RESUMEN
Arhgap21 is a member of the Rho GTPase activating protein (RhoGAP) family, which function as negative regulators of Rho GTPases. Arhgap21 has been implicated in adhesion and migration of cancer cells. However, the role of Arhgap21 has never been investigated in hematopoietic cells. Herein, we evaluated functional aspects of hematopoietic stem and progenitor cells (HSPC) using a haploinsufficient (Arhgap21+/-) mouse. Our results show that Arhgap21+/- mice have an increased frequency of phenotypic HSC, impaired ability to form progenitor colonies in vitro and decreased hematopoietic engraftment in vivo, along with a decrease in LSK cell frequency during serial bone marrow transplantation. Arhgap21+/- hematopoietic progenitor cells have impaired adhesion and enhanced mobilization of immature LSK and myeloid progenitors. Arhgap21+/- mice also exhibit reduced erythroid commitment and differentiation, which was recapitulated in human primary cells, in which knockdown of ARHGAP21 in CMP and MEP resulted in decreased erythroid commitment. Finally, we observed enhanced RhoC activity in the bone marrow cells of Arhgap21+/- mice, indicating that Arhgap21 functions in hematopoiesis may be at least partially mediated by RhoC inactivation.
Asunto(s)
Células de la Médula Ósea/patología , Proteínas Activadoras de GTPasa/fisiología , Haploinsuficiencia , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/patología , Proteína rhoC de Unión a GTP/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Eritroides/metabolismo , Células Eritroides/patología , Fibronectinas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína rhoC de Unión a GTP/genéticaRESUMEN
AIMS: ARHGAP21 is a Rho GTPase-activating protein (RhoGAP) that associates with many proteins and modulates several cellular functions, including actin cytoskeleton rearrangement in different tissues. However, it is unknown whether ARHGAP21 is expressed in pancreatic beta cells and its function in these cells. Herein, we assess the participation of ARHGAP21 in insulin secretion. MAIN METHODS: Neonatal mice were treated with anti-sense oligonucleotide against ARHG AP21 (AS) for 2 days, resulting in a reduction of the protein's expression of about 60% in the islets. F-actin depolimerization, insulin secretion,mRNA level of genes involved in insulin secretion, maturation and proliferation were evaluated in islets from both control and AS-treated mice. KEY FINDINGS: ARHGAP21 co-localized with actin inMIN6 beta cells and with insulin in neonatal pancreatic islets. F-actin was reduced in AS-islets, as judged by lower phalloidin intensity. Insulin secretion was increased in islets from AS-treated mice, however no differences were observed in the GSIS (glucose-stimulated insulin secretion). In these islets, the pERK1/2 was increased, as well as the gene expressions of VAMP2 and SNAP25, proteins that are present in the secretory machinery. Maturation and cell proliferation were not affected in islets from AS-treated mice. SIGNIFICANCE: In conclusion, our data show, for the first time, that ARHGAP21 is expressed and participates in the secretory process of pancreatic beta cells. Its effect is probably via pERK1/2, which modulates the rearrangement of the cytoskeleton. ARHGAP21 also controls the expression of genes that encodes proteins of the secretory machinery.
Asunto(s)
Actinas/efectos de los fármacos , Actinas/metabolismo , Proteínas Activadoras de GTPasa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Animales , Animales Recién Nacidos , ADN/biosíntesis , ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Insulina/biosíntesis , Insulina/genética , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , RatonesRESUMEN
Recently, a novel CXCL12-binding receptor, has been identified. This CXCL12-binding receptor commonly known as CXCR7 (CXC chemokine receptor 7), has lately, based on a novel nomenclature, has received the name ACKR3 (atypical chemokine receptor 3). In this study, we aimed to investigate the expression of CXCR7 in leukemic cells, as well as its participation in CXCL12 response. Interesting, we clearly demonstrated that CXCR7 is highly expressed in acute lymphoid leukemic cells compared with myeloid or normal hematopoietic cells and that CXCR7 contributed to T-acute lymphoid leukemic cell migration induced by CXCL12. Moreover, we showed that the cellular location of CXCR7 varied among T-lymphoid cells and this finding may be related to their migration capacity. Finally, we hypothesized that CXCR7 potentiates CXCR4 response and may contribute to the maintenance of leukemia by initiating cell recruitment to bone marrow niches that were once occupied by normal hematopoietic stem cells.
Asunto(s)
Quimiocina CXCL12/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Bencilaminas , Western Blotting , Médula Ósea/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Ciclamas , Femenino , Citometría de Flujo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Células K562 , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferencia de ARN , Receptores CXCR/sangre , Receptores CXCR/genética , Receptores CXCR4/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937 , Adulto JovenRESUMEN
OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors.
Asunto(s)
Células de la Médula Ósea/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Síndromes Mielodisplásicos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Quinasa 2 de Adhesión Focal/análisis , Proteína-Tirosina Quinasas de Adhesión Focal/análisis , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Reacción en Cadena de la Polimerasa , Pronóstico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Factores de Riesgo , Estadísticas no Paramétricas , Adulto Joven , Dominios Homologos src/fisiologíaRESUMEN
OBJECTIVE: The aim of this study was to evaluate the expression of protein tyrosine kinase 2 and protein tyrosine phosphatase non-receptor type 11, which respectively encode focal adhesion kinase protein and src homology 2 domain-containing protein-tyrosine phosphatase 2, in hematopoietic cells from patients with myelodysplastic syndromes. METHODS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions were analyzed by quantitative polymerase chain reaction in bone marrow cells from patients with myelodysplastic syndromes and healthy donors. RESULTS: Protein tyrosine kinase 2 and tyrosine phosphatase non-receptor type 11 expressions did not significantly differ between normal cells and myelodysplastic cells. CONCLUSIONS: Our data suggest that despite the relevance of focal adhesion kinase and src homology 2 domain-containing protein-tyrosine phosphatase 2 in hematopoietic disorders, their mRNA expression do not significantly differ between total bone marrow cells from patients with myelodysplastic syndromes and healthy donors. .
Asunto(s)
Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Células de la Médula Ósea/metabolismo , /metabolismo , Síndromes Mielodisplásicos/metabolismo , /análisis , /análisis , Proteína-Tirosina Quinasas de Adhesión Focal/análisis , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Síndromes Mielodisplásicos/genética , Reacción en Cadena de la Polimerasa , Pronóstico , /metabolismo , Factores de Riesgo , Estadísticas no Paramétricas , Dominios Homologos src/fisiologíaRESUMEN
Glioblastoma multiforme is highly aggressive and is the most common glial tumor type. Although there have been advances in treatment, the average survival expectancy is 12-15 months. Several genes have been shown to influence glioblastoma progression. In the present work, we demonstrate that the RhoGTPase Activating Protein 21 (ARHGAP21) is expressed in the nuclear and perinuclear regions of several cell lines. In T98G and U138MG, glioblastoma derived cell lines, ARHGAP21 interacts with the C-terminal region of Focal Adhesion Kinase (FAK). ARHGAP21 depletion by shRNAi in T98G cells alters cellular morphology and increases: FAK phosphorylation states and activation of downstream signaling; the activity state of Cdc42; the production of metalloproteinase 2 (MMP-2) and cell migration rates. These modifications were found to be mainly due to the loss of ARHGAP21 action on FAK and, consequently, the activation of downstream effectors. These results suggest not only that ARHGAP21 might act as a tumor suppressor gene, but also indicate that ARHGAP21 might be a master regulator of migration having a crucial role in controlling the progression of different tumor types.
Asunto(s)
Movimiento Celular/fisiología , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Glioblastoma/metabolismo , Transducción de Señal/fisiología , Actinas/metabolismo , Animales , Línea Celular Tumoral , Forma de la Célula , Proteína Sustrato Asociada a CrK/metabolismo , Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteínas Activadoras de GTPasa/genética , Glioblastoma/patología , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Interferencia de ARN , Proteína de Unión al GTP cdc42/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCzeta. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCzeta and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCzeta constructions demonstrated that ARHGAP21 associates with PKCzeta-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCzeta phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCzeta is phosphorylated by mechanical stimuli. These results suggest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKCzeta signaling pathways, developing an important function during cardiac stress.