Insulin-like Growth Factor Binding Proteins

Diabetes, Digestive, Kidney Diseases

Investigators

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Abstract

Insulin-like growth factor (IGF)-I and -II are small proteins chemically related to insulin that stimulate cell survival and proliferation by binding to signaling IGF-I receptors. The IGFs also bind with high affinity to a family of six secreted IGF binding proteins (IGFBPs), forming biologically inactive complexes that cannot activate IGF-I receptors. Some of the IGFBPs, notably IGFBP-3, also can act by direct, IGF-independent mechanisms to stimulate apoptosis and inhibit cell proliferation. During the past year, our ongoing studies of the regulation and biological role of the IGFBPs have focused on the regulation of IGFBP-1 transcription by post-translational modification of the transcription factor Foxo1, and the IGF-independent mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells. (i) Regulation of IGFBP-1 transcription by post-translational modification of Foxo1. The FOXO subgroup of forkhead transcription factors controls the expression of many genes involved in fundamental cellular processes including genes encoding enzymes involved in gluconeogenesis and IGFBP-1. Insulin, the major negative regulator, stimulates the phosphorylation of three protein kinase B/Akt phosphorylation sites in Foxo1, leading to a decrease in Foxo1-stimulated transcription due to export of Foxo1 from the nucleus and decreased transactivation. We have shown that the coactivator p300 directly acetylates lysines in the carboxyl-terminal region of Foxo1 in vivo and in vitro, and potently stimulates Foxo1-induced IGFBP-1 promoter activity in transient transfection experiments. The intrinsic acetyltransferase activity of p300 is required for both functions. Our results suggest that acetylation of Foxo1 by p300 is responsible, at least in part, for its increased transactivation potency, although a contribution from histone acetylation cannot be excluded. Insulin enhances p300 acetylation of Foxo1 but, in contrast to the importance of acetylation in increasing the transcriptional activity of Foxo1 in the absence of insulin, a further increase in acetylation in response to insulin does not overcome the inhibition of Foxo1 transcriptional activity induced by insulin-stimulated phosphorylation. (ii) IGF-independent mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells. IGFBP-3, a secreted protein, has the intrinsic ability to induce apoptosis directly without binding IGFs. We previously reported that an IGFBP-3 mutant protein that does not bind IGF-I or IGF-II (6m-IGFBP-3) induces apoptosis in PC-3 human prostate carcinoma cells as effectively as wild-type IGFBP-3. Studies have been initiated to understand how IGFBP-3 induces apoptosis in these cells. IGFBP-3 contains a C-terminal nuclear localization signal, and it has been proposed that nuclear localization of IGFBP-3 is required for it to induce apoptosis. To test this hypothesis, we constructed plasmids fusing yellow fluorescent protein (YFP) to mature IGFBP-3 lacking a signal peptide so that the protein would not be secreted. We also mutated the nuclear localization signal, 228-KGRKR-232, to MDGEA, alone or in combination with the 6m mutation of the IGF binding site. Biochemical fractionation and in vivo imaging studies of PC-3 cells expressing these constructs showed that YFP-IGFBP-3 and YFP-MDGEA are not detectable in the extracellular media. YFP-IGFBP-3 localized predominantly to the nucleus, whereas YFP-MDGEA localized predominantly to the cytoplasm. Apoptosis, measured by increased labeling with annexin V using flow cytometry, was induced in PC-3 cells expressing YFP-IGFBP-3 or YFP-MDGEA, and was prevented by incubation with the pan-caspase inhibitor z-VAD-fmk. Similar results were obtained using double mutant constructs (6m/MDGEA) containing the 6m-IGFBP-3 mutation. Tetrapeptide inhibitors of initiator caspases 8 and 9 inhibited apoptosis induced by these constructs, suggesting that cross-talk occurs between the two initiator caspase pathways. Our results indicate that non-secreted IGFBP-3 can induce apoptosis in prostate cancer cells by intracrine mechanisms in an IGF-independent manner. Both caspase 8 and caspase 9 initiator pathways are involved, but nuclear localization is not required.