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 molecular mechanisms by which insulin inhibits IGFBP-1 transcription and the IGF-independent mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells. (i) Insulin inhibition of Foxo1-stimulated IGFBP-1 transcription. Transcription of IGFBP-1, like the hepatic gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, is inhibited by insulin. The decreased transcription results from inhibition of the transcriptional activity of the Forkhead box class O (FOXO) subfamily of transcription factors, proteins that bind to a response element in the proximal promoter of the IGFBP-1 gene to stimulate transcription. Insulin stimulates the phosphorylation of the FOXO proteins at three consensus phosphorylation sites for protein kinase B (PKB)/Akt, a serine-threonine protein kinase that is activated by phosphatidylinositol 3-kinase following its activation by insulin receptors. When these sites are phosphorylated, FOXO proteins are exported from the nucleus and FOXO-stimulated transcription is inhibited. It has been proposed that insulin inhibition of Foxo1-stimulated IGFBP-1 transcription is due solely to the exclusion of the transcription factor from the nucleus. To test this hypothesis, we mutated a critical leucine residue in the leucine-rich nuclear export signal of Foxo1 to alanine, and transfected wild-type and mutant Foxo1 into H4IIE rat hepatoma cells. Immunofluorescence microscopy showed that after insulin treatment, wild-type Foxo1 redistributed to the cytoplasm whereas the mutant Foxo1 remained predominantly in the nucleus. Despite retention of the mutant transcription factor in the nucleus, transcription stimulated by the Foxo1 mutant could still be inhibited by insulin. These results demonstrate that insulin can inhibit Foxo1-stimulated transcription by some other mechanism(s) when nuclear export is prevented. In support of this conclusion, we have shown that insulin can inhibit Foxo1-stimulated transactivation, and others have shown that insulin-stimulated phosphorylation of Foxo1 inhibits its binding to DNA. We also have examined whether the transcriptional activity of Foxo1 can be modified by other post-translational modifications besides phosphorylation. We observed that the coactivator p300 directly acetylates lysines in the carboxyl-terminal region of Foxo1 in vivo and in vitro, and that overexpression of p300 potently stimulates Foxo1-induced transcription of IGFBP-1. The intrinsic acetyltransferase activity of p300 is required for both functions, suggesting that acetylation of Foxo1 by p300 may be responsible, at least in part, for its increased transcriptional activity. Insulin further stimulates the acetylation of Foxo1 by p300 but, unlike phosphorylation, acetylation plays only a minor role in insulin inhibition of Foxo1 transcriptional activity. (ii) Mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells. We previously reported that an IGFBP-3 mutant protein that does not bind IGF-I or IGF-II (6m-IGFBP-3) can induce apoptosis in PC-3 prostate carcinoma cells. To begin to understand the initial mechanisms by which IGFBP-3 acts, we examined early events in the induction of apoptosis in these cells. Apoptosis results from the proteolysis of critical cell proteins by efffector caspases, which are activated from inactive precursors by either the caspase 8 or caspase 9 initiator caspase pathway. Caspase 8 is activated by ligands that interact with death receptors causing them to aggregate, triggering the aggregation of the adapter protein FADD and procaspase 8, leading to its autoactivation. Activation of caspase 9 is triggered by the release of cytochrome C from the intermembrane space of mitochondria, forming complexes in the cytosol in which procaspase 9 is activated. Activation of either the caspase 8 or caspase 9 initiator caspase pathways may be able to activate effector caspases sufficiently to induce apoptosis. In some cases, however, the signal must be amplified by cross-activation of the other initiator pathway. Both the caspase 8 and caspase 9 pathways are involved in the induction of apoptosis in PC-3 cells by wild-type IGFBP-3 and 6m-IGFBP-3. Tetrapeptide inhibitors of caspase 8 or caspase 9 completely inhibited apoptosis induced by 6m-IGFBP-3. Decreased induction of apoptosis by 6m-IGFBP-3 also was observed in stable transfectants of PC-3 cells that overexpress dominant negative FADD (FADD-DN) to selectively inhibit the activation of caspase 8, or the anti-apoptotic protein Bcl-2 to stabilize mitochondria, preventing the release of cytochrome c and the activation of caspase 9. FADD-dependent activation of apoptosis cannot occur by cross-activation from the mitochondrial / caspase 9 pathway, suggesting that IGFBP-3 might act first by triggering caspase 8 activation. Consistent with this hypothesis, caspase 8 activity increased rapidly following addition of 6m-IGFBP-3, reaching its peak at 1 h. Strong support for the hypothesis that the induction of caspase 8 by IGFBP-3 was FADD-dependent came from the observation that the activity was greatly decreased in PC-3-FADD-DN cells, but occurred normally in PC-3 cells that overexpressed Bcl-2. We conclude that 6m-IGFBP-3 triggers apoptosis in PC-3 cells by FADD-dependent activation of caspase 8, and that the caspase 8 signal must be amplified by cross-activation of the mitochondrial pathway to activate effector caspases sufficiently to induce apoptosis. The molecular mechanisms by which IGFBP-3 activates FADD remain to be elucidated. 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 in PC-3 human prostate cancers, 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 mutated the nuclear localization signal, KGRKR, to MDGEA, alone or in combination with the 6m-IGFBP-3 mutation of the IGF-binding site. Following transient transfection, we confirmed that YFP-IGFBP-3 remained cell-associated, whereas a fusion protein consisting of a secretory form of IGFBP-3 fused to the N-terminus of YFP (pre-IGFBP-3-YFP) was recovered almost exclusively in the media. Fluorescence microscopy of live cells and biochemical fractionation followed by western blotting confirmed that wild-type YFP-IGFBP-3 localized to the nucleus, whereas transfected YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Cell death was examined by flow cytometry, with non-viable cells identified by staining with Annexin V-APC and 7-AAD (7-Amino-Actinomycin D). Caspase-dependent death was induced by both YFP-MDGEA and YFP-6m/MDGEA, indicating that nuclear localization of IGFBP-3 is not necessary for it to induce apoptosis. Although it has been thought that secretion of IGFBP-3 is required for it to induce apoptosis, presumably by allowing it to interact with putative IGFBP-3 receptors to stimulate signal transduction pathways or be internalized, our results suggest that non-secreted IGFBP-3 also can act intracellularly to induce apoptosis.
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