RESUMEN
Prostaglandin E(2) (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3ß and ß-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs.
Asunto(s)
Dinoprostona/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/fisiología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Células Cultivadas , Dinoprostona/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/metabolismoRESUMEN
Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16(Ink4a) or p19(Arf) deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.
Asunto(s)
Proteínas Portadoras/metabolismo , Inestabilidad Genómica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Apoptosis , Células de la Médula Ósea/metabolismo , Ciclo Celular , Ensayo de Unidades Formadoras de Colonias , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Proteínas de Unión al GTP , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Angiopoietin-1 (Angpt1) signaling via the Tie2 receptor regulates vascular and hematopoietic systems. To investigate the role of Angpt1-Tie2 signaling in hematopoiesis, we prepared conditionally inducible transgenic (Tg) mice expressing a genetically engineered Angpt1, cartridge oligomeric matrix protein (COMP)-Angpt1. The effects of COMP-Angpt1 overexpression in osteoblasts on hematopoiesis were then investigated by crossing COMP-Angpt1 Tg mice with Col1a1-Cre Tg mice. Interestingly, peripheral blood analyses showed that 4 week (wk)-old (but not 8 wk-old) Col1a1-Cre+/COMP-Angpt1+ mice had a lower percentage of circulating B cells and a higher percentage of myeloid cells than Col1a1-Cre-/COMP-Angpt1+ (control) mice. Although there were no significant differences in the immunophenotypic hematopoietic stem and progenitor cell (HSPC) populations between Col1a1-Cre+/COMP-Angpt1+ and control mice, lineage(-)Sca-1(+)c-Kit(+) (LSK) cells isolated from 8 wk-old Col1a1-Cre+/COMP-Angpt1+ mice showed better long-term bone marrow reconstitution ability. These data indicate that Angpt1-Tie2 signaling affects the differentiation capacity of hematopoietic lineages during development and increases the stem cell activity of HSCs.
Asunto(s)
Angiopoyetina 1/metabolismo , Linaje de la Célula , Hematopoyesis , Células Madre Hematopoyéticas/citología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Angiopoyetina 1/genética , Animales , Vasos Sanguíneos/anomalías , Células de la Médula Ósea/citología , Separación Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Pérdida del Embrión/genética , Pérdida del Embrión/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteínas Matrilinas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/metabolismo , Receptor TIE-2 , Transducción de SeñalRESUMEN
Diabetes is known to increase the risk of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Here we treat male STAM (STelic Animal Model) mice, which develop diabetes, NASH and HCC associated with dysbiosis upon low-dose streptozotocin and high-fat diet (HFD), with insulin or phlorizin. Although both treatments ameliorate hyperglycemia and NASH, insulin treatment alone lead to suppression of HCC accompanied by improvement of dysbiosis and restoration of antimicrobial peptide production. There are some similarities in changes of microflora from insulin-treated patients comorbid with diabetes and NASH. Insulin treatment, however, fails to suppress HCC in the male STAM mice lacking insulin receptor specifically in intestinal epithelial cells (ieIRKO), which show dysbiosis and impaired gut barrier function. Furthermore, male ieIRKO mice are prone to develop HCC merely on HFD. These data suggest that impaired gut insulin signaling increases the risk of HCC, which can be countered by restoration of insulin action in diabetes.
Asunto(s)
Carcinoma Hepatocelular , Diabetes Mellitus Experimental , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Masculino , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patología , Hígado/patología , Carcinoma Hepatocelular/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Disbiosis/complicaciones , Disbiosis/patología , Neoplasias Hepáticas/patología , Insulina , Ratones Endogámicos C57BL , Dieta Alta en Grasa/efectos adversos , Modelos Animales de EnfermedadRESUMEN
Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad(+)c-Kit(+) and N-cad(+) endothelial protein C receptor (EPCR)(+) HSCs co-localized with Lyve-1(+) sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad(+) HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR(+) long-term (LT)-HSCs were enriched in the N-cad(+)Lin(-)Sca-1(+)c-Kit(+) (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad(+) LSK cells versus N-cad(-) LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad(+) LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.
Asunto(s)
Médula Ósea/fisiología , Cadherinas/metabolismo , Feto/fisiología , Hematopoyesis Extramedular , Células Madre Hematopoyéticas/fisiología , Hígado/embriología , Animales , Cadherinas/análisis , Movimiento Celular , Regulación hacia Abajo , Células Endoteliales/fisiología , Femenino , Glicoproteínas/análisis , Glicoproteínas/metabolismo , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BLRESUMEN
During insulin resistance, lipid uptake by the liver is promoted by peroxisome proliferator-activated protein (PPAR) γ upregulation, leading to hepatic steatosis. Insulin, however, does not directly regulate adipogenic gene expression in liver, and the mechanisms for its upregulation in obesity remain unclear. Here, we show that the Irs2 locus, a critical regulator of insulin actions, encodes an antisense transcript, ASIrs2, whose expression increases in obesity or after refeeding in liver, reciprocal to that of Irs2. ASIrs2 regulates hepatic Pparg expression, and its suppression ameliorates steatosis in obese mice. The human ortholog AL162497.1, whose expression is correlated with that of hepatic PPARG and the severity of non-alcoholic steatohepatitis (NASH), shows genomic organization similar to that of ASIrs2. We also identified HARS2 as a potential binding protein for ASIrs2, functioning as a regulator of Pparg. Collectively, our data reveal a functional duality of the Irs2 gene locus, where reciprocal changes of Irs2 and ASIrs2 in obesity cause insulin resistance and steatosis.
Asunto(s)
Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Animales , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/genética , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/complicaciones , Obesidad/genética , PPAR gamma/genéticaRESUMEN
In diabetic pathology, insufficiency in ß-cell mass, unable to meet peripheral insulin demand, and functional defects of individual ß-cells in production of insulin are often concurrently observed, collectively causing hyperglycemia. Here we show that the phosphorylation of ERK1/2 is significantly decreased in the islets of db/db mice as well as in those of a cohort of subjects with type 2 diabetes. In mice with abrogation of ERK signaling in pancreatic ß-cells through deletion of Mek1 and Mek2, glucose intolerance aggravates under high-fat diet-feeding conditions due to insufficient insulin production with lower ß-cell proliferation and reduced ß-cell mass, while in individual ß-cells dampening of the number of insulin exocytosis events is observed, with the molecules involved in insulin exocytosis being less phosphorylated. These data reveal bifunctional roles for MEK/ERK signaling in ß-cells for glucose homeostasis, i.e., in regulating ß-cell mass as well as in controlling insulin exocytosis in individual ß-cells, thus providing not only a novel perspective for the understanding of diabetes pathophysiology but also a potential clue for new drug development for diabetes treatment.
Asunto(s)
Glucemia/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Homeostasis , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Animales , Línea Celular , Dieta Alta en Grasa , Exocitosis , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de SeñalRESUMEN
Repeated cell divisions and aging impair stem cell function. However, the mechanisms by which this occurs are not fully understood. Here we show that protection of telomeres 1A (Pot1a), a component of the Shelterin complex that protects telomeres, improves haematopoietic stem cell (HSC) activity during aging. Pot1a is highly expressed in young HSCs, but declines with age. In mouse HSCs, Pot1a knockdown increases DNA damage response (DDR) and inhibits self-renewal. Conversely, Pot1a overexpression or treatment with POT1a protein prevents DDR, maintained self-renewal activity and rejuvenated aged HSCs upon ex vivo culture. Moreover, treatment of HSCs with exogenous Pot1a inhibits the production of reactive oxygen species, suggesting a non-telomeric role for Pot1a in HSC maintenance. Consistent with these results, treatment with exogenous human POT1 protein maintains human HSC activity in culture. Collectively, these results show that Pot1a/POT1 sustains HSC activity and can be used to expand HSC numbers ex vivo.Repeated cell divisions induce DNA damage in haematopoietic stem cells (HSC) and telomeres are sensitive to this damage. Here, the authors show in murine HSCs that the telomere binding protein POT1a inhibited the production of reactive oxygen species, and rejuvenated aged HSCs.