular phase tissues. In luteal phase tissues, genes associated with chromatin remodeling, inflammation, angiogenesis, oxidative stress, and immune cell regulation were predominately expressed. Using samples from additional patients' tissues, select genes were confirmed by quantitative real-time PCR; immunohistochemical staining was also done to examine protein levels. This is the first microarray analysis comparing gene expression in endocervix tissues in cycling women. This study identified key genes and molecular pathways that were differentially regulated during the menstrual cycle.

Paracrine activation of WNT/ß-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/ß-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of ß-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of ß-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/ß-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Identification of FISH biomarkers to detect chromosome abnormalities associated with prostate adenocarcinoma in tumour and field effect environment.

BACKGROUND: To reduce sampling error associated with cancer detection in prostate needle biopsies, we explored the possibility of using fluorescence in situ hybridisation (FISH) to detect chromosomal abnormalities in the histologically benign prostate tissue from patients with adenocarcinoma of prostate. METHODS: Tumour specimens from 33 radical prostatectomy (RP) cases, histologically benign tissue from 17 of the 33 RP cases, and 26 benign prostatic hyperplasia (BPH) control cases were evaluated with Locus Specific Identifier (LSI) probes MYC (8q24), LPL (8p21.22), and PTEN (10q23), as well as with centromere enumerator probes CEP8, CEP10, and CEP7. A distribution of FISH signals in the tumour and histologically benign adjacent tissue was compared to that in BPH specimens using receiver operating characteristic curve analysis. RESULTS: The combination of MYC gain, CEP8 Abnormal, PTEN loss or chromosome 7 aneusomy was positive in the tumour area of all of the 33 specimens from patients with adenocarcinomas, and in 88% of adjacent histologically benign regions (15 out of 17) but in only 15% (4 out of 26) of the benign prostatic hyperplasia control specimens. CONCLUSIONS: A panel of FISH markers may allow detection of genomic abnormalities that associate with adenocarcinoma in the field adjacent to and surrounding the tumour, and thus could potentially indicate the presence of cancer in the specimen even if the cancer focus itself was missed by biopsy and histology review.

Atypical Systemic Neonatal Herpes Diagnosed Incidentally in Twin Infants.

Neonatal herpes simplex virus (HSV) infections are serious infections that usually occur in the first few weeks of life. Infants generally present with mucocutaneous lesions, central nervous system infection, and/or systemic disease. In this case report, we describe a set of twins that had unexpected presentations of neonatal HSV. Twin A was diagnosed incidentally on routine eye exam, and Twin B was diagnosed only because his twin was found to be infected; both infants were still hospitalized and were beyond 1 month of age. These twins exhibited atypical manifestations that diverge from the 3 main categories of neonatal HSV and expand our understanding of the spectrum of disease.

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

The post-adjustable gastric band abdominoplasty.

Serious complications associated with post-laparoscopic adjustable gastric band (LAGB) abdominoplasty have been reported in the medical literature. Furthermore, others have noted aesthetic problems with closure of the umbilicus due to apparatus port proximity. Currently, no clinical protocol or formal industry guidance for LAGB apparatus management during abdominoplasty is available in the medical literature. In this article, the authors describe their procedure for safe LAGB apparatus management during abdominoplasty and illustrate key surgical principles by presenting unique cases from their series of 20 patients treated with this technique.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Enhancement of a multianalyte serum biomarker panel to identify lymph node metastases in non-small cell lung cancer with circulating autoantibody biomarkers.

We recently reported the development of a multianalyte serum algorithm to identify nodal status in non-small cell lung cancer (NSCLC) patients facing an anatomic resection with curative intent. This study aims to enhance the overall performance characteristics of this test by adding autoantibody biomarkers identified through immunoproteomic discovery. More specifically, we used sera from 20 NSCLC patients to probe 2-D immunoblots of HCC827 lysates for tumor-associated autoantigens. Relevant differences in immunoreactivity associated with pathological nodal status were then identified via tandem mass spectrometry. Identified autoantigens were then developed into Luminex immunobead assays alongside a series of autoantigen targets relevant to early-disease detection. These assays were then used to measure circulating autoantibody levels in the identical cohort of NSCLC patients used in our original study. This strategy identified 11 autoantigens found primarily in patients with disease progression to the locoregional lymph nodes. Custom Luminex-based "direct-capture" assays (25 total; including autoantibody targets relevant to early-disease detection) were assembled to measure autoantibody levels in sera from 107 NSCLC patients. Multivariate classification algorithms were then used to identify the optimal combination of biomarkers when considered collectively with our original 6-analyte serum panel. The new algorithm resulting from this analysis consists of TNF-α, TNF-RI, MIP-1α and autoantibodies against Ubiquilin-1, hydroxysteroid-(17-ß)-dehydrogenase, and triosephosphate isomerase. The inclusion of autoantibody biomarkers provided a dramatic improvement in the overall test performance characteristics, relative to the original test panel, including an 11% improvement in the classification efficiency.

PTEN, RASSF1 and DAPK site-specific hypermethylation and outcome in surgically treated stage I and II nonsmall cell lung cancer patients.

The primary objective of this study is to identify prognostic site-specific epigenetic changes in surgically treated Stage I and II nonsmall cell lung cancer (NSCLC) patients by quantifying methylation levels at multiple CpG sites within each gene promoter. Paraffin-embedded tumors from stage Ib, IIa and IIb in training and validation groups of 75 and 57 surgically treated NSCLC patients, respectively, were analyzed for p16, MGMT, RASSF1, RASSF5, CDH1, LET7, DAPK and PTEN promoter hypermethylation. Hypermethylation status was quantified individually at multiple CpG sites within each promoter by pyrosequencing. Molecular and clinical characteristics with time to recurrence (TTR) and overall survival (OS) were evaluated. Overall average promoter methylation levels of MGMT and RASSF1 were significantly higher in smokers than in nonsmokers (p = 0.006 and p = 0.029, respectively). Methylation levels of the p16 promoter were significantly higher in squamous cell carcinoma than in adenocarcinoma (p = 0.020). In univariate analysis, hypermethylation of RASSF1 at CpG sites -53 and -48 and PTEN at CpG site -1310 were the significantly associated with shorter TTR (p = 0.002 and p < 0.000, respectively). Hypermethylation of PTEN at -1310 and DAPK at -1482 were most significantly associated with outcome in multivariate analysis. These results show that methylation of specific promoter CpG sites in PTEN, RASSF1 and DAPK is associated with outcome in early stage surgically treated NSCLC.

The potential predictive value of cyclooxygenase-2 expression and increased risk of gastrointestinal hemorrhage in advanced non-small cell lung cancer patients treated with erlotinib and celecoxib.

PURPOSE: Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, potentiates antitumor effects of erlotinib in preclinical studies, and COX-2 is frequently expressed in non-small cell lung cancer (NSCLC). With these observations, we designed a phase II trial to evaluate the efficacy and safety of erlotinib plus celecoxib in advanced NSCLC. EXPERIMENTAL DESIGN: Previously treated stage IIIB/IV NSCLC patients were given celecoxib at 400 mg orally twice daily and erlotinib at 150 mg orally daily until disease progression. Planned accrual was 40 patients. Tissue was collected for epidermal growth factor receptor (EGFR) analysis and COX-2 immunohistochemistry. RESULTS: Twenty-six patients were enrolled (17 men, 9 women; median age, 66 years). Eighteen and 21 patients had tissue available for EGFR analysis and COX-2 immunohistochemistry, respectively. The median progression-free survival (PFS) and overall survival were 2.0 and 9.2 months, respectively. Eleven of 21 patients tested had increased tumor COX-2 expression, which was strongly associated with prolonged PFS (P=0.048). Four patients on anticoagulation or with a history of peptic ulcer disease had grade 3/grade 4 upper gastrointestinal bleeding (GIB), prompting early study closure. Three patients with GIB had endoscopy that found peptic ulcers. CONCLUSIONS: The combination of erlotinib and celecoxib does not seem superior to erlotinib alone in unselected patients. However, longer PFS with high-tumor COX-2 expression suggests that trials of EGFR and COX-2 inhibitors may be warranted in this patient subset. GIB observed in our trial supports excluding patients with a history of peptic ulcer disease or those requiring therapeutic anticoagulation from future EGFR and COX-2 inhibitor studies.

PLIN2 Functions As a Novel Link Between Progesterone Signaling and Metabolism in Uterine Leiomyoma Cells.

CONTEXT: Uterine leiomyoma (fibroids) are the most common tumors in women. Recently, perilipin-2 (PLIN2) was identified as a critical target gene of the progesterone receptor; however, its function in the pathogenesis of fibroids is unknown. OBJECTIVE: To determine the function of PLIN2 in leiomyoma cells. DESIGN: Tissue and primary cells from leiomyoma and myometrium were analyzed. PLIN2 function in leiomyoma was assessed using small interfering RNA. RNA-sequencing was performed to identify genome-wide effects of PLIN2 depletion. Metabolic activity was measured using the Seahorse XF96 analyzer. Real-time quantitative PCR and immunoblotting were also performed. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Forty-one premenopausal women undergoing surgery for fibroids. MAIN OUTCOME MEASURES: Gene expression, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and cell proliferation. RESULTS: PLIN2 gene expression was 2.4-fold lower in leiomyoma compared with adjacent myometrium, suggesting a link between PLIN2 deficiency and fibroids. A total of 3877 genes were differentially expressed after PLIN2 knockdown. Gene ontology analysis identified metabolism as the second-highest biological process affected by PLIN2 depletion. OCR (mitochondrial respiration) and ECAR (glycolysis) were significantly upregulated after PLIN2 knockdown; PLIN2-depleted cells had a greater basal metabolic activity and higher metabolic stress response. Cell proliferation was also significantly increased after PLIN2 knockdown. CONCLUSIONS: PLIN2 depletion increases mitochondrial respiration and glycolysis, suggesting that PLIN2 is a critical regulator of metabolic function in leiomyoma cells. PLIN2 deficiency also reprograms leiomyoma cells to a proproliferative phenotype. These findings introduce metabolomics as an area to explore to better understand leiomyoma tumorigenesis.

Progesterone receptor integrates the effects of mutated MED12 and altered DNA methylation to stimulate RANKL expression and stem cell proliferation in uterine leiomyoma.

Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.

Selecting patients for treatment with epidermal growth factor tyrosine kinase inhibitors.

Identification of objective tumor regressions with epidermal growth factor receptor tyrosine kinases (EGFR TKI) in non-small cell lung cancer (NSCLC) patients has resulted in intense, worldwide clinical and basic research directed toward finding the optimal use of EGFR TKIs in NSCLC. EGFR TKI clinical trials have shown that higher response rates and longer survival are associated with specific patient characteristics and that using conventional chemotherapy simultaneously with EGFR TKIs in unselected patients does not increase survival. Molecular studies have revealed that EGFR-activating mutations and high EGFR gene copy number are frequently found in patients who have the best outcomes with EGFR TKIs. More recent studies suggest that KRAS mutations may identify the subset of patients who have the worst outcome with the EGFR TKI treatment. Currently, investigators are trying to determine the optimal approach to selecting patients for treatment with EGFR TKIs. Studies that have evaluated the potential predictive value of clinical features and/or molecular profiles in EGFR TKI-treated NSCLC patients are discussed in this review.

RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth.

Context: Uterine leiomyomas are the most common type of gynecologic tumor in women. Objective: To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Design: Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Setting: Research laboratory. Patients: Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. Interventions: None. Main Outcome Measure: Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. Results: RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Conclusions: Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

X-ray structure of the T. aquaticus FtsY:GDP complex suggests functional roles for the C-terminal helix of the SRP GTPases.

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 A X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain "module" are regulated in the context of the two SRP GTPases.

Structure of a GDP:AlF4 complex of the SRP GTPases Ffh and FtsY, and identification of a peripheral nucleotide interaction site.

The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF4. This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Context: Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. Objective: To define differential gene expression and signaling pathways in leiomyoma cell populations. Design: Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Setting: Research laboratory. Patients: Eight African American women. Interventions: None. Main Outcomes Measures: Gene expression patterns, cell proliferation, and differentiation. Results: A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. Conclusions: IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Estrogen receptor ß regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

OBJECTIVE: To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. DESIGN: Case-control. SETTING: University research setting. PATIENT(S): Premenopausal women. INTERVENTION(S): Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. MAIN OUTCOME MEASURE(S): Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) ß. RESULT(S): Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERß-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERß based on small interfering RNA knockdown and chromatin immunoprecipitation of ERß followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. CONCLUSION(S): ERß leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis.