RESUMEN
Sprouty proteins are established modifiers of receptor tyrosine kinase (RTK) signaling and play important roles in vasculogenesis, bone morphogenesis, and renal uteric branching. Little is understood, however, concerning possible roles for these molecular adaptors during hematopoiesis. Within erythroid lineage, Spry1 was observed to be selectively and highly expressed at CFU-e to erythroblast stages. In analyses of possible functional roles, an Mx1-Cre approach was applied to conditionally delete Spry1. At steady state, Spry1 deletion selectively perturbed erythroid development and led to reticulocytosis plus heightened splenic erythropoiesis. When challenged by hemolysis, Spry1-null mice exhibited worsened anemia and delayed recovery. During short-term marrow transplantation, Spry1-null donor marrow also failed to efficiently rescue the erythron. In each anemia model, however, hyperexpansion of erythroid progenitors was observed. Spry function depends on phosphorylation of a conserved N-terminal PY motif. Through an LC-MS/MS approach, Spry1 was discovered to be regulated via the erythropoietin receptor (EPOR), with marked EPO-induced Spry1-PY53 phosphorylation observed. When EPOR signaling pathways were analyzed within Spry1-deficient erythroid progenitors, hyperactivation of not only Erk1,2 but also Jak2 was observed. Studies implicate Spry1 as a novel regulator of erythropoiesis during anemia, transducer of EPOR signals, and candidate suppressor of Jak2 activity.
Asunto(s)
Eritropoyesis , Eritropoyetina/metabolismo , Janus Quinasa 2/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Eritropoyetina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Anemia/genética , Anemia/metabolismo , Animales , Trasplante de Médula Ósea , Células Cultivadas , Activación Enzimática , Eritroblastos/citología , Eritroblastos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfoproteínas/genética , Reticulocitos/citología , Reticulocitos/metabolismoRESUMEN
Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit(-)CD71(high)Ter119(-) "stage E2" proerythroblast pool first is described, which (unlike its Kit(+) "stage E1" progenitors, or maturing Ter119(+) "stage E3" progeny) proved to selectively expand â¼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1âE2âE3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.
Asunto(s)
Anemia , Proliferación Celular , Eritroblastos/citología , Células Precursoras Eritroides/citología , Eritropoyetina/farmacología , Alelos , Animales , Trasplante de Médula Ósea , Comunicación Celular/fisiología , Eritropoyesis , Ratones , Ratones Endogámicos C57BL , Receptores de Eritropoyetina/genética , Bazo/citología , Células del EstromaRESUMEN
Anemia as associated with numerous clinical conditions can be debilitating, but frequently can be treated via administration of epoetin-alfa, darbepoietin-alfa, or methoxy-PEG epoetin-beta. Despite the complexity of EPO-EPO receptor interactions, the development of interesting EPO mimetic peptides (EMPs) also has been possible. CNTO 530 is one such novel MIMETIBODY Fc-domain dimeric EMP fusion protein. In a mouse model, single-dose CNTO 530 (unlike epoetin-alfa or darbepoietin-alfa) bolstered red cell production for up to 1 month. In 5-fluorouracil and carboplatin-paclitaxel models, CNTO 530 also protected against anemia with unique efficiency. These actions were not fully accounted for by half-life estimates, and CNTO 530 signaling events therefore were studied. Within primary bone marrow erythroblasts, kinetics of STAT5, ERK, and AKT activation were similar for CNTO 530 and epoetin-alfa. p70S6K activation by CNTO 530, however, was selectively sustained. In vivo, CNTO 530 uniquely stimulated the enhanced formation of PODXL(high)CD71(high) (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow-resident Kit(neg)CD71(high)Ter119(neg) progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Eritroblastos/efectos de los fármacos , Eritropoyetina/análogos & derivados , Hematínicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anemia/patología , Animales , Células de la Médula Ósea/fisiología , Recuento de Células , Células Cultivadas , Evaluación Preclínica de Medicamentos , Eritroblastos/fisiología , Eritropoyesis/efectos de los fármacos , Eritropoyetina/química , Femenino , Hematínicos/química , Ratones , Ratones Endogámicos C57BL , Imitación Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPO-regulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wild-type EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPO-dependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit(pos)CD71(high)Ter119(neg) erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus FoxO3a, and EPOR/PY343/STAT5-dependent stimulation of Pim1, Pim3, Irs2 plus Serpina-3G, and Trb3 as new antiapoptotic effectors.
Asunto(s)
Apoptosis/fisiología , Eritroblastos/fisiología , Regulación de la Expresión Génica/fisiología , Receptores de Eritropoyetina/fisiología , Animales , Supervivencia Celular , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G(1)-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho beta-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of beta-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of beta-catenin and CREB pathways.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclina D1/genética , Proteínas del Citoesqueleto/metabolismo , Gastrinas/metabolismo , Neoplasias Gástricas/metabolismo , Transactivadores/metabolismo , Proteínas de Pez Cebra , Ciclina D1/biosíntesis , Fase G1/fisiología , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Fase S/fisiología , Transducción de Señal/fisiología , Transcripción Genética , Proteínas Wnt , beta CateninaRESUMEN
Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia). Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.
Asunto(s)
Eritroblastos/metabolismo , Eritropoyesis/genética , Isoformas de Proteínas/genética , ARN Mensajero/genética , Receptores de Eritropoyetina/genética , Receptores del Factor de Necrosis Tumoral/genética , Transcriptoma , Animales , Médula Ósea/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Eritroblastos/citología , Eritropoyetina/metabolismo , Eritropoyetina/farmacología , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Isoformas de Proteínas/metabolismo , Receptores de Eritropoyetina/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de SeñalRESUMEN
Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.
Asunto(s)
Alelos , Policitemia/genética , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Secuencia de Aminoácidos , Brefeldino A/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patología , Eritropoyetina/farmacología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ligandos , Modelos Biológicos , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Receptores de Eritropoyetina/químicaRESUMEN
Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) following exposure to PPARgamma-specific ligands resulted in growth inhibition in various carcinoma cell lines. Our aim was to elucidate the pathway of PPARgamma2 activation-mediated modulation of cyclin D1 transcription in mouse hepatocytes. To address this we utilized stable control and PPARgamma hepatocyte cell lines created via retroviral overexpression utilizing AML-12 hepatocytes. Addition of PPARgamma ligand troglitazone (TZD) activated PPARgamma2 in proliferating hepatocytes and resulted in growth arrest accompanied by a down-regulation of proliferating cell nuclear antigen, cyclin D1, and beta-catenin expression. Furthermore activation of PPARgamma2 attenuated cyclin D1 promoter activity indicating a transcriptional regulation of cyclin D1. Since beta-catenin plays a pivotal role in regulating cyclin D1 transcription, we studied whether PPARgamma2-mediated inhibition of cyclin D1 transcription involved beta-catenin. Interestingly overexpression of either wild-type or S37A mutant beta-catenin was unable to rescue PPARgamma2-mediated suppression of cyclin D1 transcription, whereas overexpression of cAMP-response element-binding protein (CREB) was capable of antagonizing this inhibitory effect of PPARgamma2. Additionally pretreatment with okadaic acid antagonized PPARgamma2-mediated inhibition of cyclin D1 transcription without any effect on beta-catenin expression. These studies also showed a TZD-mediated inhibition of total and phospho-CREB(Ser133) levels, CREB promoter activity, and cAMP-response element-mediated transcription in PPARgamma hepatocytes. Pretreatment of PPARgamma hepatocytes with okadaic acid, however, maintained higher total and phospho-CREB(Ser133) levels in the presence of TZD. These results indicated that PPARgamma2 activation inhibited cyclin D1 transcription in hepatocytes via CREB-dependent and beta-catenin-independent pathways.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclina D1/biosíntesis , Proteínas del Citoesqueleto/metabolismo , Hepatocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Western Blotting , División Celular , Línea Celular , Cromanos/farmacología , AMP Cíclico/metabolismo , Ciclina D1/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Humanos , Hipoglucemiantes/farmacología , Ligandos , Hígado/metabolismo , Luciferasas/metabolismo , Ratones , Mutación , Ácido Ocadaico/farmacología , Plásmidos/metabolismo , Serina/química , Tiazolidinedionas/farmacología , Transfección , Troglitazona , beta CateninaRESUMEN
The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to the family of nuclear hormone receptors and consists of two isotypes, PPARgamma1 and PPARgamma2. Our earlier studies have shown that troglitazone (TZD)-mediated activation of PPARgamma2 in hepatocytes inhibits growth and attenuates cyclin D1 transcription via modulating CREB levels. Because this process of growth inhibition was also associated with an inhibition of beta-catenin expression at a post-translational level, our aim was to elucidate the mechanism involved. beta-Catenin is a multifunctional protein, which can regulate cell-cell adhesion by interacting with E-cadherin and other cellular processes via regulating target gene transcription in association with TCF/LEF transcription factors. Two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways, one involving glycogen synthase kinase 3beta (GSK3beta) and the other involving p53-Siah-1, degrade excess beta-catenin in normal cells. Our immunofluorescence and Western blot studies indicated a TZD-dependent decrease in cytoplasmic and membrane-bound beta-catenin, indicating no increase in its membrane translocation. This was associated with a reduction in E-cadherin expression. PPARgamma2 activation inhibited GSK3beta kinase activity, and pharmacological inhibition of GSK3beta activity was unable to restore beta-catenin expression following PPARgamma2 activation. Additionally, this beta-catenin degradation pathway was operative in cells, with inactivating mutations of both APC and p53. Inhibition of the proteasomal pathway inhibited PPARgamma2-mediated degradation of beta-catenin, and incubation with TZD increased ubiquitination of beta-catenin. We conclude that PPARgamma2-mediated suppression of beta-catenin levels involves a novel APC/GSK3beta/p53-independent ubiquitination-mediated proteasomal degradation pathway.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Proteínas del Citoesqueleto/metabolismo , PPAR gamma/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transactivadores/metabolismo , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genes APC , Humanos , Complejos Multienzimáticos/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , beta CateninaRESUMEN
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.