Differential gene expression profile in omental adipose tissue in women with polycystic ovary syndrome

10 pages, 2 figures, 5 tables.CONTEXT: The polycystic ovary syndrome (PCOS) is frequently associated with visceral obesity, suggesting that omental adipose tissue might play an important role in the pathogenesis of the syndrome. OBJECTIVE: The objective was to study the expression profiles of omental fat biopsy samples obtained from morbidly obese women with or without PCOS at the time of bariatric surgery. DESIGN: This was a case-control study. SETTINGS: We conducted the study in an academic hospital. PATIENTS: Eight PCOS patients and seven nonhyperandrogenic women submitted to bariatric surgery because of morbid obesity. INTERVENTIONS: Biopsy samples of omental fat were obtained during bariatric surgery. MAIN OUTCOME MEASURE: The main outcome measure was high-density oligonucleotide arrays. RESULTS: After statistical analysis, we identified changes in the expression patterns of 63 genes between PCOS and control samples. Gene classification was assessed through data mining of Gene Ontology annotations and cluster analysis of dysregulated genes between both groups. These methods highlighted abnormal expression of genes encoding certain components of several biological pathways related to insulin signaling and Wnt signaling, oxidative stress, inflammation, immune function, and lipid metabolism, as well as other genes previously related to PCOS or to the metabolic syndrome. CONCLUSION: The differences in the gene expression profiles in visceral adipose tissue of PCOS patients compared with nonhyperandrogenic women involve multiple genes related to several biological pathways, suggesting that the involvement of abdominal obesity in the pathogenesis of PCOS is more ample than previously thought and is not restricted to the induction of insulin resistance.This work was supported by PI020578, PI020741, PI050341, PI050551, RCMN C03/08, and RGDM 03/212 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, and Grants 08.6/0021/2003 and GR/SAL/0137/2004 from the Consejería de Educación y Cultura, Comunidad de Madrid, Spain.Peer reviewe

Functional Inactivation of CXC Chemokine Receptor 4–mediated Responses through SOCS3 Up-regulation

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2+ flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Gαi pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system

Comparative and functional genomics of the protozoan parasite Babesia divergens highlighting the invasion and egress processes

Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.This work was funded by grants from Ministerio de Economía y Competitividad from Spain (AGL2010-21774 and AGL2014-56193 R to EM and LMG). ES was awarded a research fellowship from Plan Estatal de Investigación Científica y Técnica y de Innovación, Ministerio de Economía y Competitividad, Spain (http://www.mineco.gob.es/portal/site/mineco/). Work in CL’s laboratory is funded by a grant from the National Institutes of Health (https://www.nih.gov/) NIH- 1R01HL140625-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

Gβγ dimers released in response to thyrotropin activate phosphoinositide 3-kinase and regulate gene expression in thyroid cells

18 pages, 10 figures.Signaling by TSH through its receptor leads to the dissociation of trimeric G proteins into Galpha and Gbetagamma. Galphas activates adenylyl cyclase, which increases cAMP levels that induce several effects in the thyroid cell, including transcription of the sodium-iodide symporter (NIS) gene through a mechanism involving Pax8 binding to the NIS promoter. Much less is known about the function of Gbetagamma in thyroid differentiation, and therefore we studied their role in TSH signaling. Gbetagamma overexpression inhibits NIS promoter activation and reduces NIS protein accumulation in response to TSH and forskolin. Conversely, inhibition of Gbetagamma-dependent pathways increases NIS promoter activity elicited by TSH but does not modify forskolin-induced activation. Gbetagamma dimers are being released from the Gs subfamily of proteins, because cholera toxin mimics the effects elicited by TSH, whereas pertussis toxin has no effect on NIS promoter activity. We also found that TSH stimulates Akt phosphorylation in a phosphoinositide 3-kinase (PI3K)-dependent and cAMP-independent manner. This is mediated by Gbetagamma, because its overexpression or specific sequestration, respectively, increased or reduced phosphorylated Akt levels upon TSH stimulation. Gbetagamma sequestration increases NIS protein levels induced by TSH and Pax8 binding to the NIS promoter, which is also increased by PI3K inhibition. This is, at least in part, caused by Gbetagamma-mediated Pax8 exclusion from the nucleus that is attenuated when PI3K activity is blocked. These data unequivocally demonstrate that Gbetagamma released by TSH action stimulate PI3K, inhibiting NIS gene expression in a cAMP-independent manner due to a decrease in Pax8 binding to the NIS promoter.This work was supported by grants from the Dirección General de Investigación BFU2004-03169, SAF-2007-60614 (MEC) and from FIS of the Instituto de Salud Carlos III (PI042374, PI041216 and RD06/0020/0060). B.G. was a recipient of a postdoctoral contract of the Comunidad de Madrid (Grant CAMGR/SAL/0773/2004), and M.A.Z holds an FPI predoctoral fellowship from the Spanish Ministry of Education and Science.Peer reviewe

DARPP-32 (dopamine and 3',5'-cyclic adenosine monophosphate-regulated neuronal phosphoprotein) is essential for the maintenance of thyroid differentiation

14 pages, 10 figures.-- Part of this work was presented at the Meeting of the European Thyroid Association, Edinburgh, Scotland, United Kingdom, 2003, and was awarded the M. Köning Prize financed by Organon (Oss, The Netherlands).Coordination of events leading to differentiation is mediated by the concerted action of multiple signal transduction pathways. In general, the uncoupling of mechanisms linking differentiation to cell cycle exit is a hallmark of cancer, yet the identity and regulation of molecules integrating signal transduction pathways remains largely unknown. One notable exception is DARPP-32 (dopamine and cAMP-regulated neuronal phosphoprotein, molecular mass, 32 kDa), a third messenger that integrates multiple signaling pathways in the brain. Thyroid cells represent an excellent model for understanding the coupling of signal transduction pathways leading to both proliferation and differentiation. The cooperative action of IGF-I and TSH together, but not alone, enable thyroid cells to proliferate while maintaining their differentiated state. How signaling downstream from these molecules is integrated is not known. Here we show that DARPP-32 expression is targeted by TSH and IGF-I in thyrocytes. Significantly, dedifferentiated, tumoral, or Ras-transformed thyrocytes fail to express DARPP-32 whereas short interfering RNA-mediated silencing of DARPP-32 expression in normally differentiated thyroid cells results in loss of differentiation markers such as thyroid transcription factor 1, Pax8, thyroglobulin, and the Na/I symporter. Consistently, DARPP-32 reexpression in ras-transformed cells results in reactivation of the otherwise silent thyroglobulin and thyroperoxidase promoter. Thus, DARPP-32 is critical for the maintenance of thyroid differentiation by TSH and IGF-I, and loss of DARPP-32 expression may be a characteristic of thyroid cancer. Our results also raise the possibility that DARPP-32 may play a similar role in the maintenance of differentiation of a range of other cell types.This work was supported by Dirección General de Investigación Científica y Técnica, Grant BFU2004-03169, GR/SAL 0773/2004 from Comunidad de Madrid and Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (RCMN-C03/08, RCGC C03/10) and PI041216. M.A.Z. was the recipient of a fellowship from the Ministerio de Educación y Ciencia (Spain).Peer reviewe

Key signaling pathways in thyroid cancer

Whole genome sequencing approaches have provided unprecedented insights into the genetic lesions responsible for the onset, progression and dedifferentiation of various types of thyroid carcinomas. Through these efforts, the MAPK and PI3K signaling cascades have emerged as the main activation pathways implicated in thyroid tumorigenesis. The nature of these essential pathways is highly complex, with hundreds of components, multiple points of crosstalk, different subcellular localizations and with the ability to potentially regulate many cellular processes. Small-molecule inhibitors targeting key kinases of these pathways hold great promise as novel therapeutics and several have reached clinical trials. However, while some remarkable responses have been reported, the development of resistance remains a matter of concern and limits the benefit for patients. In this review, we discuss the latest findings on the major components of the MAPK and PI3K pathways, including their mechanisms of activation in physiological and pathological contexts, their genetic alterations with respect to the different types of thyroid carcinomas and the more relevant drugs designed to block their activity.Supported by the following grants: GCB14142311CRES from the Spanish Foundation against Cancer (F-AECC), SAF2013-44709-R and SAF2016-75531-R from the Ministry of Economy and Competitiveness, RD12/0036/0030 and Ciberonc from the Carlos III Health Institute, (Spain), and the European Regional Development Fund (FEDER).Peer reviewe

FOXO1 controls thyroid cell proliferation in response to TSH and IGF-I and is involved in thyroid tumorigenesis

TSH and insulin/IGF-I synergistically induce the proliferation of thyroid cells mainly through the cAMP and phosphatidylinositol 3-kinase (PI3K) pathways. However, the events involved in this cooperative induction remain unknown, and molecules that are potentially controlled by both TSH and IGF-I are interesting candidates as integrators of both stimuli. The finding that the PI3K pathway is frequently activated in thyroid malignancies has attracted attention to this pathway in the thyroid field. One of the targets of PI3K is Forkhead box O (FoxO)-1, a widely expressed transcription factor involved in a variety of cellular processes such as differentiation, proliferation, and apoptosis. Here we show that FoxO1 is highly expressed in differentiated rat thyroid cells and human thyroid tissue compared with human thyroid tumor-derived cells and surgically removed thyroid tumors, in which its expression is reduced. In differentiated cells, TSH/cAMP treatment decreases FoxO1 mRNA and protein levels through proteasome activation, whereas both TSH and IGF-I control FoxO1 localization by promoting a rapid exclusion from the nucleus in an Akt-dependent manner. FoxO1 can control p27KIP1 expression in differentiated and tumor cells of the thyroid. Furthermore, FoxO1 reexpression in tumor cells promotes a decrease in their proliferation rate, whereas FoxO1 interference in differentiated cells increases their proliferation. These data point to an important role of FoxO1 in mediating the effects of TSH and IGF-I on thyroid cell proliferation and provide a link between loss of FoxO1 expression and the uncontrolled proliferation of thyroid tumor cells. © 2013 by The Endocrine Society.This work was supported by Grant BFU-2010-16025 from the Dirección General de Proyectos de Investigación; Grant RD06/0020/0060 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, and Grant S2011/BMD-2328 TIRONET project from the Comunidad de Madrid (Spain). M.A.Z. was supported by a contract associated with a SAF-2007-60164 project.Peer Reviewe

β-catenin signaling is required for RAS-driven thyroid cancer through PI3K activation

Mutations in β-catenin are traditionally described as late events in thyroid cancer progression. However, the functional implications of β-catenin dysregulation in the context of tumor initiating events remain unclear. The aim of this work was to investigate whether the two main oncogenic drivers in thyroid cancer, RAS and BRAF, could activate the Wnt/β-catenin pathway. Expression of HRAS but not BRAF in thyroid cells induced β-catenin nuclear localization, increased β-catenin-dependent transcriptional activity and inhibited GSK3β. In a panel of human thyroid cancer cell lines representative of the main genetic events in thyroid cancer, β-catenin activation was highly dependent on PI3K/AKT activity through its phosphorylation at S552, but not on MAPK. Silencing of β-catenin expression in cell lines led to a dramatic reduction in proliferation due to an induction of senescence, which was concordant with a reduction in tumor size in nude mice. Moreover, β-catenin silencing suppressed the expression of EMT-related genes and reduced the invasive capacity of the tumor cells. In conclusion, this work demonstrates that RAS-driven tumors induce PI3K/ AKT-dependent β-catenin activation.This work was supported by grants SAF2013-44709-R from the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER), RD12/0036/0030 from Instituto de Salud Carlos III (ISCIII), S2011/BMD-2328 from Comunidad de Madrid, and GCB14142311CRES from Fundación Española contra el Cancer (AECC). AS-P was supported by predoctoral FPU fellowship from the Ministerio de Economía y Competitividad and MA-Z has a research contract of AECC (GCB14142311CRES) .Peer Reviewe

Role of the IQGAP1 scaffold protein in thyroid cancer

Resumen del póster presentado al 6th Symposium on Biomedical Research: Advances and Perspectives in Molecular Endocrinology "In Homage to Gabriella Morreale", celebrado en el Instituto de Investigaciones Biomédicas Alberto Sols (IIBM-CSIC) el 31 de mayo de 2019.The IQGAP family of scaffold proteins comprises three members which have been described to interact with numerous proteins in several biological processes and participate in the signaling cascade of multiple pathways. Among these proteins IQGAP1 has been the most deeply studied, having characterized its function as a scaffold protein in the MAPK pathway and its interaction with actin leading to regulation of the cytoskeleton. Because of these functions it has been studied in multiple malignancies, where it has been assigned a tumorigenic effect. We have studied IQGAP1 expression using different stablished cell lines of human thyroid carcinomas. Our results indicate that these carcinoma cell lines have variable levels of IQGAP1 but in general these levels are higher when compared to the normal thyroid cell line control. The results obtained are in agreement with the TCGA and Oncomine databases. Using IQGAP1 silenced cell lines we tried to elucidate how this protein was affecting tumor hallmarks such as proliferation, invasion, cell migration and epithelial mesenchymal transition. The effects of silencing IQGAP1 were different depending on the cell line and the oncogenic signature. While in some of them silencing produced an increase in the migration rate of the cells, others had the opposite behavior. We also found these differences when studying EMT markers such as E-cadherin and N-cadherin. Furthermore, cell viability was unaffected by IQGAP1 silencing and consequently the size of the primary tumor was not altered when cells were xenografted in a chicken embryo model. Importantly IQGAP1 silencing greatly decreased the ability of the tumor cells to intravasate and metastasize to distant organs such as lungs and brain. Further studies will focus on trying to discover the underlying mechanisms responsible for the differences observed between cell lines. Our results indicate a role forIQGAP1 in thyroid tumorigenesis and underscore the importance of defining the oncogenic signature and particularities of a given tumor type in order to design effective therapeutic strategies.This work was supported by grants SAF2016-75531-R from MINECO, Spain and PI14/01980 from ISCIII, Spain (FEDER); B2017/BMD-3724 from CAM and GCB14142311CRES from AECC.Peer reviewe