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    1. Obesity Induced a Leptin-Notch Signaling Axis in Breast Cancer

      Monica Battle, Corey Gillespie, Alexander Quarshie, Viola Lanier, Tia Harmon, Kaamilah Wilson, Marta Torroella-Kouri and Ruben R. Gonzalez-Perez


      Abstract

      To investigate whether obesity induces a leptin–Notch signaling axis in breast cancer (BC), leptin-induced Notch was determined in human MCF-7 and MDA-MB231 and mouse E0771 cells and, in E0771-BC hosted by syngeneic lean and diet-induced-obesity (DIO) C57BL/6J female mice. Lean and DIO-mice were treated for three weeks with leptin inhibitor (PEG-LPrA2) one week after the inoculation of E0771 cells. Leptin induced Notch1, 3 and 4 in BC cells, but Notch2 expression showed opposite pattern in MCF-7 compared to MDA-MB231 cells. Notch loss-of-function [DAPT and dominant negative (R218H) RBP-Jk (CSL/CBF1)] showed that a functional leptin-Notch signaling axis was involved in the proliferation and migration of E0771 cells. E0771-BC onset was affected by obesity [lean mice: 7/10 (70%) vs DIO-mice: 11/12 (92%); Pearson Chi2: P=0.06]. PEG-LPrA2 significantly reduced BC growth [untreated: 19/42; (45%) vs treated: 8/42 (19%); Pearson Chi2: p=0.008]. PEG-LPrA2 did not influence the caloric intake of mice, but increased carcass and/or body weights of lean and DIO-mice inoculated with E0771 cells, which could be related to the improvement of health conditions (less aggressive disease). Importantly, BC from obese mice had higher levels of Notch3, JAG-1 and survivin than lean mice. Inhibition of leptin signaling reduced protein levels of Notch (NICD1, NICD4, Notch3, JAG1 and survivin) and significantly decreased mRNA expression of Notch receptors, ligands, and targets. PEG-LPrA's effects were more prominent in DIO-mice. Present data suggest that leptin induces Notch, which could be involved in the reported higher incidence and aggressiveness and, poor prognosis of BC in obese patients. © 2013 Wiley Periodicals, Inc.

    2. Leptin’s Pro-Angiogenic Signature in Breast Cancer

      Ruben Rene Gonzalez-Perez, Viola Lanier and Gale Newman

      Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, United States

      Abstract

      Obesity is linked to increased incidence of breast cancer. The precise causes and mechanisms of these morbid relationships are unknown. Contradictory data on leptin angiogenic actions have been published. However, accumulating evidence would suggest that leptin’s pro-angiogenic effects in cancer play an essential role in the disease. Leptin, the main adipokine secreted by adipose tissue, is also abnormally expressed together with its receptor (OB-R) by breast cancer cells. Leptin induces proliferation and angiogenic differentiation of endothelial cells upregulates VEGF/VEGFR2 and transactivates VEGFR2 independent of VEGF. Leptin induces two angiogenic factors: IL-1 and Notch that can increase VEGF expression. Additionally, leptin induces the secretion and synthesis of proteases and adhesion molecules needed for the development of angiogenesis. Leptin’s paracrine actions can further affect stromal cells and tumor associated macrophages, which express OB-R and secrete VEGF and IL-1, respectively. A complex crosstalk between leptin, Notch and IL-1 (NILCO) that induces VEGF/VEGFR2 is found in breast cancer. Leptin actions in tumor angiogenesis could amplify, be redundant and/or compensatory to VEGF signaling. Current failure of breast cancer anti-angiogenic therapies emphasizes the necessity of targeting the contribution of other pro-angiogenic factors in breast cancer. Leptin’s impact on tumor angiogenesis could be a novel target for breast cancer, especially in obese patients. However, more research is needed to establish the importance of leptin in tumor angiogenesis. This review is focused on updated information on how leptin could contribute to tumor angiogenesis.

    3. Leptin–cytokine crosstalk in breast cancer

      Gale Newman , Ruben Rene Gonzalez-Perez

      Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA 30310, United States

      Abstract
      Despite accumulating evidence suggesting a positive correlation between leptin levels, obesity, post-menopause and breast cancer incidence, our current knowledge on the mechanisms involved in these relationships is still incomplete. Since the cloning of leptin in 1994 and its receptor (OB-R) 1 year later by Friedman’s laboratory (Zhang et al., 1994) and Tartaglia et al. (Tartaglia et al., 1995), respectively, more than 22,000 papers related to leptin functions in several biological systems have been published (Pubmed, 2012). The ob gene product, leptin, is an important circulating signal for the regulation of body weight. Additionally, leptin plays critical roles in the regulation of glucose homeostasis, reproduction, growth and the immune response. Supporting evidence for leptin roles in cancer has been shown in more than 1000 published papers, with almost 300 papers related to breast cancer (Pubmed, 2012). Specific leptin-induced signaling pathways are involved in the increased levels of inflammatory, mitogenic and pro-angiogenic factors in breast cancer. In obesity, a mild inflammatory condition, deregulated secretion of proinflammatory cytokines and adipokines such as IL-1, IL-6, TNF-α and leptin from adipose tissue, inflammatory and cancer cells could contribute to the onset and progression of cancer. We used an in silico software program, Pathway Studio 9, and found 4587 references citing these various interactions. Functional crosstalk between leptin, IL-1 and Notch signaling (NILCO) found in breast cancer cells could represent the integration of developmental, proinflammatory and pro-angiogenic signals critical for leptin-induced breast cancer cell proliferation/migration, tumor angiogenesis and breast cancer stem cells (BCSCs). Remarkably, the inhibition of leptin signaling via leptin peptide receptor antagonists (LPrAs) significantly reduced the establishment and growth of syngeneic, xenograft and carcinogen-induced breast cancer and, simultaneously decreased the levels of VEGF/VEGFR2, IL-1 and Notch. Inhibition of leptin–cytokine crosstalk might serve as a preventative or adjuvant measure to target breast cancer, particularly in obese women. This review is intended to present an update analysis of leptin actions in breast cancer, highlighting its crosstalk to inflammatory cytokines and growth factors essential for tumor development, angiogenesis and potential role in BCSC.

    4. Potential Role of Leptin Signaling in DMBA-induced Mammary Tumors by Non-Responsive C57BL/6J Mice Fed a High-Fat Diet

      Gillespie C, Quarshie A, Penichet M and Gonzalez-Perez RR

      Abstract
      Environmental carcinogens, High-Fat Diet (HFD) and elevated levels of leptin correlate to increase breast cancer incidence. To test whether these factors could affect the development of Mammary Tumors (MT) via DMBA (7,12-dimethylbenz[a]anthracene) challenge, we used C57BL/6J mice that are non-responsive to develop MT in absence of hormonal stimulation. C57BL/6J female mice without hormonal stimulation were fed HFD (55% Kcal-fat) and low-fat diets (10% Kcal-fat) received DMBA (oral gavage: 1 mg/weekly) for 6 weeks. To test whether leptin signaling is involved in DMBA-MT development, a potent inhibitor, pegylated leptin peptide receptor antagonist (PEG-LPrA2; half-life 66 hours), was used for 30 weeks. As expected, irrespective of PEG-LPrA2 treatment, lean mice fed with low-fat diet did not develop MT. However, HFD induced obesity and significantly stimulated earlier onset (within 18 weeks) and marginally increased the incidence of MT (21%; 3/14) in DIO-mice (diet-induced-obesity). It appears that leptin signaling may be involved in DMBA-induced mammary carcinogenesis in obese mice because no evidence of MT was found in DIO-mice treated with PEG-LPrA2 (0% incidence; 0/14; Wilcoxon-Breslow test Chi2, p=0.03). Interestingly, PEG-LPrA2 treatment did not apparently affect body weight or food intake, but reduced protein levels of several molecules related to breast cancer [Aryl hydrocarbon Receptor (AhR), leptin receptor (OBR), interleukin 1 receptor type I (IL-1R tI), hypoxia-induced factor 1 alpha (HIF-1α), Jagged1 (JAG1) and Notch1 activated (NICD1)] within the mammary glands. Our findings reinforce the idea that obesity induced by HFD is an additional risk factor for chemical-induced breast carcinogenesis. The present study reveals some potential mechanisms involving leptin in the effects of HFD and adiposity on mammary chemical-induced carcinogenesis. Overall, present data suggest that the inhibition of leptin signaling might be a new way to prevent breast cancer induced by chemical carcinogens, especially in obese individuals.

    5. Regulation of Angiogenesis in Human Cancer via Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2)

      Biochim Biophys Acta. 2012 Jan 24;1825(2):207-222

    6. Oncogenic role and therapeutic target of leptin signaling in breast cancer and cancer stem cells

      Guo S, Liu M, Wang G, Torroella-Kouri M, Gonzalez-Perez RR.

      Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310

      Abstract

      Significant correlations between obesity and incidence of various cancers have been reported. Obesity, considered a mild inflammatory process, is characterized by a high level of secretion of several cytokines from adipose tissue. These molecules have disparate effects, which could be relevant to cancer development. Among the inflammatory molecules, leptin, mainly produced by adipose tissue and overexpressed with its receptor (Ob-R) in cancer cells is the most studied adipokine. Mutations of leptin or Ob-R genes associated with obesity or cancer are rarely found. However, leptin is an anti-apoptotic molecule in many cell types, and its central roles in obesity-related cancers are based on its pro-angiogenic, pro-inflammatory and mitogenic actions. Notably, these leptin actions are commonly reinforced through entangled crosstalk with multiple oncogenes, cytokines and growth factors. Leptin-induced signals comprise several pathways commonly triggered by many cytokines (i.e., canonical: JAK2/STAT; MAPK/ERK1/2 and PI-3K/AKT1 and, non-canonical signaling pathways: PKC, JNK and p38 MAP kinase). Each of these leptin-induced signals is essential to its biological effects on food intake, energy balance, adiposity, immune and endocrine systems, as well as oncogenesis. This review is mainly focused on the current knowledge of the oncogenic role of leptin in breast cancer. Additionally, leptin pro-angiogenic molecular mechanisms and its potential role in breast cancer stem cells will be reviewed. Strict biunivocal binding-affinity and activation of leptin/Ob-R complex makes it a unique molecular target for prevention and treatment of breast cancer, particularly in obesity contexts

    7. Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

      Guo, Shanchun and Gonzalez-Perez RR (2011) Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer. PLoS ONE 6(6): e21467. doi:10.1371/journal.pone.0021467

      Abstract
      High levels of pro-angiogenic factors, leptin, IL-1, Notch and VEGF (ligands and receptors), are found in breast cancer, which is commonly correlated with metastasis and lower survival of patients. We have previously reported that leptin induces the growth of breast cancer and the expression of VEGF/VEGFR-2 and IL-1 system. We hypothesized that Notch, IL-1 and leptin crosstalk outcome (NILCO) plays an essential role in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer. To test this hypothesis, leptin's effects on the expression and activation of Notch signaling pathway and VEGF/VEGFR-2/IL-1 were determined in mouse (4T1, EMT6 and MMT) breast cancer cells. Remarkably, leptin up-regulated Notch1-4/JAG1/Dll-4, Notch target genes: Hey2 and survivin, together with IL-1 and VEGF/VEGFR-2. RNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that it was linked to leptin-activated JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. Interestingly, leptin upregulatory effects on cell proliferation/migration and pro-angiogenic factors Notch, IL-1 and VEGF/VEGFR-2 were abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, blockade of IL-1R tI inhibited leptin-induced Notch, Hey2 and survivin as well as VEGF/VEGFR-2 expression. These data suggest leptin is an inducer of Notch (expression/activation) and IL-1 signaling modulates leptin effects on Notch and VEGF/VEGFR-2. We show for the first time that a novel unveiled crosstalk between Notch, IL-1 and leptin (NILCO) occurs in breast cancer. Leptin induction of proliferation/migration and upregulation of VEGF/VEGFR-2 in breast cancer cells were related to an intact Notch signaling axis. NILCO could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin-induced cell proliferation/migration and regulation of VEGF/VEGFR-2 in breast cancer. Targeting NILCO might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis.

      © 2011 Guo, Gonzalez-Perez.

    8. Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

      Garonna E, Botham KM, Birdsey GM, Randi AM, Gonzalez-Perez RR, et al. 2011 Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells. PLoS ONE April 2011 | Volume 6 | Issue 4 | : e18823. doi:10.1371/journal.pone.0018823

      Abstract
      Background: The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.

      Conclusions/Significance
      We conclude that a functional endothelial p38MAPK/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals.

      © 2011 Garonna et al.

    9. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer
      Volume 1815, issue 2, April 2011, pages 197-213

      Role of Notch and its oncogenic signaling crosstalk in breast cancer.
      Guo S, Liu, M, Gonzalez-Perez RR.

      Abstract
      Background: The Notch signaling plays a key role in cell differentiation, survival, and proliferation through diverse mechanisms. Notch signaling is also involved in vasculogenesis and angiogenesis. Moreover, Notch expression is regulated by hypoxia and inflammatory cytokines (IL-1, IL-6 and leptin). Entangled crosstalk between Notch and other developmental signaling (Hedgehog and Wnt), and signaling triggered by growth factors, estrogens and oncogenic kinases, could impact on Notch targeted genes. Thus, alterations of the Notch signaling can lead to a variety of disorders, including human malignancies. Notch signaling is activated by ligand binding, followed by ADAM/tumor necrosis factor-α-converting enzyme (TACE) metalloprotease and γ-secretase cleavages that produce the Notch intracellular domain (NICD). Translocation of NICD into the nucleus induces the transcriptional activation of Notch target genes. The relationships between Notch deregulated signaling, cancer stem cells and the carcinogenesis process reinforced by Notch crosstalk with many oncogenic signaling pathways suggest that Notch signaling may be a critical drug target for breast and other cancers. Since current status of knowledge in this field changes quickly, our insight should be continuously revised. In this review, we will focus on recent advancements in identification of aberrant Notch signaling in breast cancer and the possible underlying mechanisms, including potential role of Notch in breast cancer stem cells, tumor angiogenesis, as well as its crosstalk with other oncogenic signaling pathways in breast cancer. We will also discuss the prognostic value of Notch proteins and therapeutic potential of targeting Notch signaling for cancer treatment.

      Copyright 2010. Published by Elsevier B.V.
      PMID: 21193018 [PubMed - as supplied by publisher]

    10. Br J Cancer. 2010 Dec. 7
      Leptin pro-angiogenetic signature in breast cancer is linked to IL-1 signaling.

      Zhou W, Guo S, Gonzalez-Perez RR.

      Microbiology, Biochemistry and Immunology
      Morehouse School of Medicine
      720 Westview Drive
      Atlanta, GA 30310
      USA
      Email: rgonzalez@msm.edu

      Abstract
      Background: Leptin and interleukin-1 (IL-1) upregulate vascular endothelial growth factor (VEGF), promote angiogenesis and are related to worse prognosis of breast cancer. However, it is unknown whether leptin regulates IL-1, and whether these effects are related to leptin-induction of VEGF/VEGFR2 in breast cancer.Methods:Several genetic and pharmacological approaches were used to determine the mechanisms involved in leptin regulation of IL-1 system (IL-1α, IL-1β, IL-1Ra and IL-1R tI) and the impact of IL-1 signalling on leptin-induced VEGF/VEGFR2 expression in mouse mammary cancer 4T1 cells (a model that resembles invasive and highly metastatic human breast cancer).Results:Leptin increased protein and mRNA levels of all components of the IL-1 system. IL-1 upregulation involved leptin activation of JAK2/STAT3, MAPK/ERK 1/2, PI-3K/AKT1, PKC, p38 and JNK. Leptin-induced phosphorylation of mTOR/4E-BP1 increased IL-1β and IL-1Ra expression, but downregulated IL-1α. Leptin upregulation of IL-1α promoter was linked to SP1 and NF-κB transcription factors. In addition, leptin receptor (Ob-Rb) was upregulated by leptin. Interestingly, leptin upregulation of VEGF/VEGFR2 was partially mediated by IL-1/IL-1R tI signalling.Conclusions:We show for the first time that leptin induces several signalling pathways to upregulate the translational and transcriptional expression of IL-1 system in breast cancer cells. Moreover, leptin upregulation of VEGF/VEGFR2 was impaired by IL-1 signalling blockade. These data suggest that leptin pro-angiogenic signature in breast cancer is linked to, or regulated, in part by IL-1 signalling.British Journal of Cancer advance online publication, 7 December 2010; doi:10.1038/sj.bjc.6606013 www.bjcancer.com.

      PMD: 21139583 [PubMed - as supplied by the publisher]

    11. Int J Oncol, 2010 Oct;37(4):891-900

      Blood monocytes from mammary tumor-bearing mice: Early targets of tumor-induced immune suppression?

      Caso R, Silvera R, Carrio R, Iragavarapu-Charyulu V, Gonzalez-Perez RR, Torroella-kouri M.

      Abstract
      We have previously shown that peritoneal macro- phages from mice bearing advanced D1-DMBA3 mammary tumors are impaired in their inflammatory functions but are not alternatively activated either and are less differentiated than the ones from normal mice. However, little is known about whether similar defects exist in their precursor stages as blood monocytes. We examined if blood monocytes from mammary tumor-bearing mice are already altered in their activation profiles before becoming macrophages and whether they correspond to inflammatory or resident monocyte sub- types. Much effort is currently devoted to reversing macro- phage adverse traits in tumor hosts; as these cells reside within tissues, access is limited. Blood monocytes could be better targeted and manipulated by less invasive means. In the present study, mononuclear cells were isolated from whole blood of D1-DMBA-3 mammary tumor-bearing and normal BALB/c mice and CD115+ monocytes were analyzed. Our results show that there is an increase in circulating monocytes in tumor hosts; these monocytes exhibit a reduced expression of several myeloid differentiation markers such as CD115, F4/80, CD68 and CD11b. Moreover, downregulation of MHC II, CD62L and the proangiogenic marker Tie-2 are observed in these cells, whereas Gr-1 and Ly6C are upregulated. Furthermore, gene microarray analysis performed for the first time in blood monocytes from tumor hosts indicates that they express a mixture of pro-inflammatory and anti- inflammatory cytokines and chemokines. Interestingly, CCR2 and CX3CR1, which are crucial in monocyte definition as inflammatory or resident, respectively, are both upregulated. Importantly, complement proteins are enhanced whereas nitric oxide production is decreased and there is no measurable arginase activity detected in these cells. Collectively, our study represents the first comprehensive analysis of blood monocytes from tumor-bearing mice; we conclude that these cells are neither completely inflammatory nor suppressive and are less differentiated, similar to the macrophages they later become.

    12. Biochim Biophys Acta. 2010 Aug;1806(1):108-121. Epub 2010 May 11.

      Vascular endothelial growth factor receptor-2 in breast cancer.

      Guo S, Colbert LS, Fuller M, Zhang Y, Gonzalez-Perez RR.

      Microbiology, Biochemistry and Immunology
      Morehouse School of Medicine
      720 Westview Drive
      Atlanta, GA 30310
      USA
      Email: rgonzalez@msm.edu

      Abstract
      Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR was structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival. Copyright © 2010 Elsevier B.V. All rights reserved.

      PMID: 20462514 [PubMed - as supplied by publisher]PMCID: PMC2885515 [Available on 2011/8/1]

    13. Cell Signal. 2010 Sep;22(9):1350-62. Epub 2010 May 11.

      Leptin upregulates VEGF in breast cancer via canonic and non-canonical signalling pathways and NFkappaB/HIF-1alpha activation.

      Gonzalez-Perez RR, Xu Y, Guo S, Watters A, Zhou W, Leibovich SJ.

      Department of Microbiology, Biochemistry and Immunology
      Morehouse School of Medicine
      720 Westview Drive
      Atlanta, GA 30310
      USA
      Email: rgonzalez@msm.edu

      Abstract
      High levels of VEGF and leptin are strongly linked to worse prognosis of breast cancer. Leptin signalling upregulates VEGF in human and mouse mammary tumor cells (MT), but the specific molecular mechanisms are largely unknown. Pharmacologic and genetic approaches were used to dissect the mechanism of leptin regulation of VEGF protein and mRNA in MT (4T1, EMT6 and MMT). A series of VEGF-promoter Luc-reporters (full-length and transcription factor-binding deletions) were transfected into MT to analyze leptin regulation of VEGF transcription. Deletion analysis of VEGF promoter and RNA knockdown shows that HIF-1alpha and NFkappaB are essentials for leptin regulation of VEGF. Leptin activation of HIF-1alpha was mainly linked to canonic (MAPK, PI-3K) and non-canonic (PKC, JNK and p38 MAP) signalling pathways. Leptin non-canonic signalling pathways (JNK, p38 MAP and to less extent PKC) were linked to NFkappaB activation. SP1 was involved in leptin regulation of VEGF in 4T1 cells. AP1 was not involved and AP2 repressed leptin-induced increase of VEGF. Overall, these data suggest that leptin signalling regulates VEGF mainly through HIF-1alpha and NFkappaB. These results delineate a comprehensive mechanism for leptin regulation of VEGF in MT. Disruption of leptin signalling could be used as a novel way to treat breast cancer. Copyright (c) 2010 Elsevier Inc. All rights reserved.

      PMID: 20466060 [PubMed - in process

    14. Breast Cancer Res. 2009;11(3):R36. Epub 2009 Jun 16.

      Leptin-signaling inhibition results in efficient anti-tumor activity in estrogen receptor positive or negative breast cancer.

      Rene Gonzalez R, Watters A, Xu Y, Singh UP, Mann DR, Rueda BR, Penichet ML.

      Department of Microbiology, Biochemistry and Immunology
      Morehouse School of Medicine
      720 Westview Drive
      Atlanta, GA 30310
      USA
      Email: rgonzalez@msm.edu

      Abstract
      INTRODUCTION: We have shown previously that treatment with pegylated leptin peptide receptor antagonist 2 (PEG-LPrA2) reduced the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor type 2 (VEGFR2) and growth of 4T1-breast cancer (BC) in syngeneic mice. In this investigation, PEG-LPrA2 was used to evaluate whether the inhibition of leptin signaling has differential impact on the expression of pro-angiogenic and pro-proliferative molecules and growth of human estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) BC xenografts hosted by immunodeficient mice. METHODS: To test the contribution of leptin signaling to BC growth and expression of leptin-targeted molecules, PEG-LPrA2 treatment was applied to severe immunodeficient mice hosting established ER+ (MCF-7 cells; ovariectomized/supplemented with estradiol) and ER- (MDA-MB231 cells) BC xenografts. To further assess leptin and PEG-LPrA2 effects on ER+ and ER- BC, the expression of VEGF and VEGFR2 (protein and mRNA) was investigated in cell cultures. RESULTS: PEG-LPrA2 more effectively reduced the growth of ER+ (>40-fold) than ER- BC (twofold) and expression of pro-angiogenic (VEGF/VEGFR2, leptin/leptin receptor OB-R, and IL-1 receptor type I) and pro-proliferative molecules (proliferating cell nuclear antigen and cyclin D1) in ER+ than in ER-BC. Mouse tumor stroma in ER+ BC expressed high levels of VEGF and leptin that was induced by leptin signaling. Leptin upregulated the transcriptional expression of VEGF/VEGFR2 in MCF-7 and MDA-MB231 cells. CONCLUSIONS: These results suggest that leptin signaling plays an important role in the growth of both ER+ and ER- BC that is associated with the leptin regulation of pro-angiogenic and pro-proliferative molecules. These data provide support for the potential use of leptin-signaling inhibition as a novel treatment for ER+ and ER- BC.

      PMCID: PMC2716504
      PMID: 19531256 [PubMed - indexed for MEDLINE]

    15. Int J Cancer. 2008 Dec 15;123(12):2782-90.

      Leptin regulation of proangiogenic molecules in benign and cancerous endometrial cells.

      Carino C, Olawaiye AB, Cherfils S, Serikawa T, Lynch MP, Rueda BR, Gonzalez RR.

      Boston Biomedical Research Institute (BBRI)
      Watertown, MA
      USA

      Abstract
      Several proangiogenic/proinflammatory factors involved in endometrial cancer are regulated by leptin, but the signaling mechanisms responsible for these leptin-induced actions are largely unknown. Here, we report that in benign (primary and HES) and cancerous-endometrial epithelial cells (EEC) (An3Ca, SK-UT2 and Ishikawa), leptin in a dose-dependent manner regulates vascular endothelial growth factor, (VEGF); interleukin-1 beta, (IL-1beta); leukemia inhibitory factor, (LIF) and their respective receptors, VEGFR2, IL-1R tI and LIFR.  Remarkably, leptin induces a greater increase in VEGF/VEGFR2 and LIF levels in cancer than in benign cells. However, IL-1beta was only increased by leptin in benign primary-EEC. Cancer-EEC expressed higher levels of leptin receptor (full-length OB-Rb and short isoforms) in contrast to benign primary-EEC.  Leptin-mediated activation of JAK2 (janus kinase 2) was upstream to the activation of PI-3K (phosphatidylinositol-3 kinase) and/or MAPK (mitogen-activated protein kinase) signaling pathways. Leptin induction of cytokines/receptors generally involved JAK2 and MAPK activation, but PI-3K phosphorylation was required for leptin increase of LIF, IL-1/IL-1R tI.  Leptin-mediated activation of mTOR (mammalian target of Rapamycin), mainly linked to MAPK, played a central role in leptin regulation of all cytokines and  receptors. These results suggest that leptin's effects are cell-specific and could confer a proliferative or cell survival advantage or possibly promote endometrial thickness. Leptin's effects on proangiogenic molecules were more evident in malignant versus benign cells and may imply that there is an underlying shift in leptin-induced cell signaling pathways in endometrial cancer cells. (c) 2008 Wiley-Liss, Inc.

      PMID: 18798554 [PubMed - indexed for MEDLINE]

    16. Endocrinology. 2008 Feb;149(2):506-14. Epub 2007 Oct 25.

      Ablation of leptin signaling disrupts the establishment, development, and maintenance of endometriosis-like lesions in a murine model.

      Styer AK, Sullivan BT, Puder M, Arsenault D, Petrozza JC, Serikawa T, Chang S, Hasan T, Gonzalez RR, Rueda BR.

      Massachusetts General Hospital
      Vincent Center for Reproductive Biology
      Department of Obstetrics and Gynecology
      Boston, MA 02114
      USA

      Abstract
      Leptin, a 16-kDa cytokine, has been implicated in several reproductive processes and disorders. Notably, elevated leptin levels in the peritoneal fluid of women with mild endometriosis has been demonstrated, suggesting a role for this cytokine in the early stages of disease establishment. To gain insight into the functional significance of leptin during the initial requisite proliferative and neovascularization events involved in endometriosis, we investigated the effect of disruption of in vivo leptin signaling on the establishment and/or maintenance of an endometriosis-like lesion in a syngeneic immunocompetent mouse model of endometriosis. Findings of this study show that the disruption of leptin signaling by ip injection of the pegylated leptin peptide receptor antagonist (LPrA) impairs the establishment of endometriosis-like lesions (derived from uteri of C57BL/6 female siblings) and results in a reduction of viable organized glandular epithelium, vascular endothelial growth factor-A expression, and mitotic activity. LPrA treatment resulted in a significant reduction of microvascular density in endometriosis-like lesions after continuous and acute courses. Endometriosis-like lesions (derived from tissue with functional leptin receptor) of Lepr(db) hosts (nonfunctional leptin receptor) were phenotypically similar to those of LPrA-treated mice. Our results confirm that leptin signaling is a necessary component in lesion proliferation, early vascular recruitment, and maintenance of neoangiogenesis in a murine model of endometriosis.

      PMCID: PMC2219296
      PMID: 17962343 [PubMed - indexed for MEDLINE]

    17. J Biol Chem. 2006 Sep 8;281(36):26320-8. Epub 2006 Jul 6.

      Leptin signaling promotes the growth of mammary tumors and increases the expression of vascular endothelial growth factor (VEGF) and its receptor type two (VEGF-R2).

      Gonzalez RR, Cherfils S, Escobar M, Yoo JH, Carino C, Styer AK, Sullivan BT, Sakamoto H, Olawaiye A, Serikawa T, Lynch MP, Rueda BR.

      Boston Biomedical Research Institute
      Watertown, Massachusetts 02472
      USA
      Email: rgonzalez@msm.edu

      Abstract
      To gain insight into the mechanism(s) by which leptin contributes to mammary tumor (MT) development we investigated the effects of leptin, kinase inhibitors, and/or leptin receptor antagonists (LPrA2) on 4T1 mouse mammary cancer cells in vitro and LPrA2 on 4T1-MT development in vivo. Leptin increases the expression of vascular endothelial growth factor (VEGF), its receptor (VEGF-R2), and cyclin D1 through phosphoinositide 3-kinase, Janus kinase 2/signal transducer and activator of transcription 3, and/or extracellular signal-activated kinase 1/2 signaling pathways. In contrast to leptin-induced levels of cyclin D1 the changes in VEGF or VEGF-R2 were more dependent on specific signaling pathways. Incubation of 4T1 cells with anti-VEGF-R2 antibody increased leptin-mediated VEGF expression suggesting an autocrine/paracrine loop. Pretreatment of syngeneic mice with LPrA2 prior to inoculation with 4T1 cells delayed the development and slowed the growth of MT (up to 90%) compared with controls. Serum VEGF levels and VEGF/VEGF-R2 expression in MT were significantly lower in mice treated with LPrA2.  Interestingly, LPrA2-induced effects were more pronounced in vivo than in vitro suggesting paracrine actions in stromal, endothelial, and/or inflammatory cells that may impact the growth of MT. Although all the mechanism(s) by which leptin contributes to tumor development are unknown, it appears leptin stimulates an increase in cell numbers, and the expression of VEGF/VEGF-R2. Together, these results provide further evidence suggesting leptin is a MT growth-promoting factor. The inhibition of leptin signaling could serve as a potential adjuvant therapy for treatment of breast cancer and/or provide a new target for the designing strategies to prevent MT development.

      PMID: 16825198 [PubMed - indexed for MEDLINE]

    18. Fertil Steril. 2005 Mar;83(3):587-93.

      Differential expression of endometrial integrins and progesterone receptor during the window of implantation in normo-ovulatory women treated with clomiphene citrate.

      Palomino WA, Fuentes A, González RR, Gabler F, Boric MA, Vega M, Devoto L.

      Institute for Maternal and Child Research
      School of Medicine
      University of Chile
      Santiago, Chile
      Email: palomino@email.unc.edu

      Abstract
      OBJECTIVE: To assess the effect of clomiphene citrate (CC) on endometrial epithelial integrins and P receptors (PR) during the window of implantation.
      DESIGN: Controlled, prospective, clinical study.
      SETTING: Teaching hospital and university research laboratory.
      PATIENT(S): Thirty-one fertile, normo-ovulatory women participated in this trial. Thirteen women exhibited a CC-stimulated cycle with 50 mg on days 5-9, and 18 women with spontaneous menstrual cycles served as controls.
      INTERVENTION(S): Endometrial biopsies in the midluteal phase.
      MAIN OUTCOME MEASURE(S): Immunohistochemical determination and endometrial cellular localization of alpha1, alpha v, beta3, and alpha4 epithelial integrins and PR during the window of implantation. The staining intensity was assessed by a semiquantitative index (HSCORE) and compared by nonparametric Mann-Whitney test.
      RESULT(S): Higher plasma levels of P and E2 and delayed histologic dating of the endometrium (38%) were features of CC-treated women. In addition, a low epithelial beta3 integrin expression and persistent PR were observed in glandular epithelial cells of "out-of-phase" endometrial biopsies from CC-treated women. In contrast, in "in-phase" biopsies, neither epithelial PR nor beta3 integrin were different from spontaneous control cycles. There was no difference in the expression of alpha1, alpha v, and alpha4 between the groups studied.
      CONCLUSION(S): The administration of clomiphene produces aberrant endometrial beta3 integrin expression in conjunction with a failure in the down-regulation of PR during the window of implantation in a significant number of normo-ovulatory women, notwithstanding the higher plasma P levels. Therefore, CC might affect the expression of endometrial receptivity markers.

      PMID: 15749485 [PubMed - indexed for MEDLINE]

    19. Endocrinology. 2004 Aug;145(8):3850-7. Epub 2004 May 13.

      Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling.

      Gonzalez RR, Rueda BR, Ramos MP, Littell RD, Glasser S, Leavis PC.

      Boston Biomedical Research Institute
      Watertown, Massachusetts 02472
      USA
      Email: gonzalezr@bbri.org

      Abstract
      Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24-48 h in a medium containing insulin (5 microg/ml) and leptin (3, 10, and 62 nm) or IL-1beta (0.6, 3, and 10 nm) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1beta also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin's role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).

      PMID: 15142989 [PubMed - indexed for MEDLINE]

    20. Endocrinology. 2005 Feb;146(2):694-701. Epub 2004 Nov 11.

      Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process.

      Ramos MP, Rueda BR, Leavis PC, Gonzalez RR.

      Boston Biomedical Research Institute
      64 Grove Street
      Watertown, Massachusetts 02472
      USA

      Abstract
      Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.

      PMID: 15539553 [PubMed - indexed for MEDLINE]

    21. Endocrine. 2003 Jul;21(2):185-95.

      A peptide derived from the human leptin molecule is a potent inhibitor of the leptin receptor function in rabbit endometrial cells.

      Gonzalez RR, Leavis PC.

      Boston Biomedical Research Institute (BBRI)
      Watertown, MA 02111
      USA
      Email: gonzalezr@bbri.org

      Abstract
      In this article we show that rabbit endometrial cells express leptin receptor and that human leptin triggers phosphorylation of signal transducer and activator of transcription 3 and up-regulates the expression of interleukin- 1 receptor type I as was previously found in human endometrial cells. Interestingly, leptin also upregulates the secretion of leukemia inhibitory factor and expression of its receptor by rabbit endometrial cells. Analysis of a structural model of the leptin-leptin receptor complex suggested that helices I and III of the human leptin structure were likely sites of interaction with the cytokine binding domain of leptin receptor. Accordingly, we synthesized a peptide (LPA-2) comprising helix III (residues 70-95) and investigated its ability to inhibit leptin receptor function. The effects of LPA-2 were assayed in rabbit endometrial cells, and an antileptin receptor antibody and a scrambled version of LPA-2 were used as positive and negative controls, respectively. LPA-2 binds specifically and with high affinity (Ki ~ 0.6 x 10-10 M) to leptin receptor and is a potent inhibitor of its functions in rabbit endometrial cells. Because leukemia inhibitory factor and interleukin- 1 have been implicated in embryo implantation, our results raise the possibility that the LPA-2-induced inhibition of leptin receptor may be exploited to study the actions of leptin in endometrium and in other tissues under conditions characterized by abnormal leptin production.

      PMID: 12897384 [PubMed - indexed for MEDLINE]

    22. Mol Hum Reprod. 2003 Mar;9(3):151-8.

      Leptin regulation of the interleukin-1 system in human endometrial cells.

      Gonzalez RR, Leary K, Petrozza JC, Leavis PC.

      Boston Biomedical Research Institute
      64 Grove Street
      Watertown, MA 02472
      USA
      Email: gonzalezr@bbri.org

      Abstract
      We have previously shown that (i). leptin and leptin receptor (Ob-R) are expressed in the human endometrium, and (ii). leptin secretion is regulated in blastocyst and endometrial epithelial cell (EEC) co-cultures. Interleukin-1beta (IL-1beta) up-regulates leptin and Ob-R, and both cytokines up-regulate beta3 integrin expression in EEC. In the present investigation we examined the effect of leptin on the expression of the IL-1 system in EEC and endometrial stromal cells (ESC) cultured in a medium containing insulin, leptin or IL-1beta (0-3 nmol/l). Leptin stimulated IL-1 antagonist (IL-1Ra), IL-1beta secretion and expression of IL-1 receptor type I (IL-1R tI) in both cell types. IL-1beta and IL-1Ra secretion were down-regulated by IL-1R tI blockade using specific antibodies. Interestingly, leptin partially neutralized this effect. The blockade of Ob-R neutralized the effects of both leptin and IL-1beta on expression of the IL-1beta system and beta3 integrin and on phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that leptin regulates the IL-1 system and that the blockade of functional Ob-R impairs leptin and IL-1beta functions at the endometrial level. Leptin could be an important molecule for implantation and a molecular mediator for actions of the IL-1 system. The fact that leptin, in the absence of IL-1, can trigger the expression of markers of endometrial receptivity and of the invasive trophoblast phenotype (as does IL-1), suggest that leptin could substitute for these IL-1 functions during the implantation process.

      PMID: 12606591 [PubMed - indexed for MEDLINE]

    23. Mol Cell Endocrinol. 2002 Jan 25;186(2):137-41.

      Control of human luteal steroidogenesis.

      Devoto L, Kohen P, Vega M, Castro O, González RR, Retamales I, Carvallo P, Christenson LK, Strauss JF.

      Departamento de Obstetricia y Ginecología
      Facultad de Medicina
      Instituto de Investigaciones Materno Infantil
      IDIMI y Universidad de Chile
      PO Box 226-3
      Santiago, Chile
      Email: ldevoto@machi.med.uchile.cl

      Abstract
      The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.

      PMID: 11900886 [PubMed - indexed for MEDLINE]

    24. Endocrine. 2001 Oct;16(1):21-8.

      Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures.

      Gonzalez RR, Leavis P.

      Boston Biomedical Research Institute
      Watertown, MA 02472
      USA
      Email: gonzalezr@bbri.org

      Abstract
      Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

      PMID: 11822823 [PubMed - indexed for MEDLINE]

    25. Early Pregnancy. 2001 Apr;5(2):132-43.

      Abnormal pattern of integrin expression at the implantation window in endometrium from fertile women treated with clomiphene citrate and users of intrauterine device.

      Gonzalez RR, Palomino A, Vantman D, Gabler F, Devoto L.

      Boston Biomedical Research Institute, BBRI
      64 Grove St.
      Watertown, MA 02472
      USA

      Abstract
      Determine quantitative expression of endometrial integrins that reflect receptivity during implantation window in fertile women treated with clomiphene citrate (CC), and in intrauterine device users (IUD) as compared to fertile controls. Comparative study of the quantitative expression of a1, a4, av and b3 integrins in epithelial and stromal cells in mid-secretory endometrium of CC treated fertile women, IUD users and controls. All subjects included in this study had regular and ovulatory menstrual cycles. Subjects: Ten women treated with a daily dose of 50 mg of CC. Six women T-Cu device users and nine fertile controls. Age ranges for all groups were similar, 29-41 years old (mean 36.3). Tissue samples were taken at the mid-secretory phase or implantation window. A histological dating of the endometrial biopsies was assessed according to Noyes criteria. Ovulation was assessed by repeated transvaginal ultrasonography. The expression of a1, a4, aV and b3 integrins in dispersions of epithelial (EEC) and stromal (ESC) cells isolated from endometrial biopsies was quantitatively determined by flow cytometry using specific monoclonal antibodies.  Immunohistochemistry was also used to detect integrin expression. Biopsies from CC-treated women had a high incidence of out-of-phase endometria. Interestingly, CC-treated women over-expressed a1, aV and b3-ESC integrins and under-expressed b3-EEC subunit (P<0.05). IUD users over-expressed the a1-EEC and under-expressed a4-ESC (P<0.05) at the time of the implantation window. CC treatment in fertile women provokes a high frequency of out-of-phase endometrium and desynchronises the expression of endometrial integrins at the implantation window. The epithelial b3 integrin was under-expressed in all CC-treated patients. The T-Cu intrauterine device alters endometrial receptivity by a different mechanism independent of the expression of the epithelial b3 integrin. However, both CC and IUD use alter the expression of some epithelial and stromal integrins during the implantation window.

      PMID: 11753526 [PubMed - indexed for MEDLINE]

    26. J Clin Endocrinol Metab. 2001 Nov;86(11):5633-9.

      Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

      Devoto L, Kohen P, Gonzalez RR, Castro O, Retamales I, Vega M, Carvallo P, Christenson LK, Strauss JF 3rd.

      Instituto de Investigaciones Materno Infantil
      IDIMI y Departamento de Obstetricia y Ginecología
      Facultad de Medicina
      Universidad de Chile, Santiago
      Email: ldevoto@machi.med.uchile.cl

      Abstract
      The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL.  Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase.  Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

      PMID: 11701746 [PubMed - indexed for MEDLINE]

    27. Endocrine. 2001 Jul;15(2):157-64.

      Effects of leptin, interleukin-1alpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype: integrins and metalloproteinases.

      Gonzalez RR, Devoto L, Campana A, Bischof P.

      Boston Biomedical Research Institute
      Watertown, MA 02472
      USA
      Email: gonzalezr@bbri.org

      Abstract
      Phenotypic changes of integrin and metalloproteinase secretion of the invasive human cytotrophoblast are regulated by cytokines and growth factors, but how this occurs is not completely understood. We used 24-h cytotrophoblast cultures from first trimester pregnancies to investigate the effects of leptin and cytokines on the expression of the alpha2, alpha5, and alpha6 integrin subunits and on the activity of metalloproteinase-2 (gelatinase A) and metalloproteinase-9 (gelatinase B). The alpha2 subunit was marginally upregulated by leptin and interleukin-1alpha (IL-1alpha). All compounds tested upregulated, in some degree, the alpha5 expression. The a6 integrin subunit was massively upregulated, by leptin, interleukins, and transforming growth factor-beta. None of the factors tested affected metalloproteinase-2 activity, but the activity of metalloproteinase-9 was upregulated by leptin and IL-1alpha. In conclusion, leptin and IL-1alpha actively induce some of the changes that cytotrophoblasts undergo to achieve a more invasive phenotype. A novel role for leptin is proposed during early pregnancy: leptin might be an autocrine/paracrine regulator of cytotrophoblast invasiveness during implantation and placentation.

      PMID: 11720241 [PubMed - indexed for MEDLINE]

    28. J Clin Endocrinol Metab. 2000 Dec;85(12):4883-8.

      Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst.

      González RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simon C.

      Instituto de Investigaciones Materno Infantil
      Facultad de Medicina
      Hospital San Borja Arriarán
      Universidad de Chile
      Santiago, Chile

      Abstract
      Embryonic implantation is a crucial event for the human reproductive function. Cytokines and paracrine molecules have been proposed as putative local regulators of this process. The leptin or the OB protein has been linked to the reproductive function and inflammatory response. In the present study, we describe for the first time the expression of leptin and leptin receptor (long form) in the secretory endometrium and that endometrial leptin secretion is regulated in vitro by the human blastocyst. Leptin and leptin receptor messenger RNA and protein were identified in secretory endometrium and in cultured endometrial epithelial cells (EECs) by RT-PCR, Western blot, and immunohistochemistry. The concentrations of immunoreactive leptin secreted by human embryos alone or cocultured with EECs were also assessed. We found that human blastocysts secrete significantly higher levels of leptin than arrested embryos. In contrast, leptin concentrations secreted by arrested embryos cocultured with EECs were significantly higher than blastocysts cocultured with EECs. These findings suggest that the human endometrium is a site for local production and a target tissue for circulating leptin. Expression of leptin and its functional receptor in the endometrium and regulation of endometrial leptin secretion by the human embryo suggests that the leptin system may be implicated in the human implantation process.

      PMID: 11134157 [PubMed - indexed for MEDLINE]

    29. Hum Reprod Update. 2000 May-Jun;6(3):290-300.

      Leptin and reproduction.

      González RR, Simón C, Caballero-Campo P, Norman R, Chardonnens D, Devoto L, Bischof P.

      Institute of Maternal and Child Research (IDIMI)
      Hospital San Borja Arriaran
      Faculty of Medicine
      University of Chile
      Santiago, Chile

      Abstract
      Leptin, the product of the ob gene, is a small peptide molecule synthesized by white adipocytes with an important role in the regulation of body fat and food intake. Leptin and leptin receptor mRNA were first detected in the brain and hypothalamus but now their ubiquitous presence has been demonstrated. Leptin receptor signal transduction involves the activation of signal transducer and activator of transcription (STAT)-3, a member of the transcription family of proteins. Leptin is regulated by hormones and cytokines, interleukin-1, tumour necrosis factor-alpha and transforming growth factor-beta, linking this molecule with the inflammatory response. In addition, emerging evidence has demonstrated that this molecule is related to reproductive function. This small protein is present in the ovary and decidua, in mature oocytes and during embryonic development and trophoblast invasion. Animal models have demonstrated that leptin-deficient ob/ob mice are sterile; however, fertility can be restored by exogenous leptin. In addition, embryos implanted in STAT-3-deficient mice degenerate rapidly and are the target disruption of STAT-3-provoked embryonic lethality. Leptin acts as a novel placental hormone participating in the control of fetal growth and development. Leptin could be a modulator for invasive features of cytotrophoblast cells. We postulate that leptin may have an autocrine/paracrine role in human implantation and placentation.

      PMID: 10874574 [PubMed - indexed for MEDLINE]

    30. Hum Reprod. 1999 Oct;14(10):2485-92.

      A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal.

      Gonzalez RR, Palomino A, Boric A, Vega M, Devoto L.

      Institute of Maternal and Child Research (IDIMI)
      School Of Medicine
      University Of Chile
      Santiago, Chile

      Abstract
      The expression of integrin molecules alpha1beta1, alpha4beta1 and alphaVbeta3 within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. The present investigation examines by flow cytometric analysis the concentrations of alpha1, alpha4, alphaV and beta3 integrin subunits in endometrial stromal (ESC) and epithelial cells (EEC) in two groups of women throughout the menstrual cycle: normal fertile women (n = 27) and women with unexplained infertility (n = 26). Integrin concentrations in endometrial cells were calculated in relative fluorescence units against a negative cellular control. The assessment of integrin subunits detected the protein in ESC and EEC from the late proliferative to the late secretory phase.  In both groups of women, the alpha1 was the highest integrin expressed in ESC and EEC throughout the menstrual cycle. All women exhibited low concentrations of alpha4-EEC at the time of the implantation window. Infertile women expressed lower concentrations of the alpha4-ESC during the proliferative and early secretory phase while lower concentrations of the alpha1-ESC were seen during the late secretory phase. Interestingly, the infertile women expressed lower concentrations of beta3-EEC in the early, mid-secretory and late secretory phases (P < 0.05). Infertile women also expressed lower concentrations of alpha1-EEC and alphaV-EEC during the late secretory phase (P < 0.05). It can be concluded that the quantitative determination of beta3-EEC by flow cytometry confirmed its potential feature as a marker of endometrial receptivity at the time of the implantation window. In addition, the defective expression of the alpha1-ESC found in the late secretory phase might be associated with the poor fertility outcome of women with unexplained infertility.

      PMID: 10527974 [PubMed - indexed for MEDLINE]

    31. Clin Chim Acta. 1991 Mar 29;197(3):159-70.

      An enzyme immunoassay for determining total thyroxine in human serum using an ultramicroanalytical system.

      Gonzalez RR, Robaina R, Rodriguez ME, Blanca S

      National Institute of Endocrinology
      Hospital Fajardo
      Vedado, Havana, Cuba

      Abstract
      A simple, rapid competitive ultramicroElisa assay has been developed for the measurement of total thyroxine (T4) using only 10 microliters of serum. Our novel UME is based on fluorescence measurements of the hydrolytic product of 4-methyl-umbelliferyl-beta-D-galactopyranoside. T4-beta-Galactosidase conjugates  and monoclonal antibodies were immobilized on polyvinyl plates, with sodium salicylate used as a blocking agent for thyroxine binding protein. The analytical steps were carried out using a semiautomated batch-assay system entitled "SUMA" (system for ultramicroanalysis). The T4 assay was completed in 2 h, with a measuring range of 24-386 nmol/L. The intra-assay coefficient of variation (CV) was 4.6-6.9% and the inter-assay C.V. 9.0-12.4% depending on the T4 concentrations. Percentage recovery ranged from 99.2-111%. Regression analysis showed a good correlation with an established radioimmunoassay (n = 121, r =0.946, P less than 0.01).

      PMID: 1904803 [PubMed - indexed for MEDLINE]

    32. Clin Chem. 1990 Sep;36(9):1667-72.

      Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper.

      Gonzalez RR, Mäentausta O, Solyom J, Vihko R.

      Department of Clinical Chemistry
      University of Oulu, Finland

      Abstract
      We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

      PMID: 2208708 [PubMed - indexed for MEDLINE]