RESUMEN
Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.
Asunto(s)
Neoplasias Óseas/metabolismo , Núcleo Celular/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Osteosarcoma/metabolismo , Transporte de Proteínas/fisiología , Fraccionamiento Celular/métodos , Citoplasma/metabolismo , Endocitosis/fisiología , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacocinética , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Unión Proteica , Proteínas Recombinantes/farmacocinética , Células Tumorales CultivadasRESUMEN
We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3.
Asunto(s)
Cromatografía de Afinidad , Fibroblastos/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Somatomedinas/metabolismo , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
IGF-binding protein-3 (IGFBP-3) is a modulator of the IGF-signaling pathway and was described as an anti-cancer agent in prostate cancer. The molecular mechanisms underlying these effects remained, however, largely undefined. We analyzed the influence of recombinant IGFBP-3 on cell proliferation of PC3, Du145, and LNCaP prostate cancer cells. As expected, IGFBP-3 inhibited IGF-stimulated cell proliferation by blocking IGF-mediated proliferation signals, but we observed an IGF-independent inhibitory effect of IGFBP-3 on prostate cancer cell proliferation in long-term cultures. We further investigated the influence of IGFBP-3 on adhesion, motility, and invasion of prostate cancer cells using adhesion assays, live-cell imaging techniques, and matrigel invasion measurements. There was a clear inhibitory effect of IGFBP-3 on tumor cell adhesion to extracellular matrix components in the presence and absence of IGF, whereas cell-cell adhesion was not affected. The same inhibitory effect of IGFBP-3 was determined on cell motility when real-time cell movements were followed. In addition, IGFBP-3 was able to inhibit tumor cell invasion through matrigel. In summary, we show that IGFBP-3 inhibits proliferation, adhesion, migration, and invasion processes of prostate tumor cells. These newly described mechanisms of IGFBP-3 can be of importance for tumor progression and support a role of IGFBP-3 in therapeutic settings.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Neoplasias de la Próstata/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Masculino , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
The superoxide radical (O(2)(-)) has long been considered a major cause of aging. O(2)(-) in cytosolic, extracellular, and mitochondrial pools is detoxified by dedicated superoxide dismutase (SOD) isoforms. We tested the impact of each SOD isoform in Caenorhabditis elegans by manipulating its five sod genes and saw no major effects on life span. sod genes are not required for daf-2 insulin/IGF-1 receptor mutant longevity. However, loss of the extracellular Cu/ZnSOD sod-4 enhances daf-2 longevity and constitutive diapause, suggesting a signaling role for sod-4. Overall, these findings imply that O(2)(-) is not a major determinant of aging in C. elegans.