Publications by authors named "Joan-Marc Servitja"

28 Publications

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Alpha1-antitrypsin ameliorates islet amyloid-induced glucose intolerance and β-cell dysfunction.

Mol Metab 2020 07 27;37:100984. Epub 2020 Mar 27.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain. Electronic address:

Objective: Pancreatic β-cell failure is central to the development and progression of type 2 diabetes (T2D). The aggregation of human islet amyloid polypeptide (hIAPP) has been associated with pancreatic islet inflammation and dysfunction in T2D. Alpha1-antitrypsin (AAT) is a circulating protease inhibitor with anti-inflammatory properties. Here, we sought to investigate the potential therapeutic effect of AAT treatment in a mouse model characterized by hIAPP overexpression in pancreatic β-cells.

Methods: Mice overexpressing hIAPP (hIAPP-Tg) in pancreatic β-cells were used as a model of amyloid-induced β-cell dysfunction. Glucose homeostasis was evaluated by glucose tolerance tests and insulin secretion assays. Apoptosis and amyloid formation was assessed in hIAPP-Tg mouse islets cultured at high glucose levels. Dissociated islet cells were cocultured with macrophages obtained from the peritoneal cavity.

Results: Nontreated hIAPP-Tg mice were glucose intolerant and exhibited impaired insulin secretion. Interestingly, AAT treatment improved glucose tolerance and restored the insulin secretory response to glucose in hIAPP-Tg mice. Moreover, AAT administration normalized the expression of the essential β-cell genes MafA and Pdx1, which were downregulated in pancreatic islets from hIAPP-Tg mice. AAT prevented the formation of amyloid deposits and apoptosis in hIAPP-Tg islets cultured at high glucose concentrations. Since islet macrophages mediate hIAPP-induced β-cell dysfunction, we investigated the effect of AAT in cocultures of macrophages and islet cells. AAT prevented hIAPP-induced β-cell apoptosis in these cocultures without reducing the hIAPP-induced secretion of IL-1β by macrophages. Remarkably, AAT protected β-cells against the cytotoxic effects of conditioned medium from hIAPP-treated macrophages. Similarly, AAT also abrogated the cytotoxic effects of exogenous proinflammatory cytokines on pancreatic β-cells.

Conclusions: These results demonstrate that treatment with AAT improves glucose homeostasis in mice overexpressing hIAPP and protects pancreatic β-cells from the cytotoxic actions of hIAPP mediated by macrophages. These results support the use of AAT-based therapies to recover pancreatic β-cell function for the treatment of T2D.
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http://dx.doi.org/10.1016/j.molmet.2020.100984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186564PMC
July 2020

The type 2 diabetes-associated HMG20A gene is mandatory for islet beta cell functional maturity.

Cell Death Dis 2018 02 15;9(3):279. Epub 2018 Feb 15.

Department of Cell Regeneration and Advanced Therapies, Andalusian Center of Molecular Biology and Regenerative Medicine-CABIMER, Junta de Andalucia-University of Pablo de Olavide-University of Seville-CSIC, Seville, Spain.

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.
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http://dx.doi.org/10.1038/s41419-018-0272-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833347PMC
February 2018

Stress-Induced MicroRNA-708 Impairs β-Cell Function and Growth.

Diabetes 2017 12 2;66(12):3029-3040. Epub 2017 Oct 2.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

The pancreatic β-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets. We identified miR-708 as the most upregulated miRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response. miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of / mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin () as a potential glucose-regulated target of miR-708. Indeed, expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in / mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of β-cells, miR-708 overexpression impaired glucose-stimulated insulin secretion (GSIS), which was recovered by overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed β-cell proliferation and induced β-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of β-cell function and growth by repressing stress-induced miR-708.
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http://dx.doi.org/10.2337/db16-1569DOI Listing
December 2017

Late-stage differentiation of embryonic pancreatic β-cells requires Jarid2.

Sci Rep 2017 09 14;7(1):11643. Epub 2017 Sep 14.

Diabetes and Obesity Research Laboratory, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Rosselló 149-153, 08036, Barcelona, Spain.

Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing β-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived β-cells.
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http://dx.doi.org/10.1038/s41598-017-11691-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599523PMC
September 2017

Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

FASEB J 2017 12 15;31(12):5296-5306. Epub 2017 Aug 15.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, Barcelona, Spain;

Human islet amyloid polypeptide (hIAPP) aggregation is associated with β-cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether treatment with chemical chaperone 4-phenylbutyrate (PBA) ameliorates hIAPP-induced β-cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic β cells counteracted impaired glucose homeostasis and restored glucose-stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased β-cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring β-cell function and preventing amyloid formation in mice expressing hIAPP in β cells, highlighting the therapeutic potential of PBA for the treatment of T2D.-Montane, J., de Pablo, S., Castaño, C., Rodríguez-Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz-Vizán, G., Sanchez-Martinez, M., Nonell-Canals, A., Parrizas, M., Servitja, J.-M., Novials, A. Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.
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http://dx.doi.org/10.1096/fj.201700236RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690378PMC
December 2017

Mitochondrial Dynamics Mediated by Mitofusin 1 Is Required for POMC Neuron Glucose-Sensing and Insulin Release Control.

Cell Metab 2017 Jun;25(6):1390-1399.e6

Neuronal Control of Metabolism (NeuCoMe) Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain; CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain. Electronic address:

Proopiomelanocortin (POMC) neurons are critical sensors of nutrient availability implicated in energy balance and glucose metabolism control. However, the precise mechanisms underlying nutrient sensing in POMC neurons remain incompletely understood. We show that mitochondrial dynamics mediated by Mitofusin 1 (MFN1) in POMC neurons couple nutrient sensing with systemic glucose metabolism. Mice lacking MFN1 in POMC neurons exhibited defective mitochondrial architecture remodeling and attenuated hypothalamic gene expression programs during the fast-to-fed transition. This loss of mitochondrial flexibility in POMC neurons bidirectionally altered glucose sensing, causing abnormal glucose homeostasis due to defective insulin secretion by pancreatic β cells. Fed mice lacking MFN1 in POMC neurons displayed enhanced hypothalamic mitochondrial oxygen flux and reactive oxygen species generation. Central delivery of antioxidants was able to normalize the phenotype. Collectively, our data posit MFN1-mediated mitochondrial dynamics in POMC neurons as an intrinsic nutrient-sensing mechanism and unveil an unrecognized link between this subset of neurons and insulin release.
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http://dx.doi.org/10.1016/j.cmet.2017.05.010DOI Listing
June 2017

BACE2 suppression promotes β-cell survival and function in a model of type 2 diabetes induced by human islet amyloid polypeptide overexpression.

Cell Mol Life Sci 2017 08 23;74(15):2827-2838. Epub 2017 Mar 23.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), C/Rosselló 149-153, 5th floor, 08036, Barcelona, Spain.

BACE2 (β-site APP-cleaving enzyme 2) is a protease expressed in the brain, but also in the pancreas, where it seems to play a physiological role. Amyloidogenic diseases, including Alzheimer's disease and type 2 diabetes (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. In T2D, islet amyloid polypeptide (IAPP) deposits have been shown to be a pathogenic key feature of the disease. The aim of the present study was to investigate the effect of BACE2 modulation on β-cell alterations in a mouse model of T2D induced by IAPP overexpression. Heterozygous mice carrying the human transcript of IAPP (hIAPP-Tg) were used as a model to study the deleterious effects of IAPP upon β-cell function. These animals showed glucose intolerance and impaired insulin secretion. When crossed with BACE2-deficient mice, the animals presented a significant improvement in glucose tolerance accompanied with an enhanced insulin secretion, as compared to hIAPP-Tg mice. BACE2 deficiency also partially reverted gene expression changes observed in islets from hIAPP-Tg mice, including a set of genes related to inflammation. Moreover, homozygous hIAPP mice presented a severe hyperglycemia and a high lethality rate from 8 weeks onwards due to a massive destruction of β-cell mass. This process was significantly reduced when crossed with the BACE2-KO model, improving the survival rate of the animals. Altogether, the absence of BACE2 ameliorates glucose tolerance defects induced by IAPP overexpression in the β-cell and promotes β-cell survival. Thus, targeting BACE2 may represent a promising therapeutic strategy to improve β-cell function in T2D.
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http://dx.doi.org/10.1007/s00018-017-2505-1DOI Listing
August 2017

Maternal Exposure to Bisphenol-A During Pregnancy Increases Pancreatic β-Cell Growth During Early Life in Male Mice Offspring.

Endocrinology 2016 Nov 13;157(11):4158-4171. Epub 2016 Sep 13.

Institute of Bioengineering (M.G.-A. T.B., B.M., S.V., I.Q., A.Na.) and Department of Applied Biology (P.A.-M.), Miguel Hernández University of Elche, 03202 Elche, Alicante, Spain; Diabetes and Obesity Research Laboratory (J.-M.S., A.No.), Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic de Barcelona, 08036 Barcelona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (M.G.-A., P.A.-M., J.-M.S., T.B.-B., B.M., S.V.-P., A.No., I.Q., A.Na.), Spain; and Department of Basic Sciences of Health (G.M.-G.), Area of Biochemistry and Molecular Biology, Rey Juan Carlos University, 28922 Alcorcon, Madrid, Spain.

Alterations during development of metabolic key organs such as the endocrine pancreas affect the phenotype later in life. There is evidence that in utero or perinatal exposure to bisphenol-A (BPA) leads to impaired glucose metabolism during adulthood. However, how BPA exposure during pregnancy affects pancreatic β-cell growth and function in offspring during early life has not been explored. We exposed pregnant mice to either vehicle (control) or BPA (10 and 100 μg/kg·d, BPA10 and BPA100) and examined offspring on postnatal days (P) P0, P21, P30, and P120. BPA10 and BPA100 mice presented lower birth weight than control and subsequently gained weight until day 30. At that age, concentration of plasma insulin, C-peptide, and leptin were increased in BPA-exposed animals in the nonfasting state. Insulin secretion and content were diminished in BPA10 and maintained in BPA100 compared with control. A global gene expression analysis indicated that genes related with cell division were increased in islets from BPA-treated animals. This was associated with an increase in pancreatic β-cell mass at P0, P21, and P30 together with increased β-cell proliferation and decreased apoptosis. On the contrary, at P120, BPA-treated animals presented either equal or decreased β-cell mass compared with control and altered fasting glucose levels. These data suggest that in utero exposure to environmentally relevant doses of BPA alters the expression of genes involved in β-cell growth regulation, incrementing β-cell mass/area, and β-cell proliferation during early life. An excess of insulin signaling during early life may contribute to impaired glucose tolerance during adulthood.
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http://dx.doi.org/10.1210/en.2016-1390DOI Listing
November 2016

Protein disulfide isomerase ameliorates β-cell dysfunction in pancreatic islets overexpressing human islet amyloid polypeptide.

Mol Cell Endocrinol 2016 Jan 1;420:57-65. Epub 2015 Dec 1.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain. Electronic address:

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits in islets of type 2 diabetic patients. hIAPP misfolding and aggregation is one of the factors that may lead to β-cell dysfunction and death. Endogenous chaperones are described to be important for the folding and functioning of proteins. Here, we examine the effect of the endoplasmic reticulum chaperone protein disulfide isomerase (PDI) on β-cell dysfunction. Among other chaperones, PDI was found to interact with hIAPP in human islet lysates. Furthermore, intrinsically recovered PDI levels were able to restore the effect of high glucose- and palmitate-induced β-cell dysfunction by increasing 3.9-fold the glucose-stimulated insulin secretion levels and restoring insulin content up to basal control values. Additionally, PDI transduction decreased induced apoptosis by glucolipotoxic conditions. This approach could reveal a new therapeutic target and aid in the development of strategies to improve β-cell dysfunction in type 2 diabetic patients.
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http://dx.doi.org/10.1016/j.mce.2015.11.018DOI Listing
January 2016

Islet amyloid polypeptide exerts a novel autocrine action in β-cell signaling and proliferation.

FASEB J 2015 Jul 25;29(7):2970-9. Epub 2015 Mar 25.

*Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain; and Department of Metabolism, Nutrition and Hormones, Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain

The toxic effects of human islet amyloid polypeptide (IAPP) on pancreatic islets have been widely studied. However, much less attention has been paid to the physiologic actions of IAPP on pancreatic β cells, which secrete this peptide together with insulin upon glucose stimulation. Here, we aimed to explore the signaling pathways and mitogenic actions of IAPP on β cells. We show that IAPP activated Erk1/2 and v-akt murine thymoma viral oncogene homolog 1 (Akt) at the picomolar range (10-100 pM) in mouse pancreatic islets and MIN6 β cells cultured at low glucose concentrations. In contrast, IAPP decreased the induction of these pathways by high glucose levels. Consistently, IAPP induced a 1.7-fold increase of β-cell proliferation at low-glucose conditions, whereas it reduced β-cell proliferation at high glucose levels. Strikingly, the specific antagonist of the IAPP receptor AC187 (100 nM) decreased the activation of Erk1/2 and Akt and reduced β-cell proliferation by 24% in glucose-stimulated β cells, uncovering a key role of endogenously released IAPP in β-cell responses to glucose. We conclude that exogenously added IAPP exerts a dual effect on β-cell mitogenic signaling and proliferation, depending on the glucose concentration. Importantly, secreted IAPP contributes to the signaling and mitogenic response of β cells to glucose through an autocrine mechanism.
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http://dx.doi.org/10.1096/fj.15-270553DOI Listing
July 2015

Circulating miR-192 and miR-193b are markers of prediabetes and are modulated by an exercise intervention.

J Clin Endocrinol Metab 2015 Mar 22;100(3):E407-15. Epub 2014 Dec 22.

Diabetes and Obesity Research Laboratory (M.P., L.B., Y.E., A.G.-F., S.C., S.M., P.M.G.-R., J.-M.S., A.N.), Institut d'Investigacions Biomèdiques August Pi i Sunyer, 08036 Barcelona, Spain; Biomedical Research Center in Diabetes and Associated Metabolic Disorders (Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas) (M.P., L.B., Y.E., A.G.-F., S.C., S.M., P.M.G.-R., J.-M.S., A.N.), 08036 Barcelona, Spain; Department of Endocrinology (E.G.-B.), Hospital Clínic, 08036 Barcelona, Spain; and Department of Physiological Sciences (R.C.), and National Institute of Physical Education of Catalonia (J.A.C.), University of Barcelona, 08038 Barcelona, Spain.

Context: Diabetes is frequently diagnosed late, when the development of complications is almost inevitable, decreasing the quality of life of patients. However, early detection of affected individuals would allow the implementation of timely and effective therapies.

Objective: Here we set to describe the profile of circulating microRNAs (miRNAs) in prediabetic patients with the intention of identifying novel diagnostic and therapeutic tools.

Design: We used real-time RT-PCR to measure the abundance of 176 miRNAs in serum of a cohort of 92 control and prediabetic individuals with either impaired fasting glucose or impaired glucose tolerance, as well as newly diagnosed diabetic patients. We validated the results in a second cohort of control and prediabetic subjects undergoing a therapeutic exercise intervention, as well as in a mouse model of glucose intolerance.

Results: We identified two miRNAs, miR-192 and miR-193b, whose abundance is significantly increased in the prediabetic state but not in diabetic patients. Strikingly, these miRNAs are also increased in plasma of glucose-intolerant mice. Moreover, circulating levels of miR-192 and miR-193b return to baseline in both prediabetic humans and glucose-intolerant mice undergoing a therapeutic intervention consisting in chronic exercise, which succeeded in normalizing metabolic parameters.

Conclusions: Our data show that the pattern of circulating miRNAs is modified by defects in glucose metabolism in a similar manner in mice and humans. This circulating miRNA signature for prediabetes could be used as a new diagnostic tool, as well as to monitor response to intervention.
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http://dx.doi.org/10.1210/jc.2014-2574DOI Listing
March 2015

Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic β-cells.

FASEB J 2015 Jan 23;29(1):95-104. Epub 2014 Oct 23.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Barcelona, Spain

BACE2 (β-site APP-cleaving enzyme 2) is a protease localized in the brain, where it appears to play a role in the development of Alzheimer disease (AD). It is also found in the pancreas, although its biologic function is not fully known. Amyloidogenic diseases, including AD and type 2 diabetes mellitus (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. Islet amyloid polypeptide (IAPP) deposits are a key pathogenic feature of T2D. Within this context, we found by global gene expression profiling that BACE2 was up-regulated in the rat pancreatic β-cell line INS1E stably transfected with human IAPP gene (hIAPP-INS1E). Glucose-stimulated insulin secretion (GSIS) in hIAPP-INS1E cells was 30% lower than in INS1E cells. Additionally, INS1E cells transfected with a transient overexpression of BACE2 showed a 60% decrease in proliferation, a 3-fold increase in reactive oxygen species production, and a 25% reduction in GSIS compared to control cells. Remarkably, silencing of endogenous BACE2 in hIAPP-INS1E cells resulted in a significant improvement in GSIS (3-fold increase vs. untransfected cells), revealing the significant role of BACE2 expression in β-cell function. Thus, BACE2 inhibition may be useful to recover insulin secretion in hIAPP-INS1E defective cells and may be proposed as a therapeutic target for T2D.
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http://dx.doi.org/10.1096/fj.14-255489DOI Listing
January 2015

Chaperones ameliorate beta cell dysfunction associated with human islet amyloid polypeptide overexpression.

PLoS One 2014 10;9(7):e101797. Epub 2014 Jul 10.

Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic de Barcelona, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain.

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101797PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092029PMC
March 2015

Thyroid hormones promote endocrine differentiation at expenses of exocrine tissue.

Exp Cell Res 2014 Apr 4;322(2):236-48. Epub 2014 Feb 4.

Inserm U845, Research Center Growth and Signaling, Faculté de Médecine, Université Paris Descartes, Sorbone Paris Cité, Necker Hospital, Paris, France; Diabetes and Obesity Research Laboratory, Institut d׳Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. Electronic address:

Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.
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http://dx.doi.org/10.1016/j.yexcr.2014.01.030DOI Listing
April 2014

Nurr1 protein is required for N-methyl-D-aspartic acid (NMDA) receptor-mediated neuronal survival.

J Biol Chem 2012 Mar 31;287(14):11351-62. Epub 2012 Jan 31.

Institut de Neurociencies and Departament de Bioquímica i Biología Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.
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http://dx.doi.org/10.1074/jbc.M111.272427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322860PMC
March 2012

Gene expression dynamics after murine pancreatitis unveils novel roles for Hnf1α in acinar cell homeostasis.

Gut 2012 Aug 23;61(8):1187-96. Epub 2011 Sep 23.

Grup de Recerca en Patologia Pancreàtica Exocrina, Institut de Recerca Hospital Universitari Vall d'Hebron, Pg Vall d'Hebron 119-129, Barcelona 08035, Spain.

Objectives: During pancreatitis, specific transcriptional programmes govern functional regeneration after injury. The objective of this study was to analyse the dynamic regulation of pancreatic genes and the role of transcriptional regulators during recovery from pancreatitis.

Design: Wild-type and genetically modified mice (Hnf1α(-/-) and Ptf1a(+/-)) were used. After caerulein or L-arginine induced pancreatitis, blood or pancreata were processed for enzymatic assays, ELISA, histology, immunohistochemistry, western blotting and quantitative reverse transcriptase-PCR. Nr5a2 promoter reporter and chromatin immunoprecipitation assays for Hnf1α were also performed.

Results: After caerulein pancreatic injury, expression of acinar and endocrine genes rapidly decreased, but eventually recovered, depicting distinct cell-type-specific patterns. Pdx1 and Hnf1α mRNAs underwent marked downregulation, matching endocrine/exocrine gene expression profiles. Ptf1a, Pdx1 and Hnf1α protein levels were also reduced and recovered gradually. These changes were associated with transient impairment of exocrine and endocrine function, including abnormal glucose tolerance. On l-arginine pancreatitis, changes in Ptf1a, Pdx1 and Hnf1α gene and protein expression were recapitulated. Reduced Hnf1α and Ptf1a levels after pancreatitis coincided with increased acinar cell proliferation, both in Hnf1α(-/-) and Ptf1a(+/-) mice. Moreover, Hnf1α(-/-) mice had reduced Ptf1a protein as well as transcripts for Ptf1a and digestive enzymes. Dispersed acini from Hnf1α(-/-) mice showed suboptimal secretory responses to caerulein. Bioinformatics analysis did not support a role for Hnf1α as a direct regulator of digestive enzyme genes. Instead, it was found that Hnf1α binds to, and regulates, the promoter of Nr5a2, coding an orphan nuclear receptor that regulates acinar gene expression.

Conclusions: Dynamic changes in gene expression occur on pancreatitis induction, determining altered exocrine and endocrine function. This analysis uncovers roles for Hnf1α in the regulation of acinar cell determination and function. This effect may be mediated, in part, through direct regulation of Nr5a2.
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http://dx.doi.org/10.1136/gutjnl-2011-300360DOI Listing
August 2012

EuroDia: a beta-cell gene expression resource.

Database (Oxford) 2010 Oct 12;2010:baq024. Epub 2010 Oct 12.

Vital-IT, SIB Swiss Institute of Bioinformatics, Genopode Building, CH-1015 Lausanne, Switzerland.

Type 2 diabetes mellitus (T2DM) is a major disease affecting nearly 280 million people worldwide. Whilst the pathophysiological mechanisms leading to disease are poorly understood, dysfunction of the insulin-producing pancreatic beta-cells is key event for disease development. Monitoring the gene expression profiles of pancreatic beta-cells under several genetic or chemical perturbations has shed light on genes and pathways involved in T2DM. The EuroDia database has been established to build a unique collection of gene expression measurements performed on beta-cells of three organisms, namely human, mouse and rat. The Gene Expression Data Analysis Interface (GEDAI) has been developed to support this database. The quality of each dataset is assessed by a series of quality control procedures to detect putative hybridization outliers. The system integrates a web interface to several standard analysis functions from R/Bioconductor to identify differentially expressed genes and pathways. It also allows the combination of multiple experiments performed on different array platforms of the same technology. The design of this system enables each user to rapidly design a custom analysis pipeline and thus produce their own list of genes and pathways. Raw and normalized data can be downloaded for each experiment. The flexible engine of this database (GEDAI) is currently used to handle gene expression data from several laboratory-run projects dealing with different organisms and platforms. Database URL: http://eurodia.vital-it.ch.
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http://dx.doi.org/10.1093/database/baq024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963318PMC
October 2010

Hnf1alpha (MODY3) controls tissue-specific transcriptional programs and exerts opposed effects on cell growth in pancreatic islets and liver.

Mol Cell Biol 2009 Jun 16;29(11):2945-59. Epub 2009 Mar 16.

Genomic Programming of Beta-Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

Heterozygous HNF1A mutations cause pancreatic-islet beta-cell dysfunction and monogenic diabetes (MODY3). Hnf1alpha is known to regulate numerous hepatic genes, yet knowledge of its function in pancreatic islets is more limited. We now show that Hnf1a deficiency in mice leads to highly tissue-specific changes in the expression of genes involved in key functions of both islets and liver. To gain insights into the mechanisms of tissue-specific Hnf1alpha regulation, we integrated expression studies of Hnf1a-deficient mice with identification of direct Hnf1alpha targets. We demonstrate that Hnf1alpha can bind in a tissue-selective manner to genes that are expressed only in liver or islets. We also show that Hnf1alpha is essential only for the transcription of a minor fraction of its direct-target genes. Even among genes that were expressed in both liver and islets, the subset of targets showing functional dependence on Hnf1alpha was highly tissue specific. This was partly explained by the compensatory occupancy by the paralog Hnf1beta at selected genes in Hnf1a-deficient liver. In keeping with these findings, the biological consequences of Hnf1a deficiency were markedly different in islets and liver. Notably, Hnf1a deficiency led to impaired large-T-antigen-induced growth and oncogenesis in beta cells yet enhanced proliferation in hepatocytes. Collectively, these findings show that Hnf1alpha governs broad, highly tissue-specific genetic programs in pancreatic islets and liver and reveal key consequences of Hnf1a deficiency relevant to the pathophysiology of monogenic diabetes.
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http://dx.doi.org/10.1128/MCB.01389-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682018PMC
June 2009

Functional targets of the monogenic diabetes transcription factors HNF-1alpha and HNF-4alpha are highly conserved between mice and humans.

Diabetes 2009 May 2;58(5):1245-53. Epub 2009 Feb 2.

Genomic Programming of Beta-cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain.

Objective: The evolutionary conservation of transcriptional mechanisms has been widely exploited to understand human biology and disease. Recent findings, however, unexpectedly showed that the transcriptional regulators hepatocyte nuclear factor (HNF)-1alpha and -4alpha rarely bind to the same genes in mice and humans, leading to the proposal that tissue-specific transcriptional regulation has undergone extensive divergence in the two species. Such observations have major implications for the use of mouse models to understand HNF-1alpha- and HNF-4alpha-deficient diabetes. However, the significance of studies that assess binding without considering regulatory function is poorly understood.

Research Design And Methods: We compared previously reported mouse and human HNF-1alpha and HNF-4alpha binding studies with independent binding experiments. We also integrated binding studies with mouse and human loss-of-function gene expression datasets.

Results: First, we confirmed the existence of species-specific HNF-1alpha and -4alpha binding, yet observed incomplete detection of binding in the different datasets, causing an underestimation of binding conservation. Second, only a minor fraction of HNF-1alpha- and HNF-4alpha-bound genes were downregulated in the absence of these regulators. This subset of functional targets did not show evidence for evolutionary divergence of binding or binding sequence motifs. Finally, we observed differences between conserved and species-specific binding properties. For example, conserved binding was more frequently located near transcriptional start sites and was more likely to involve multiple binding events in the same gene.

Conclusions: Despite evolutionary changes in binding, essential direct transcriptional functions of HNF-1alpha and -4alpha are largely conserved between mice and humans.
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http://dx.doi.org/10.2337/db08-0812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671044PMC
May 2009

Distinct roles of HNF1beta, HNF1alpha, and HNF4alpha in regulating pancreas development, beta-cell function and growth.

Endocr Dev 2007 ;12:33-45

Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Endocrinology, Hospital Clínic de Barcelona, Barcelona , Spain.

Mutations in the genes encoding transcriptional regulators HNF1beta (TCF2), HNF1alpha (TCF1), and HNF4alpha cause autosomal dominant diabetes (also known as maturity-onset diabetes of the young). Herein, we review what we have learnt during recent years concerning the functions of these regulators in the developing and adult pancreas. Mouse studies have revealed that HNF1beta is a critical regulator of a transcriptional network that controls the specification, growth, and differentiation of the embryonic pancreas. HNF1beta mutations in humans accordingly often cause pancreas hypoplasia. By contrast, HNF1alpha and HNF4alpha have been shown to regulate the function of differentiated beta-cells. HNF1alpha and HNF4alpha mutations in patients thus cause decreased glucose-induced insulin secretion that leads to a progressive form of diabetes. HNF4alpha mutations paradoxically also cause in utero and neonatal hyperinsulinism, which later evolves to decreased glucose-induced secretion. Recent studies show that Hnf4alpha deficiency in mice causes not only abnormal insulin secretion, but also an impairment of the expansion of beta-cell mass that normally occurs during pregnancy. In line with this finding, we present data that Hnf1alpha-/- beta-cells expressing SV40 large T antigen show a severe impairment of proliferation and failure to form tumours. Collectively, these findings implicate HNF1beta as a regulator of pancreas organogenesis and differentiation, whereas HNF1alpha and HNF4alpha primarily regulate both growth and function of islet beta-cells.
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http://dx.doi.org/10.1159/000109603DOI Listing
December 2007

Putting pancreatic cell plasticity to the test.

J Clin Invest 2007 Apr;117(4):859-62

Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Endocrinology Unit, Hospital Clínic de Barcelona, Barcelona, Spain.

Diabetes results from the absolute or relative deficiency of insulin-producing beta cells. The prospect that non-beta pancreatic cells could be harnessed to become beta cells has led to interest in understanding the plasticity of pancreatic cells. Recent studies, however, have shown that adult beta cells are largely derived from preexisting beta cells. In this issue of the JCI, Desai et al. show that acinar cells, the major cell type in the pancreas, do not contribute to new beta cells formed during pancreatic regeneration (see the related article beginning on page 971). These studies suggest that the fate of adult pancreatic cell lineages is immutable. However, also in this issue of the JCI, Collombat et al. demonstrate that inducing a single transcription factor named Arx in adult beta cells causes these cells to undergo massive transdifferentiation into alpha and pancreatic polypeptide endocrine cells (see the related article beginning on page 961). This finding points to an unexpected plasticity of postnatal pancreatic endocrine cells.
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http://dx.doi.org/10.1172/JCI31749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1838951PMC
April 2007

Persistent activation of Rac1 in squamous carcinomas of the head and neck: evidence for an EGFR/Vav2 signaling axis involved in cell invasion.

Carcinogenesis 2007 Jun 18;28(6):1145-52. Epub 2007 Jan 18.

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

The poor prognosis associated with head and neck squamous cell carcinoma (HNSCC) is primarily due to both local invasion and the regional and/or distant metastatic spread. Recent findings have provided evidence that the acquisition of a motile and invasive phenotype by cancer cells involves the dysregulated function of key intracellular molecular mechanisms together with aberrant signaling events initiated by the surrounding microenvironment. These intrinsic and extrinsic biochemical pathways in turn often converge to stimulate the activity of members of the Rho family of Ras-related guanosine triphosphate (GTP)-binding proteins, including RhoA, Rac and Cdc42, which control the organization of the actin cytoskeleton thereby regulating cell adhesion, polarity and motility. In this study, we examined the status of activation of these GTPases in a representative collection of HNSCC cell lines. Surprisingly, we found that most HNSCC cells exhibit remarkably high levels of GTP-bound Rac1. Further analysis revealed that the activation of Rac1 in these HNSCC cells could be due to two independent signaling events, an epidermal growth factor receptor (EGFR)-based autocrine loop that leads to the activation of the Rac1 exchange factor Vav2 and an EGFR/Vav2-independent pathway that arises as a consequence of the oncogenic mutation of the H-ras proto-oncogene. Indeed, we provide evidence that the EGFR/Vav2/Rac1 axis is a crucial pathway for the acquisition of motile and invasive properties of most HNSCC cells. These findings shed light onto the molecular mechanisms involved in HNSCC cell invasion, and may reveal new therapeutic opportunities to halt the metastatic spread of these aggressive malignancies.
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http://dx.doi.org/10.1093/carcin/bgm008DOI Listing
June 2007

The small GTPase Rac1 links the Kaposi sarcoma-associated herpesvirus vGPCR to cytokine secretion and paracrine neoplasia.

Blood 2004 Nov 1;104(9):2903-11. Epub 2004 Jul 1.

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Dr, Bldg 30, Rm 212, Bethesda, MD 20892-4330, USA.

Kaposi sarcoma (KS) is a multifocal angioproliferative neoplasm strictly dependent on angiogenic growth factors and cytokines and invariably associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8). A G protein-coupled receptor encoded by KSHV (vGPCR) is able to initiate KS-like tumors when targeted to the vascular endothelium of mice. Analogous to human KS, vGPCR sarcomagenesis involves the paracrine secretion of angiogenic growth factors and proinflammatory molecules from vGPCR-expressing cells. Here we demonstrate that vGPCR up-regulates expression and secretion of critical KS cytokines by stimulating key transcription factors, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and nuclear factor of activated T cells (NFAT), through the activation of the small G protein Rac1. Inhibition of Rac1 blocked vGPCR-induced transcription and secretion of KS cytokines, including interleukin-6 (IL-6), IL-8, and growth-regulated oncogene alpha (GROalpha), in vitro and reduced vGPCR tumorigenesis in vivo. Moreover, endothelial-specific infection with the constitutively active Rac1QL induced vascular lesions in mice that were remarkably similar to early vGPCR experimental lesions. These results identify Rac1 as a key mediator of vGPCR paracrine neoplasia, suggesting that this small G protein and its downstream effectors may represent suitable therapeutic targets for the treatment of KS.
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http://dx.doi.org/10.1182/blood-2003-12-4436DOI Listing
November 2004

Thrombin protease-activated receptor-1 signals through Gq- and G13-initiated MAPK cascades regulating c-Jun expression to induce cell transformation.

J Biol Chem 2003 Nov 3;278(47):46814-25. Epub 2003 Sep 3.

Oral and Pharyngeal Cancer Branch, NIDCR/National Institutes of Health, Building 30, Room 211, 9000 Rockville Pike, Bethesda, MD 20892-4330, USA.

Although the ability of G protein-coupled receptors to stimulate normal and aberrant cell growth has been intensely investigated, the precise nature of the molecular mechanisms underlying their transforming potential are still not fully understood. In this study, we have taken advantage of the potent mitogenic effect of thrombin and the focus-forming activity of one of its receptors, protease-activated receptor-1, to dissect how this receptor coupled to Galphai, Galphaq/11, and Galpha12/13 transduces signals from the membrane to the nucleus to initiate transcriptional events involved in cell transformation. Using endogenous and transfected thrombin receptors in NIH 3T3 cells, ectopic expression of muscarinic receptors coupled to Galphaq and Galphai, and chimeric G protein alpha subunits and murine fibroblasts deficient in Galphaq/11, and Galpha12/13, we show here that, although coupling to Galphai is sufficient to induce ERK activation, the ability to couple to Galphaq and/or Galpha13 is necessary to induce c-jun expression and cell transformation. Furthermore, we show that Galphaq and Galpha13 can initiate the activation of MAPK cascades, including JNK, p38, and ERK5, which in turn regulate the activity of transcription factors controlling expression from the c-jun promoter. We also present evidence that c-Jun and the kinases regulating its expression are integral components of the transforming pathway initiated by protease-activated receptor-1.
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http://dx.doi.org/10.1074/jbc.M305709200DOI Listing
November 2003

Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src.

J Biol Chem 2003 Sep 16;278(36):34339-46. Epub 2003 Jun 16.

Oral and Pharyngeal Cancer Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.

The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.
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http://dx.doi.org/10.1074/jbc.M302960200DOI Listing
September 2003

Metabotropic glutamate receptors activate phospholipase D in astrocytes through a protein kinase C-dependent and Rho-independent pathway.

Neuropharmacology 2003 Feb;44(2):171-80

Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that mediate phospholipase D (PLD) activation in brain, but the mechanism underlying this response remains unclear. Here we used primary cultures of astrocytes as a cell model to explore the mechanism that links mGluRs to PLD. Glutamate activated both phospholipase C (PLC) and PLD with equal potency and this effect was mimicked by L-cysteinesulfinic acid, a putative neurotransmitter previously shown to activate mGluRs coupled to PLD, but not PLC, in adult brain. PLD activation by glutamate was dependent on Ca(2+) mobilization and fully blocked by both protein kinase C (PKC) inhibitors and PKC down-regulation, suggesting that PLD activation is secondary to PLC stimulation. Furthermore, brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation, partially inhibited the activation of PLD by glutamate. By contrast, pretreatment of astrocytes with Clostridium difficile toxin B, which inactivates small G proteins of the Rho family (Rho, Rac, and Cdc42), had no effect on PLD stimulation by glutamate. Taken together, these results indicate that PLD activation by mGluRs in astrocytes is dependent on PKC and small G proteins of the ARF family, but does not require Rho proteins.
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http://dx.doi.org/10.1016/s0028-3908(02)00361-1DOI Listing
February 2003

Potent activation of RhoA by Galpha q and Gq-coupled receptors.

J Biol Chem 2002 Jul 16;277(30):27130-4. Epub 2002 May 16.

Oral and Pharyngeal Cancer Branch, NIDCR/National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-4330, USA.

Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.
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http://dx.doi.org/10.1074/jbc.M204715200DOI Listing
July 2002

Regulation of p73 by c-Abl through the p38 MAP kinase pathway.

Oncogene 2002 Jan;21(6):974-9

Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4330, USA.

p73 is a novel member of the p53 family of tumor suppressor proteins which is involved in cellular differentiation, tumor suppression, and the response to genotoxic stress. The molecular mechanisms regulating p73 activity are still poorly understood. Recently, p73 was found to be a target of the enzymatic activity of c-Abl, a non-receptor tyrosine kinase that potently activated in response to DNA damage. Here, we present evidence that c-Abl induces the phosphorylation of p73 in threonine residues adjacent to prolines, and that the p38 MAP kinase pathway mediates this response. Furthermore, we found that activation of p38 is sufficient to enhance the stability of p73, and that the transcriptional activation of p73 by c-Abl requires the activity of p38. These findings indicate that members of the MAP kinases superfamily of signaling molecules can regulate p73, and support a role for the p38 MAP kinase in a novel biochemical pathway by which c-Abl regulates this p53-related molecule.
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http://dx.doi.org/10.1038/sj.onc.1205134DOI Listing
January 2002