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1.
Blood ; 128(6): 839-51, 2016 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-27288519

RESUMEN

Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells.


Asunto(s)
Eritropoyesis , Janus Quinasa 2/genética , Mutación , Policitemia/genética , Animales , Secuencia de Bases , Eritrocitos/patología , Exones , Hierro/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Policitemia/metabolismo , Policitemia/fisiopatología
2.
Exp Hematol ; 43(8): 599-608, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26209551

RESUMEN

Major progress has been recently made in understanding the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in one of four genes-JAK2, MPL, CALR, and CSF3R-can be found in the vast majority of patients with MPN and represent driver mutations that can induce the MPN phenotype. Hyperactive JAK/STAT signaling appears to be the common denominator of MPN, even in patients with CALR mutations and the so-called "triple-negative" MPN, where the driver gene mutation is still unknown. Mutations in epigenetic regulators, transcription factors, and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. The central role of JAK2 in MPN allowed development of small molecular inhibitors that are in clinical use and are active in almost all patients with MPN. Advances in understanding the mechanism of JAK2 activation open new perspectives of developing the next generation of inhibitors that will be selective for the mutated forms of JAK2.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas , Mutación , Trastornos Mieloproliferativos , Proteínas de Neoplasias , Transducción de Señal/genética , Animales , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
3.
Proc Natl Acad Sci U S A ; 112(15): 4642-7, 2015 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-25825724

RESUMEN

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.


Asunto(s)
Adenosina Trifosfato/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Mutación Missense , Adenosina Trifosfato/química , Animales , Sitios de Unión/genética , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Activación Enzimática/genética , Femenino , Humanos , Immunoblotting , Janus Quinasa 2/química , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Fosforilación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Eritropoyetina/metabolismo
4.
Blood ; 125(13): 2131-40, 2015 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-25595737

RESUMEN

The acquired somatic JAK2-V617F mutation is present in >80% of patients with myeloproliferative neoplasms (MPNs). Stat3 plays a role in hematopoietic homeostasis and might influence the JAK2-V617F-driven MPN phenotype. We crossed our transgenic SclCre;V617F mice with a conditional Stat3 knockout strain and performed bone marrow transplantations into lethally irradiated recipient mice. The deletion of Stat3 increased the platelet numbers in SclCre;V617F;Stat3(fl/fl) mice compared with SclCre;V617F;Stat3(fl/+) or SclCre;V617F;Stat3(+/+) mice. Stat3 deletion also normalized JAK2-V617F-induced neutrophilia. Megakaryocyte progenitors were elevated, especially in the spleen, and a slight increase in myelofibrosis was noted. We observed increased mRNA expression levels of Stat1 and Stat1 target genes and augmented phosphorylation of Stat1 protein in bone marrow and spleen of JAK2-V617F mice after Stat3 deletion. The survival of Stat3-deficient mice expressing JAK2-V617F was reduced. Inflammatory bowel disease, previously associated with shortened survival of Stat3-deficient mice, was less prominent in the bone marrow transplantation setting, possibly by limiting deletion of Stat3 to hematopoietic tissues only. In conclusion, deletion of Stat3 in hematopoietic cells from JAK2-V617F mice did not ameliorate the course of MPN, but rather enhanced thrombocytosis and shortened the overall survival.


Asunto(s)
Neoplasias de la Médula Ósea/mortalidad , Células Madre Hematopoyéticas/metabolismo , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/mortalidad , Factor de Transcripción STAT3/genética , Trombocitosis/genética , Sustitución de Aminoácidos , Animales , Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Fenilalanina/genética , Factor de Transcripción STAT3/metabolismo , Valina/genética
5.
Blood ; 123(25): 3943-50, 2014 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-24820309

RESUMEN

The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)-V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1(-/-) double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1(+/+) mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit-erythroid and reduced colony-forming unit-megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.


Asunto(s)
Neoplasias de la Médula Ósea/genética , Eritropoyesis/genética , Janus Quinasa 2/genética , Mutación , Factor de Transcripción STAT1/genética , Trombopoyesis/genética , Animales , Western Blotting , Neoplasias de la Médula Ósea/sangre , Neoplasias de la Médula Ósea/metabolismo , Trasplante de Médula Ósea/métodos , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Interferón gamma/sangre , Janus Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Policitemia Vera/sangre , Policitemia Vera/genética , Policitemia Vera/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/metabolismo , Trombocitemia Esencial/sangre , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo
8.
Blood ; 121(7): 1188-99, 2013 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-23264594

RESUMEN

To establish a preclinical animal model for testing drugs with potential effects on myeloproliferative neoplasms (MPNs), we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2-knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelet or granulocyte levels. In a transplantation assay, JAK2-V617F(+) BM cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea (HU). HU led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized RBC and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced the phospho-Stat5 levels, whereas HU strongly increased phospho-Stat5, most likely because of the elevated erythropoietin levels in response to the HU-induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of HU and represents an adverse effect that should be avoided.


Asunto(s)
Hidroxiurea/farmacología , Janus Quinasa 2/genética , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/genética , Pirazoles/farmacología , Alelos , Sustitución de Aminoácidos , Animales , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Femenino , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación , Nitrilos , Fenotipo , Policitemia Vera/metabolismo , Policitemia Vera/patología , Pirimidinas , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos
9.
Am J Physiol Endocrinol Metab ; 304(1): E1-13, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23092914

RESUMEN

Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1ß, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1ß, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.


Asunto(s)
Adipocitos/efectos de los fármacos , Citocinas/genética , Polipéptido Inhibidor Gástrico/farmacología , Resistencia a la Insulina , Lipólisis/efectos de los fármacos , Adipocitos/metabolismo , Adulto , Fármacos Antiobesidad/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocinas/metabolismo , Polipéptido Inhibidor Gástrico/fisiología , Humanos , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lactonas/farmacología , Lipólisis/genética , Persona de Mediana Edad , FN-kappa B/metabolismo , Orlistat , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
10.
Innate Immun ; 18(1): 25-34, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21088047

RESUMEN

High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1 µg/ml LPS, IL-6 and IL-8 were induced to 19.5 ± 1.8-fold and 662.7 ± 91.5-fold (P < 0.01 vs basal), respectively. From 100 ng/ml to 1 µg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P < 0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKKß) or NF-κB inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKKß/NF-κB pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity.


Asunto(s)
Adipocitos/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipólisis/inmunología , Lipopolisacáridos/inmunología , Adipocitos/efectos de los fármacos , Adipocitos/patología , Diferenciación Celular , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Glicerol/metabolismo , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Isoproterenol/farmacología , Lipólisis/efectos de los fármacos
11.
Diabetol Metab Syndr ; 3: 16, 2011 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-21774820

RESUMEN

BACKGROUND: Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer. METHODS: Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 µg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation. RESULTS: Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05). CONCLUSIONS: Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.

12.
Arch Physiol Biochem ; 117(4): 209-14, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21338323

RESUMEN

OBJECTIVE: To study the effects of metformin on lipolysis and hormone sensitive lipase (HSL) phosphorylation in human adipocytes treated with lipolytic and inflammatory agents. METHODS: Lipolysis and phosphorylation status of HSL were assessed in subcutaneous pre-adipocytes surgically isolated from patients and differentiated into mature adipocytes in vitro. RESULTS: Stimulation for 1 h with forskolin, isoproterenol and IBMX or stimulation for 24 h with LPS, IL-1ß and TNF-α increased lipolysis (p < 0.05 vs. basal). The phosphorylation of HSL at Ser-554 was decreased while the Ser-552 phosphorylation was increased. Pre-incubation with metformin (24 h, 1 mM) inhibited forskolin-, isoproterenol-, IBMX-, LPS-, IL-1ß- and TNF-α-induced glycerol release and prevented p(Ser554)HSL decrease and p(Ser-552)HSL increase due to lipolytic and inflammatory agents. AMPKα1 is involved in metformin-induced HSL phosphorylation at Ser-552. CONCLUSION: Phosphorylation of HSL at Ser-554 inversely correlates with lipolysis and HSL phosphorylation at Ser552 in human adipocytes.


Asunto(s)
Adipocitos/efectos de los fármacos , Hipoglucemiantes , Lipólisis/efectos de los fármacos , Metformina , Fosforilación/efectos de los fármacos , Serina/metabolismo , Esterol Esterasa/metabolismo , 1-Metil-3-Isobutilxantina/efectos adversos , 1-Metil-3-Isobutilxantina/farmacología , Adipocitos/metabolismo , Adulto , Anciano , Colforsina/efectos adversos , Colforsina/farmacología , Femenino , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Inflamación/tratamiento farmacológico , Interleucina-1beta/efectos adversos , Interleucina-1beta/farmacología , Isoproterenol/efectos adversos , Isoproterenol/farmacología , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/farmacología , Masculino , Metformina/farmacología , Metformina/uso terapéutico , Persona de Mediana Edad , Cultivo Primario de Células , Factor de Necrosis Tumoral alfa/efectos adversos , Factor de Necrosis Tumoral alfa/farmacología
13.
J Clin Endocrinol Metab ; 96(2): E297-303, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21106708

RESUMEN

CONTEXT: Increased plasma levels of glucose-dependent insulinotropic polypeptide (GIP), calcitonin CT gene-related peptide (CGRP)-I, and procalcitonin (Pro-CT) are associated with obesity. Adipocytes express functional GIP receptors and the CT peptides Pro-CT and CGRP-I. However, a link between GIP and CT peptides has not been studied yet. OBJECTIVE: The objective of the study was the assessment of the GIP effect on the expression and secretion of CGRP-I and Pro-CT in human adipocytes, CGRP-I and CT gene expression in adipose tissue (AT) from obese vs. lean subjects, and plasma levels of CGRP-I and Pro-CT after a high-fat meal in obese patients. DESIGN AND PARTICIPANTS: Human preadipocyte-derived adipocytes, differentiated in vitro, were treated with GIP. mRNA expression and protein secretion of CGRP-I and Pro-CT were measured. Human CGRP-I and CT mRNA expression in AT and CGRP-I and Pro-CT plasma concentrations were assessed. RESULTS: Treatment with 1 nm GIP induced CGRP-I mRNA expression 6.9 ± 1.0-fold (P < 0.001 vs. control) after 2 h and CT gene expression 14.0 ± 1.7-fold (P < 0.001 vs. control) after 6 h. GIP stimulated CGRP-I secretion 1.7 ± 0.2-fold (P < 0.05 vs. control) after 1 h. In AT samples of obese subjects, CGRP-I mRNA expression was higher in sc AT (P < 0.05 vs. lean subjects), whereas CT expression was higher in visceral AT (P < 0.05 vs. lean subjects). CGRP-I plasma levels increased after a high-fat meal in obese patients. CONCLUSION: GIP induces CGRP-I and CT expression in human adipocytes. Therefore, elevated Pro-CT and CGRP-I levels in obesity might result from GIP-induced Pro-CT and CGRP-I release in AT and might be triggered by a high-fat diet. How these findings relate to the metabolic complications of obesity warrants further investigations.


Asunto(s)
Adipocitos/metabolismo , Péptido Relacionado con Gen de Calcitonina/biosíntesis , Calcitonina/biosíntesis , Polipéptido Inhibidor Gástrico/farmacología , Precursores de Proteínas/biosíntesis , Adipocitos/efectos de los fármacos , Adulto , Calcitonina/sangre , Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/sangre , Péptido Relacionado con Gen de Calcitonina/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Grasas de la Dieta/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/metabolismo , Obesidad Mórbida/metabolismo , Precursores de Proteínas/sangre , Precursores de Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética
14.
Innate Immun ; 17(4): 403-13, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20682585

RESUMEN

Severe systemic infections induce ubiquitous calcitonin (CALC) gene expression with release of calcitonin peptides, namely procalcitonin, calcitonin gene-related peptide and adrenomedullin. Using an in vitro model for bacterial infection, we tested the hypothesis that intracellular calcium concentration ([Ca(2+)](i)) is elevated after lipopolysaccharide (LPS) stimulation and is responsible for the LPS-mediated increase in CALC gene expression and protein secretion. In our human adipocyte model, LPS did not show any cytotoxic effects and induced increased CALC-I gene mRNA expression. Additionally, LPS provoked an elevation in [Ca(2+)](i). The LPS-induced increase in CALC-I gene mRNA was partially blocked with verapamil, an L-type calcium channel blocker and blocked almost completely with 2-aminoethoxydiphenyl borate, a blocker of store-operated calcium entry and inositol triphosphate-mediated calcium release. Treatment of cells with substances elevating [Ca(2+)]( i) led to an increased CALC-I mRNA expression level. The combination of LPS with substances raising [Ca(2+)](i) even potentiated this increase. At the same time, elevated [Ca(2+)](i) attenuated the expression level of the CALC-V gene. These findings indicate that, in human adipocytes, changes in [Ca(2+)](i) are involved in LPSregulated expression of CALC genes, thereby strengthening previous findings postulating a crucial role of intracellular calcium homeostasis in the state of bacterial infection and sepsis.


Asunto(s)
Adipocitos/metabolismo , Infecciones Bacterianas/metabolismo , Calcitonina/metabolismo , Calcio/metabolismo , Lipopolisacáridos/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/inmunología , Adipocitos/patología , Infecciones Bacterianas/inmunología , Compuestos de Boro/farmacología , Calcitonina/genética , Calcitonina/inmunología , Calcio/inmunología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Humanos , Lipopolisacáridos/inmunología , Verapamilo/farmacología
15.
Biochem Pharmacol ; 80(11): 1736-45, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-20816671

RESUMEN

The mechanisms of metformin effects on glucose transport and metabolism were investigated in human adipocytes. Human preadipocytes obtained from surgical biopsies were differentiated in vitro into adipocytes and the effects of metformin on glucose uptake, glucose oxidation and the involved signaling pathways were analyzed. Metformin (1mM, 24h) increased glucose uptake 2.3±0.2-fold (p<0.001 vs. basal) in human adipocytes, without altering cell viability and oxygen consumption. Metformin did not alter GLUT-1 mRNA expression and protein content but increased GLUT-4 mRNA expression and cellular protein content, leading to increased GLUT-4 protein content in the plasma membrane. Neither basal nor insulin-induced phosphorylation of Akt at Ser-473 and AS160 (Akt substrate of 160kDa) at Thr-642 were enhanced by metformin. Suppression of metformin-induced AMP-activated protein kinase (AMPK) activity by AMPKα1 silencing, however, reduced metformin-associated GLUT-4 expression and stimulation of glucose uptake. In addition, metformin induced glucose oxidation. In conclusion, activation of AMPKα1 without impairment of cell respiration is crucial for metformin-mediated increase in GLUT-4 protein content and glucose uptake in human adipocytes.


Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Glucosa/metabolismo , Metformina/farmacología , Adulto , Anciano , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
16.
J Steroid Biochem Mol Biol ; 117(4-5): 87-92, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19703560

RESUMEN

Protein kinases represent key nodes for the integration of multiple intracellular signalling pathways, resulting in modulation of both ligand-dependent and ligand-independent mechanisms of sex steroid receptor (sSR) signalling cascades. The proline-directed Ser/Thr kinases including mitogen-activated protein kinases and cyclin dependent kinases were especially reported to contribute to the function and activity of sSRs. The relevant effects of these kinases are well-documented but the impact of glycogen synthase kinase-3 (GSK-3), another member of this kinase family, has been underestimated. Indeed, the specific role of GSK-3 regarding the different sSRs will help to understand further the complexity of sSR signalling. So far, AR and ERalpha were identified as GSK-3 substrates. Additionally, the docking properties of GSK-3 were demonstrated to play a crucial role in sSR signal transduction. Reciprocally, GSK-3 was described as a potential target of non-genomic effects of sSRs. Therefore, GSK-3 regulates and is regulated by sSRs. This review focuses on the emerging and promising involvements of GSK-3 regarding the signalling cascade of the respective sSRs. This review represents a necessary complement of information to highlight the importance of GSK-3 regarding sSR function and activity.


Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Receptores Androgénicos/fisiología , Receptores de Estrógenos/fisiología , Animales , Humanos , Fosforilación , Transducción de Señal , Especificidad por Sustrato
17.
Endocr Relat Cancer ; 16(2): 429-41, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19153208

RESUMEN

Insulin and insulin analogs stimulate proliferation of human mammary epithelial cells. We identified and analyzed the signaling pathways related to cell proliferation induced by regular insulin and by four insulin analogs presently approved for therapeutical use. Benign and malignant mammary cell lines showing different insulin receptor (IR) and IGF-I receptor (IGF-IR) expression patterns were studied. Cell proliferation was studied by crystal violet staining (BrdU-FACS analysis). Activation of insulin and IGF signaling pathways was studied by analysis of the phosphorylation status of IGF-IR and of key signaling proteins of the phosphoinositide 3-kinase (PI3K)/Akt and MAP kinase pathways, by the use of specific PI3K and MAP kinase inhibitors, and by silencing of IR and IGF-IR. Lantus stimulated the growth of MCF7 cells, which show high IGF-IR/IR ratio, significantly at 0.3 nmol/l, while regular insulin (Actrapid and bovine insulin) and other insulin analogs (Novorapid, Humalog, and Levemir) stimulated cell growth at 1.5-15 nmol/l concentrations. No difference between Lantus and the other insulin analogs was observed regarding growth stimulation of MCF10A cells showing low IGF-IR/IR ratio. Growth stimulation of MCF7 cells by Lantus was mainly due to strong activation of the IGF-IR and the MAP kinase pathway. Regular insulin and other insulin analogs tested activated mainly the IR and the PI3K/Akt pathway. We conclude that unlike regular insulin and other insulin analogs, Lantus strongly activates the IGF-IR and the MAP kinase pathway in MCF7 cells and is a strong mitogen for cells characterized by a high-IGF-IR/IR ratio.


Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Insulina/análogos & derivados , Insulina/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Bovinos , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Immunoblotting , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas
18.
Mol Endocrinol ; 21(10): 2427-39, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17609434

RESUMEN

Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.


Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Transcripción Genética , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Serina/metabolismo , Especificidad por Sustrato , Activación Transcripcional , Ubiquitina/antagonistas & inhibidores
19.
J Biol Chem ; 280(38): 33006-14, 2005 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-16076840

RESUMEN

Like other steroid hormone receptors, estrogen receptor-alpha (ERalpha) is a substrate for protein kinases, and phosphorylation has profound effects on the function and activity of this receptor. A number of different kinases have been implicated in ERalpha regulation. In this report we show by mutational analysis and in vitro kinase assays that ERalpha is a substrate for glycogen synthase kinase-3 (GSK-3) in vitro and is phosphorylated on two sites, the Ser-102, -104, and -106 motif and Ser-118, both located in the N-terminal transcription activation function (AF-1) domain. GSK-3 forms a complex with ERalpha in vivo as demonstrated by co-immunoprecipitation from cell lysates. The GSK-3 inhibitor lithium chloride was used to determine the role of GSK-3 in phosphorylation of Ser-102, -104, and -106 and Ser-118 in vivo and to explore the role of these serines in the regulation of ERalpha function. Treatment of cells with lithium chloride resulted in dephosphorylation of Ser-104 and -106 and Ser-118, which suggests these serine residues as targets for GSK-3 in vivo. Our results further suggest that ERalpha phosphorylation by GSK-3 stabilizes ERalpha under resting conditions and modulates ERalpha transcriptional activity upon ligand binding. Inhibition and constitutive activation of GSK-3, both, resulted in inhibition of ERalpha transcriptional activity, indicating a function of active as well as inactive GSK-3 in ERalpha regulation. These findings uncover a novel mechanism for the regulation of ERalpha-mediated estrogen signaling controlled by a dual action of GSK-3.


Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular Tumoral , Proliferación Celular , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoprecipitación , Ligandos , Cloruro de Litio/farmacología , Luciferasas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Transducción de Señal , Fracciones Subcelulares/metabolismo , Transcripción Genética , Activación Transcripcional
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