RESUMO
CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Proliferação de Células , Ciclina A/metabolismo , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Imunofenotipagem , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células U937RESUMO
Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Complexo do Signalossomo COP9 , Sobrevivência Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação para Baixo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lentivirus , Camundongos , Camundongos Endogâmicos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Peptídeo Hidrolases/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Transfecção , Irradiação Corporal TotalRESUMO
Mitochondria, the dynamic energy powerhouses of the cell, have vital roles in a multitude of cellular processes including differentiation and cell survival. Tight regulation of mitochondrial dynamics, integrity and function is indispensible for preservation of homeostasis in all cells, including pluripotent stem cells. The ability to proliferate and self-renew indefinitely bestows the pluripotent embryonic stem cells (ESCs) with immense curative potential. Mechanisms that preserve mitochondrial well-being, and therefore maintain "stemness," are vital in realizing the full potential of ESCs in therapeutic regenerative medicine. However, virtually nothing is known regarding the regulation of mitochondrial dynamics and function and the relationship thereof to overall cell fate and function in pluripotent ESCs or other somatic stem cells. Using loss- and gain-of-function approaches, we show that growth factor erv1-like (Gfer) plays an essential pro-survival role in the maintenance of murine ESC pluripotency by preserving the structural and functional integrity of their mitochondria, through modulation of the key mitochondrial fission factor Drp1.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Autofagia , Diferenciação Celular , Sobrevivência Celular , Dinaminas , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/citologiaRESUMO
The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.
