RESUMO
The oncoprotein kinase Tpl2 plays an essential role in macrophage activation by the bacterial component lipopolysaccharide (LPS). In response to LPS stimulation, Tpl2 phosphorylates a downstream kinase, MEK1, leading to the activation of ERK signaling pathway. Recent studies demonstrate that the NF-kappaB1 precursor protein p105 functions as an inhibitor of Tpl2 and that the LPS-stimulated Tpl2 activation requires p105 degradation. However, how p105 inhibits the signaling function of Tpl2 is not completely understood. We show here that p105 does not inhibit the intrinsic kinase activity of Tpl2. When complexed with p105, Tpl2 remains catalytically active and uses p105 as a substrate. However, the p105-bound Tpl2 is unable to phosphorylate its physiological target, MEK1. These findings suggest that p105 functions as a competitive inhibitor of Tpl2 that blocks its access by MEK1.
Assuntos
Regulação Enzimológica da Expressão Gênica , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glutationa Transferase/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutação , Subunidade p50 de NF-kappa B/química , Subunidade p50 de NF-kappa B/genética , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.