Central glucagon-like peptide 1 receptor activation inhibits Toll-like receptor agonist-induced inflammation.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) exert anti-inflammatory effects relevant to the chronic complications of type 2 diabetes. Although GLP-1RAs attenuate T cell-mediated gut and systemic inflammation directly through the gut intraepithelial lymphocyte GLP-1R, how GLP-1RAs inhibit systemic inflammation in the absence of widespread immune expression of the GLP-1R remains uncertain. Here, we show that GLP-1R activation attenuates the induction of plasma tumor necrosis factor alpha (TNF-α) by multiple Toll-like receptor agonists. These actions are not mediated by hematopoietic or endothelial GLP-1Rs but require central neuronal GLP-1Rs. In a cecal slurry model of polymicrobial sepsis, GLP-1RAs similarly require neuronal GLP-1Rs to attenuate detrimental responses associated with sepsis, including sickness, hypothermia, systemic inflammation, and lung injury. Mechanistically, GLP-1R activation leads to reduced TNF-α via α1-adrenergic, δ-opioid, and κ-opioid receptor signaling. These data extend emerging concepts of brain-immune networks and posit a new gut-brain GLP-1R axis for suppression of peripheral inflammation.

Education of the clinical embryology laboratory professional: development of a novel program delivered in a laboratory medicine department.

Clinical embryologists are responsible for the handling, evaluation, and care of human gametes and preimplantation embryos within the context of an assisted reproductive technology laboratory. They are integral members of a team of professionals who provide care for fertility patients. Despite the increasing recognition of clinical embryologists as professionals, training requirements, continuing professional development, and appropriate credentialing have lagged in several countries. In many cases, individuals enter the profession with training limited to technical aspects provided by individual laboratory directors through an apprenticeship model. In this article, we present the rationale for rigorous formal training in clinical embryology, introduce CanEMB competencies for practicing professional clinical embryologists that are founded on CanMEDs role principles, and present a nascent Masters of Health Sciences degree program in Laboratory Medicine with a specialization in clinical embryology. This 2-year program has unique features including a Clinical Embryology Skills Development Laboratory, research capstone project, and 200-hour placement within a practicing assisted reproductive technology laboratory. Importantly, this program is delivered through a university-based Department of Laboratory Medicine and Pathobiology in partnership with a Department of Obstetrics and Gynecology. Thus, this program represents a formal acceptance of clinical embryology as a clinical laboratory science. It can be adopted elsewhere to provide a relevant, robust education that will meet current and future needs of the profession.

The adaptor protein VEPH1 interacts with the kinase domain of ERBB2 and impacts EGF signaling in ovarian cancer cells.

Upregulation of ERBB2 and activating mutations in downstream KRAS/BRAF and PIK3CA are found in several ovarian cancer histotypes. ERBB2 enhances signaling by the ERBB family of EGF receptors, and contains docking positions for proteins that transduce signaling through multiple pathways. We identified the adaptor protein ventricular zone-expressed pleckstrin homology domain-containing protein 1 (VEPH1) as a potential interacting partner of ERBB2 in a screen of proteins co-immunoprecipitated with VEPH1. In this study, we confirm a VEPH1 - ERBB2 interaction by co-immunoprecipitation and biotin proximity labelling and show that VEPH1 interacts with the juxtamembrane-kinase domain of ERBB2. In SKOV3 ovarian cancer cells, which bear a PIK3CA mutation and ERBB2 overexpression, ectopic VEPH1 expression enhanced EGF activation of ERK1/2, and mTORC2 activation of AKT. In contrast, in ES2 ovarian cancer cells, which bear a BRAFV600E mutation with VEPH1 amplification but low ERBB2 expression, loss of VEPH1 expression enabled further activation of ERK1/2 by EGF and enhanced EGF activation of AKT. VEPH1 expression in SKOV3 cells enhanced EGF-induced cell migration consistent with increased Snail2 and decreased E-cadherin levels. In comparison, loss of VEPH1 expression in ES2 cells led to decreased cell motility independent of EGF treatment despite higher levels of N-cadherin and Snail2. Importantly, we found that loss of VEPH1 expression rendered ES2 cells less sensitive to BRAF and MEK inhibition. This study extends the range of adaptor function of VEPH1 to ERBB2, and indicates VEPH1 has differential effects on EGF signaling in ovarian cancer cells that may be influenced by driver gene mutations.

Secondary cytoreductive surgery for recurrent low-grade serous ovarian carcinoma: A systematic review and meta-analysis.

OBJECTIVES: Low-grade serous ovarian cancer (LGSC) is a relatively chemo-resistant disease with limited effective treatment options for patients with recurrence. Secondary cytoreductive surgery (SCS) is commonly offered at recurrence, although any benefit this has on survival is not fully determined. This review evaluates the impact of SCS, including residual disease, on progression-free survival (PFS) and overall survival (OS) in recurrent LGSC. METHODS: A comprehensive search of Medline ALL, Embase Classic + Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Web of Science was conducted to obtain studies evaluating optimal or complete SCS versus suboptimal SCS and the amount of residual disease in recurrent LGSC. Meta-analysis was performed and PFS and OS outcomes were calculated. RESULTS: 1Of 5296 studies screened, 350 progressed to full-text review, with 9 ultimately selected for inclusion in the systematic review. Two studies met criteria for meta-analysis of PFS and of OS. The presence of visible residual disease at the conclusion of SCS negatively impacted PFS (HR = 3.51, 95% CI = 1.72-7.14), whereas SCS with no residual disease significantly improved OS (HR = 0.4, 95% CI = 0.23-0.7) in patients with recurrent LGSC. Diffuse and extensive disease distribution was inversely linked to survival. In addition, SCS as an initial treatment for recurrent LGSC was associated with superior survival in comparison to chemotherapy. A short platinum-free interval was not associated with worse survival in this cohort. CONCLUSIONS: Complete SCS, and to a lesser extent optimal SCS, are associated with improved PFS and OS in patients with recurrent LGSC. SCS may be a better initial treatment strategy than systemic chemotherapy for recurrent disease. Patients with recurrent LGSC should be evaluated for the role of SCS based on disease distribution and functional status, irrespective of the platinum-free interval. Prospective studies are needed to further study the role of SCS in patients with recurrent LGSC.

A mouse model of neoadjuvant chemotherapy followed by interval cytoreductive surgery indicates impaired efficacy of perioperative cisplatin.

BACKGROUND: Investigate the impact of interval cytoreductive surgery (ICS) on progression in an orthotopic mouse model of ovarian cancer and the impact of chemotherapy delivered at various timelines following surgery. METHODS: Luciferase-expressing ID8 murine ovarian cancer cells were implanted intra-bursally and IP to C57BL/7 mice. Once disease was established by bioluminescence, 2 cycles of neoadjuvant cisplatin were administered, and animals received either ICS (removal of the injected bursa/primary tumor) or anesthesia alone. Postsurgical chemotherapy was administered on the same day as the intervention (ICS/anesthesia), or on day 7 or day 28 following the intervention. Progression was quantified serially with in vivo bioluminescence imaging. Volume of ascitic fluid volume collected at necropsy was measured. RESULTS: Animals were matched for tumor burden at stratification. There was no accelerated growth of residual tumor after interval cytoreduction compared to controls. Animals who received chemotherapy on postoperative day (POD) 7 had better disease control compared to standard-of-care POD 28. Animals who underwent surgery had less ascites at necropsy compared to those who had anesthesia alone. CONCLUSIONS: In this animal model, surgical wounding with suboptimal cytoreduction after neoadjuvant chemotherapy did not cause accelerated expansion of residual disease. Surgical wounding appears to impair cisplatin activity when given at time of surgery.

Endometrial laminin subunit beta-3 expression associates with reproductive outcome in patients with repeated implantation failure.

PURPOSE: Endometrial laminin subunit beta-3 (LAMB3) is a candidate gene whose expression distinguishes the endometrial window of receptivity (WOR) in human. This study aims to examine endometrial LAMB3 levels in patients with repeated implantation failure (RIF), in order to assess the ability of LAMB3 to predict pregnancy outcome. METHODS: Endometrial biopsies were taken during the WOR from 21 healthy volunteers in natural menstrual cycles and from 50 RIF patients in mock cycles prior to frozen embryo transfer (FET) cycles. Immunohistochemistry (IHC) staining of LAMB3 was performed, and the H-score was correlated with the pregnancy outcome in subsequent FETs. RESULTS: In healthy volunteers, endometrial LAMB3 was demonstrated to be highly expressed during the WOR with the staining exclusively in the cytoplasm of the epithelial cells. In a discovery set of RIF patients, the LAMB3 expression level was found to be significantly higher in those who conceived compared to those who did not in subsequent FETs. A receiving operator characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.7818 (95% confidence interval 59.92-96.44%) with an H-score cutoff of 4.129 to differentiate cases with positive or negative pregnancy outcomes. This cutoff achieved an accuracy of 75% in pregnancy prediction in a following validation set of RIF patients, in which the pregnancy rate in subsequent FETs was three-fold higher when the mock cycle LAMB3 H-score was ≥ 4.129 compared to < 4.129. CONCLUSIONS: IHC measurement of endometrial LAMB3 expression could be a promising prognostic method to predict pregnancy outcome for RIF patients undergoing FETs.

Cargo small non-coding RNAs of extracellular vesicles isolated from uterine fluid associate with endometrial receptivity and implantation success.

OBJECTIVE: To optimize a method of isolating extracellular vesicles (EVs) from uterine fluid and to characterize small non-coding RNAs (sncRNAs) from the EVs, with the goal of identifying novel receptivity-associated biomarkers. DESIGN: Longitudinal study comparing sncRNA expression profiles from endometrial EVs. SETTING: University-affiliated, hospital-based fertility clinic. PATIENT(S): Healthy volunteers with no history of infertility (Group A) and women receiving controlled ovarian stimulation (COS)-in vitro fertilization treatment (Group B). INTERVENTIONS(S): In Group A, EVs were isolated from uterine fluid obtained on luteinizing hormone (LH)+2 and LH+7 in one natural menstrual cycle. In Group B, EVs were isolated from uterine fluid obtained on human chorionic gonadotropin (hCG)+2 and hCG+7 in one COS cycle. RNAs extracted from EVs were profiled using next-generation sequencing. MAIN OUTCOME MEASURE(S): Differential EV-sncRNAs between LH+2 and LH+7 (Group A), between hCG+2 and hCG+7 (Group B), and between pregnant and nonpregnant in vitro fertilization cycles (Group B). RESULT(S): Ultracentrifugation was validated as the most efficient method to isolate EVs from uterine fluid. We identified 12 endometrial EV-sncRNAs (11 microRNAs and 1 piwi-interacting RNA) as receptivity-associated transcripts conserved in both natural and COS cycles. These sncRNAs were associated strongly with biological functions related to immune response, extracellular matrix, and cell junction. Within COS cycles, we also identified a group of EV-sncRNAs that exhibited differential expression in patients who conceived versus those who did not, with hsa-miR-362-3p most robustly overexpressed in the nonpregnant patients. CONCLUSION(S): This study is the first to profile comprehensively sncRNAs in endometrial EVs from uterine fluid and identify sncRNA biomarkers of endometrial receptivity and implantation success.

The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.

The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2nd instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2nd instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2nd instar.

Increased androgen receptor levels and signaling in ovarian cancer cells by VEPH1 associated with suppression of SMAD3 and AKT activation.

Studies indicate androgens contribute to initiation or progression of epithelial ovarian cancer through poorly understood mechanisms. We provide evidence that the androgen receptor (AR) interacts in a ligand-independent manner with the putative armadillo repeat domain of ventricular zone expressed PH domain-containing 1 (VEPH1). This interaction was increased by mutation of the two nuclear receptor-interacting LxxLL motifs present within the VEPH1 armadillo repeat domain. Androgen treatment did not result in nuclear co-localization of VEPH1 with AR, suggesting that VEPH1 does not function as a nuclear co-regulatory protein. VEPH1 expression decreased SMAD3 and activated AKT levels in ovarian cancer cell lines and increased AR activity and protein levels, consistent with an impact on receptor stability. Treatment of cells with dihydrotestosterone (DHT) increased AR protein levels measured 24 h after treatment, an effect augmented in VEPH1-transfected cells, and inhibited by knock-down of endogenous VEPH1. SMAD3 overexpression decreased AR protein levels and prevented the VEPH1-dependent increase in AR; however, silencing of SMAD3 paradoxically also decreased AR levels. DHT treatment led to a rapid and sustained decrease in phosphorylated AKT (pAKT) levels that was enhanced by VEPH1 expression. Inhibition of PI3K resulted in increased AR protein levels. These studies indicate that VEPH1 acts to enhance AR activity in ovarian cancer cells by decreasing SMAD3 and pAKT levels, resulting in increased levels of AR protein.

Removing Short Wavelengths From Polychromatic White Light Attenuates Circadian Phase Resetting in Rats.

Visible light is the principal stimulus for resetting the mammalian central circadian pacemaker. Circadian phase resetting is most sensitive to short-wavelength (blue) visible light. We examined the effects of removing short-wavelengths < 500 nm from polychromatic white light using optical filters on circadian phase resetting in rats. Under high irradiance conditions, both long- (7 h) and short- (1 h) duration short-wavelength filtered (< 500 nm) light exposure attenuated phase-delay shifts in locomotor activity rhythms by (∼40-50%) as compared to unfiltered light exposure. However, there was no attenuation in phase resetting under low irradiance conditions. Additionally, the reduction in phase-delay shifts corresponded to regionally specific attenuation in molecular markers of pacemaker activation in response to light exposure, including c-FOS, Per1 and Per2. These results demonstrate that removing short-wavelengths from polychromatic white light can attenuate circadian phase resetting in an irradiance dependent manner. These results have important implications for designing and optimizing lighting interventions to enhance circadian adaptation.

Ventricular Zone Expressed PH Domain Containing 1 (VEPH1): an adaptor protein capable of modulating multiple signaling transduction pathways during normal and pathological development.

Ventricular Zone Expressed PH Domain-Containing 1 (VEPH1) is an 833-amino acid protein encoded by an evolutionarily conserved single-copy gene that emerged with pseudocoelomates. This gene has no paralog in any species identified to date and few studies have investigated the function of its encoded protein. Loss of expression of its ortholog, melted, in Drosophila results in a severe neural phenotype and impacts TOR, FoxO, and Hippo signaling. Studies in mammals indicate a role for VEPH1 in modulating TGFß signaling and AKT activation, while numerous studies indicate VEPH1 expression is altered in several pathological conditions, including cancer. Although often referred to as an uncharacterized protein, available evidence supports VEPH1 as an adaptor protein capable of modulating multiple signal transduction networks. Further studies are required to define these adaptor functions and the role of VEPH1 in development and disease progression.

Wounding promotes ovarian cancer progression and decreases efficacy of cisplatin in a syngeneic mouse model.

BACKGROUND: Primary cytoreductive surgery followed by adjuvant chemotherapy is the standard treatment for advanced epithelial ovarian cancer. The average interval between surgery and chemotherapy initiation is approximately 4-weeks at most centers; however, since surgery may accelerate residual tumor growth, a shorter interval may be more beneficial. METHODS: The murine ID8 cell model of ovarian cancer was used to examine the efficacy of cisplatin treatment administered perioperatively or 7 days after surgical wounding. Luciferase-expressing cells ID8 cells were injected intraperitoneally (i.p.) into female C57/Bl6 mice. Fourteen days post-injection, animals received an abdominal incision or anesthesia alone and received i.p. cisplatin either on the surgical day or 7 days later, or received no chemotherapy. Additional animals received cisplatin 28 days after wounding for comparison. RESULTS: Abdominal tumor mass increased 2.5-fold in wounded vs. unwounded animals as determined by bioluminescent in vivo tumor imaging. Cisplatin administered on the day of wounding decreased tumor burden by 50%, as compared to 90% in unwounded animals. Cisplatin on day 7 or day 28 decreased tumor burden by 80 and 37% respectively. CONCLUSIONS: Surgical wounding increases ovarian tumor mass and decreases perioperative cisplatin efficacy in this animal model. Administration of cisplatin 1 week after surgery was more effective than cisplatin administered perioperatively or 4 weeks after surgery.

BRCA1 Mutation Status and Follicular Fluid Exposure Alters NFκB Signaling and ISGylation in Human Fallopian Tube Epithelial Cells.

Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed epithelial ovarian cancer histotype. Other identified risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation, during which the FTE cells become transiently exposed to follicular fluid (FF). To test whether mtBRCA1 or mtBRCA2 nonmalignant FTE cells respond differently to periovulatory FF exposure than control patient FTE cells, gene expression profiles from primary FTE cultures derived from BRCA1 or BRCA2 mutation carriers or control patients were compared at baseline, 24 hours after FF exposure, and 24 hours after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Gene set enrichment analysis revealed increased NFκB and EGFR signaling at baseline in mtBRCA1 samples, with increased interferon target gene expression, including members of the ISGylation pathway, observed after recovery from FF exposure. Gene set enrichment analysis did not identify altered pathway signaling in mtBRCA2 samples. An inverse relationship between EGFR signaling and ISGylation with BRCA1 protein levels was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1 cDNA. Suppression of ISG15 and ISGylated protein levels by increased BRCA1 expression was found to be mediated by decreased NFκB signaling. These studies indicate that increased NFκB signaling associated with decreased BRCA1 expression results in increased ISG15 and protein ISGylation following FF exposure, which may be involved in predisposition to HGSOC.

The Two Faces of Adjuvant Glucocorticoid Treatment in Ovarian Cancer.

Adjuvant glucocorticoid treatment is routinely used in the treatment of ovarian cancer to mitigate the undesirable side effects of chemotherapy, thereby enhancing tolerability to higher cytotoxic drug doses and frequency of treatment cycles. However, in vitro and preclinical in vivo and ex vivo studies indicate that glucocorticoids may spare tumor cells from undergoing cell death through enhanced cell adhesion, promotion of anti-inflammatory signaling, and/or inhibition of apoptotic pathways. The implications of laboratory studies showing potential negative impact on the efficacy of chemotherapy have been long overlooked since clinical investigations have found no apparent survival detriment attributable to adjuvant glucocorticoid use. Importantly, these clinical studies were not randomized and most did not consider glucocorticoid receptor status, a vital determinant of tumor response to glucocorticoid administration. Additionally, the clinically beneficial elements of increased chemotherapy treatment adherence and dosing afforded by adjuvant glucocorticoids may offset and therefore mask their anti-chemotherapy activities. This review summarizes the current evidence on the impact of glucocorticoids in ovarian cancer and discusses the need for further research and development of alternative strategies to ameliorate untoward side effects of chemotherapy.

Collagen IV trafficking: The inside-out and beyond story.

Collagen IV networks endow basement membranes (BMs) with remarkable tensile strength and function as morphoregulatory substrata for diverse tissue-specific developmental events. A complex repertoire of intracellular and extracellular molecular interactions are required for collagen IV secretion and supramolecular assembly into BMs. These include intracellular chaperones such as Heat shock protein 47 (Hsp47) and the chaperone-binding trafficking protein Transport and Golgi organization protein 1 (Tango1). Mutations in these proteins lead to compromised collagen IV protomer stability and secretion, leading to defective BM assembly and function. In addition to intracellular chaperones, a role for extracellular chaperones orchestrating the transport, supramolecular assembly, and architecture of collagen IV in BM is emerging. We present evidence derived from evolutionarily distant model organisms that supports an extracellular collagen IV chaperone-like activity for the matricellular protein SPARC (Secreted Protein, Acidic, Rich in Cysteine). Loss of SPARC disrupts BM homeostasis and compromises tissue biomechanics and physiological function. Thus, the combined contributions of intracellular and extracellular collagen IV-associated chaperones and chaperone-like proteins are critical to ensure proper secretion and stereotypic assembly of collagen IV networks in BMs.

VEPH1 expression decreases vascularisation in ovarian cancer xenografts and inhibits VEGFA and IL8 expression through inhibition of AKT activation.

BACKGROUND: VEPH1 is amplified in several cancers including ovarian but its impact on tumour progression is unknown. Previous work has shown that VEPH1 inhibits TGFß signalling while its Drosophila ortholog increases tissue growth, raising the possibility that VEPH1 could impact tumour growth or progression. METHODS: A CRISPR approach was used to disrupt VEPH1 expression in ovarian cancer ES-2 cells, while VEPH1-negative SKOV3 cells were stably transfected with VEPH1 cDNA. The impact of altered VEPH1 expression was assessed using in vitro and in vivo assays and mechanistic studies were performed in vitro. RESULTS: VEPH1 expression in SKOV3 cells resulted in a reduced tumour growth rate associated with increased necrotic area, and decreased microvessel density and VEGF-A levels relative to tumours formed by mock-transfected cells. VEPH1 expression also decreased VEGFA and IL8 expression in SKOV3 cells and was associated with decreased activated AKT levels. These effects were not observed in ES-2 cells, which bear a BRAFV600E activating mutation that leads to constitutively increased IL8 and VEGFA expression. CONCLUSIONS: VEPH1 expression in SKOV3 ovarian cancer cells inhibits AKT activation to decrease VEGFA and IL8 expression, which leads to decreased tumour vascularisation and progression.

In vitro Autoradiographic Analysis of Regional Changes in Estrogen Receptor Alpha in the Brains of Cycling Female Rats.

BACKGROUND/AIMS: The contributions of the three principal ovarian steroid hormones (estradiol, progesterone and testosterone) to the regulation of estrogen receptor alpha (ERα) levels in the rat brain were examined during the estrous cycle. METHODS: Receptor concentrations were measured using an in vitro autoradiographic technique designed to separately quantify free, unoccupied receptors and receptors 'occupied' by (bound to) endogenous hormone. RESULTS: ERα occupation increased at proestrus and declined at estrus, reflecting changes in circulating estradiol and testosterone levels. Total ERα content followed a pattern that was the inverse of the occupation data, falling over the night of proestrus. Between 2.00 and 10.00 a.m. on the day of estrus, total ERα concentrations recovered in all brain regions except the ventromedial nucleus (VMN), in which ERα binding remained depressed at estrus. Administration of the progesterone antagonist mifepristone on the afternoon of proestrus resulted in recovery of ERα levels in the VMN by the morning of estrus, consistent with the hypothesis that the preovulatory progesterone surge selectively inhibits VMN ERα expression. Residual ERα occupation observed at estrus, when estradiol is not detectable in the serum, likely reflects intracranial aromatization of circulating androgens, since the pattern of receptor occupation observed at this stage of the cycle could be reproduced in ovariectomized rats by replacement with testosterone. CONCLUSION: These findings indicate that ERα binding in the brain fluctuates during the rat estrous cycle in a region-specific manner and suggest that local aromatization of testosterone may contribute significantly to ERα occupation when circulating estradiol levels are low.

Human ortholog of Drosophila Melted impedes SMAD2 release from TGF-ß receptor I to inhibit TGF-ß signaling.

Drosophila melted encodes a pleckstrin homology (PH) domain-containing protein that enables normal tissue growth, metabolism, and photoreceptor differentiation by modulating Forkhead box O (FOXO), target of rapamycin, and Hippo signaling pathways. Ventricular zone expressed PH domain-containing 1 (VEPH1) is the mammalian ortholog of melted, and although it exhibits tissue-restricted expression during mouse development and is potentially amplified in several human cancers, little is known of its function. Here we explore the impact of VEPH1 expression in ovarian cancer cells by gene-expression profiling. In cells with elevated VEPH1 expression, transcriptional programs associated with metabolism and FOXO and Hippo signaling were affected, analogous to what has been reported for Melted. We also observed altered regulation of multiple transforming growth factor-ß (TGF-ß) target genes. Global profiling revealed that elevated VEPH1 expression suppressed TGF-ß-induced transcriptional responses. This inhibitory effect was verified on selected TGF-ß target genes and by reporter gene assays in multiple cell lines. We further demonstrated that VEPH1 interacts with TGF-ß receptor I (TßRI) and inhibits nuclear accumulation of activated Sma- and Mad-related protein 2 (SMAD2). We identified two TßRI-interacting regions (TIRs) with opposing effects on TGF-ß signaling. TIR1, located at the N terminus, inhibits canonical TGF-ß signaling and promotes SMAD2 retention at TßRI, similar to full-length VEPH1. In contrast, TIR2, located at the C-terminal region encompassing the PH domain, decreases SMAD2 retention at TßRI and enhances TGF-ß signaling. Our studies indicate that VEPH1 inhibits TGF-ß signaling by impeding the release of activated SMAD2 from TßRI and may modulate TGF-ß signaling during development and cancer initiation or progression.

Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.