The insulin-like growth factors IGF-I and IGF-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 to a family of six secreted IGF binding proteins (IGFBPs), forming complexes that are biologically inactive because they can not bind to IGF-I receptors. In addition, some of the IGFBPs, notably IGFBP-3, can act directly and independently of binding IGFs to stimulate apoptosis and inhibit cell proliferation. During the past year, our ongoing studies of the regulation and biological role of the IGFBPs have addressed: (i) the molecular mechanisms by which insulin inhibits IGFBP-1 transcription and (ii) IGF-independent stimulation of apoptosis in human prostate cancer cells by IGFBP-3. (i) Insulin inhibition of IGFBP-1 transcription. Plasma IGFBP-1 concentration is dynamically regulated by metabolic change. Insulin is the principal regulator, acting mainly at the level of transcription. IGFBP-1 transcription is increased in diabetic rat liver and rapidly decreased by insulin treatment. The forkhead transcription factor FKHR (FOXO1) stimulates IGFBP-1 transcription. Insulin inhibits FKHR-stimulated transcription and induces the redistribution of FKHR from the nucleus to the cytoplasm, leading to the proposal that exclusion of FKHR from the nucleus is responsible for the inhibition of FKHR-stimulated transcription. To test this hypothesis, we designed mouse FKHR mutants that would be retained in the nucleus after insulin treatment and determined whether insulin could inhibit transcription stimulated by the mFKHR mutant proteins. Alanine was substituted for Leucine 375, a critical residue in the leucine-rich nuclear export sequence of mFKHR, or for Threonine 24, one of three consensus Akt phosphorylation sites that is phosphorylated in response to insulin, allowing 14-3-3 to bind to FKHR and anchor the transcription factor in the cytoplasm. H4IIE rat hepatoma cells transiently transfected with wild-type and mutant mFKHR plasmids were examined by immunofluorescence microscopy. In the absence of insulin, wild-type and mutant mFKHR were localized to the nucleus. After insulin treatment, wild-type mFKHR redistributed to the cytoplasm, whereas the mFKHR mutants remained predominantly in the nucleus. Insulin inhibited IGFBP-1 promoter activity stimulated by mutant mFKHR to the same extent as wild-type mFKHR even though the mFKHR mutants were retained in the nucleus. These results indicate that insulin can inhibit FKHR-stimulated transcription by direct inhibition of transcription as well as nuclear exclusion. The availability of alternative mechanisms to regulate FKHR-dependent transcription can provide reliable regulation of many genes involved in fundamental cell processes such as apoptosis, cell cycle regulation and DNA repair. To better understand the mechanism by which insulin inhibits FKHR-stimulated transcription, we have used a chimeric protein in which a C-terminal fragment of mFKHR containing the transactivation domain, residues 208-652, is fused to a Gal4 DNA binding domain (dbd). In transient transfection experiments in H4IIE cells, insulin inhibits Gal4 promoter activity stimulated by the Gal4 dbd-mFKHR 208-652 fusion protein. Insulin inhibition of mFKHR 208-652-stimulated transcription requires phosphatidylinositol 3-kinase but, unlike full-length mFKHR (1-652), inhibition does not require either of the two phosphorylation sites for the downstream protein kinase, Akt, present in the fragment. Inhibition occurs at the level of transactivation, and cannot be accounted for by exclusion of the transcription factor from the nucleus. Using site-directed and deletion mutagenesis, we have identified three sites that are required for insulin inhibition of mFKHR 208-652-stimulated transcription: Ser319, Ser499 and the 15-amino acid region extending from residue 350 to 364. Insulin inhibition is abolished either by double mutation of Ser319 and Ser499, or by deletion of amino acids 350-364, indicating that Ser319/Ser499 and 350-364 are involved in independent steps that are essential for insulin inhibition. Whether they are components of separate pathways or sequential steps in a single regulatory pathway remains to be determined. (ii) IGF-independent inhibition of apoptosis by IGFBP-3. IGFBP-3, the most abundant IGFBP in serum, inhibits cell proliferation and stimulates apoptosis, and has been proposed to mediate the growth inhibitory effects of potent anti-proliferative agents such as TGF-beta, retinoids, antiestrogens, vitamin D3 and p53. Elevated plasma IGFBP-3 is a negative risk factor for several common cancers. It has been assumed that IGFBP-3 acts by inhibiting the stimulation of cell survival and proliferation by IGF-I and IGF-II. IGFBP-3 also can stimulate apoptosis and inhibit cell proliferation directly. These IGF-independent actions, however, only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. To assess the general importance of IGF-independent mechanisms, we generated a human IGFBP-3 mutant, 6m-hIGFBP-3, that did not bind IGF-I or IGF-II and only can act by IGF-independent mechanisms. Alanine was substituted for six residues in the putative IGF binding site, I56/Y57/R75/L77/L80/L81. The 6m-hIGFBP-3 mutant inhibited DNA synthesis in Mv1 mink lung epithelial cells, establishing that hIGFBP-3 can inhibit Mv1 cell DNA synthesis by IGF-independent mechanisms. We then compared the ability of wild-type hIGFBP-3 and 6m-hIGFBP-3 to stimulate apoptosis in serum-deprived PC-3 human prostate cancer cells. PC-3 cells synthesize and respond to IGF-II so it was possible that IGFBP-3 might stimulate apoptosis by inhibiting IGF-II action. In fact, the 6m-hIGFBP-3 mutant stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence as wild-type hIGFBP-3, indicating that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells, and suggesting that they may play a prominent role in the anti-proliferative and anti-tumorigenic actions of IGFBP-3.
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