RESUMO
While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.
Assuntos
Envelhecimento/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Receptores de Superfície Celular/genética , Células-Tronco/metabolismo , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Metilação de DNA , DNA Mitocondrial/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Glucuronidase , Proteínas Klotho , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/patologia , Mioblastos/patologia , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Regeneração/genética , Transdução de Sinais , Células-Tronco/patologiaRESUMO
Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic factor which controls the production and differentiation of granulocytes. The G-CSF receptor (G-CSFR) belongs to the superfamily of the cytokine receptors, which transduce signals via the activation of cytosolic protein tyrosine kinases (PTK). To determine the role of specific PTK in G-CSF signaling we expressed the human G-CSFR in cell lines derived from DT40 B cells, which lack either the Src-related Lyn or Syk. Wild-type (wt) and syk-deficient cells underwent increased DNA synthesis in response to G-CSF; lyn-deficient cells did not. The purpose of these studies is to identify Lyn's downstream effectors in mediating DNA synthesis. While G-CSF stimulated Ras activity in all cell lines, G-CSF failed to induce the tyrosine phosphorylation of Shc in lyn-deficient cells. G-CSF induced a statistically significant activation of Erk1/Erk2 Kinase or p90Rsk only in the wt cells. G-CSF induced the tyrosine phosphorylation of Cbl and increased activity of PI 3-kinase in wild-type and syk-deficient, but non in lyn-deficient, cells. Inhibition of Shc by over-expression of its SH2 or PTB domains or PI 3-kinase by either treatment with wortmannin or expression of the CblY731F mutant decreased G-CSF-induced DNA synthesis. Thus, the Lyn, Cbl-PI 3-kinase, and Shc/non-Ras-dependent pathways correlate with the ability of cells to respond to G-CSF with increased DNA synthesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Ubiquitina-Proteína Ligases , Quinases da Família src/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Galinhas , DNA/biossíntese , Guanosina Trifosfato/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases/fisiologia , Proteínas Proto-Oncogênicas c-cbl , Proteínas Quinases S6 Ribossômicas , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas ras/metabolismoRESUMO
Protein tyrosine kinases play a major role in promoting cell growth, and their activity in solid tumors is well established. Inhibitors of protein tyrosine kinases are now in advanced clinical trials for the treatment of breast and brain cancers. Because Src-related PTK have been shown to be activated in leukemic cell lines, we studied their activation in human myeloid leukemia. Blasts from the majority of patients with acute leukemia showed constitutive activity of the Src kinase Lyn. In contrast, no patient samples showed constitutive activation of Jak2. Genetic and pharmacologic targeting of Lyn was used to determine its contribution to leukemic cell growth. Antisense Lyn oligonucleotide treatment resulted in the inhibition of tritiated thymidine incorporation following GM-CSF stimulation of the factor-dependent line MO7e. The Src kinase inhibitor PD166285 inhibited the growth of human leukemic cell lines and leukemic blasts. When combined with doxorubicin, an additive effect on the inhibition of leukemic cell growth occurred. These studies demonstrate the importance of Src kinases in promoting leukemic cell growth and suggests that further development of agents which target Src kinases and their inclusion in multidrug regimens are warranted for novel therapies of myeloid leukemia.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide/tratamento farmacológico , Oligonucleotídeos Antissenso/farmacologia , Piridonas/farmacologia , Pirimidinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Leucemia Mieloide/enzimologia , Leucemia Mieloide/patologia , Oligonucleotídeos Antissenso/uso terapêutico , Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
OBJECTIVE: The retroviral oncogene v-Cbl causes pre-B cell lymphomas and myeloid leukemias in mice, and its Drosophila homologue is oncogenic, causing enhanced receptor tyrosine kinase signaling. The human Cbl gene resides at 11q23. The aim of this study is to determine the effect of oncogenic Cbl on growth-regulating responses. MATERIALS AND METHODS: The oncogenic mutant of Cbl (CblDelta1-357) was transfected into factor-dependent 32Dcl3 myeloid cells. Consequently, cell survival and differentiation were measured. Lyn, Syk, MAP kinase, and phosphatidylinositol 3'(PI3')-kinase activities, protein phosphorylation, Bcl-2 promoter activity, ubiquitination, and levels of Bcl-2, Bax, Bad, and Bcl-x(L) were determined. In addition, the effect of v-Cbl on TF-1 cell survival upon granulocyte-macrophage colony-stimulating factor withdrawal was studied. RESULTS: 32Dcl3 and TF-1 cells expressing v-Cbl showed resistance to apoptosis upon growth factor withdrawal, and 32Dcl3 cells completely failed to respond to granulocyte colony-stimulating factor's induction of differentiation. Basal activities of Lyn, Syk, and PI3'-kinase were elevated in the v-Cbl line. There was neither enhanced tyrosine phosphorylation of cellular protein content, Cbl, or Jak2, nor serine phosphorylation of MAP kinase or Akt. After factor withdrawal, the level of Bcl-2 was greater in v-Cbl cells than in control cells. CONCLUSIONS: Neither increased Bcl-2 promoter activity nor decreased ubiquitination of Bcl-2 could account for increased Bcl-2 levels. v-Cbl-expressing 32Dcl3 cells were resistant to differentiation. v-Cbl suppresses apoptosis and differentiation, possibly through enhancement of Lyn, Syk, and PI3'-kinase activities and Bcl-2.