Insulin-like Growth Factor Binding Proteins

Diabetes, Digestive, Kidney Diseases

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Abstract

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 and IGF-independent mediation of transforming growth factor (TGF)-beta inhibition of cell proliferation by IGFBP-2. (i) Plasma IGFBP-1 concentration is dynamically regulated by metabolic changes. 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. An insulin response element was identified in the proximal IGFBP-1 promoter. It binds the forkhead transcription factor FKHR (Foxo1) which stimulates IGFBP-1 transcription. Similar insulin response elements are present in the promoters of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, two genes that are involved in gluconeogenesis whose transcription also is inhibited by insulin. (a) Insulin inhibition of IGFBP-1 transcription is mediated by phosphatidylinositol 3-phosphate kinase (PI-3 kinase) which activates the serine/threonine protein kinase Akt. Insulin stimulates the phosphorylation of three consensus Akt phosphorylation sites in mouse FKHR (Thr24, Ser253 and Ser316), resulting in inhibition of mFKHR-stimulated IGFBP-1 transcription and redistribution of FKHR from the nucleus to the cytoplasm. It has been proposed that insulin inhibition of transcription is due to the exclusion of FKHR from the nucleus. To test this hypothesis, we generated FKHR mutants that would remain in the nucleus of H4IIE rat hepatoma cells after insulin treatment and determined whether insulin inhibited transcription stimulated by the mutants. Alanine was substituted for Leu375, a critical residue in the leucine-rich nuclear export signal of mFKHR that is recognized by the export receptor Crm1, or for Thr 24. Phosphorylated Thr24 is a consensus binding site for the cytosolic protein 14-3-3, and binding to 14-3-3 has been proposed to sequester phosphorylated FKHR in the cytoplasm. Confocal microscopy confirmed that mFKHR containing one or both of these mutations remained predominantly in the nucleus following insulin treatment. Insulin still inhibited transcription stimulated by the mFKHR mutants. These results indicate that insulin inhibition of FKHR-stimulated transcription may occur without nuclear exclusion, and suggest that insulin may dirrectly inhibit transcription activation. (b) To study how insulin inhibits transcription activation by mFKHR, we fused the yeast Gal4 DNA binding domain to a C-terminal fragment of mFKHR, residues 208-652, that contains the transactivation domain but lacks a functional DNA binding domain. Insulin inhibited Gal4 promoter activity stimulated by the fused protein. Insulin inhibition persisted with modest truncation of the C-terminal fragment (mFKHR 317-652), but was lost after more extensive truncation (mFKHR 501-652 and 593-652). In fact, insulin enhanced transcription stimulated by mFKHR 593-652 about 5-fold. PI-3 kinase mediates insulin inhibition of transcription stimulated by mFKHR 208-652, but the Akt phosphorylation sites are not required for inhibition. Experiments are in progress to identify potential sites of insulin-dependent phosphorylation in the mFKHR 317-500 fragment that might inhibit transcription. PI-3 kinase is not involved in insulin stimulation of mFKHR 593-652-induced transcription. Moreover, alanine substitution of all 10 serines and threonines in mFKHR 593-652 had no effect on insulin stimulation, suggesting that the regulation might involve interaction of FKHR with another protein rather than direct phosphorylation of FKHR. One candidate, the histone acetyltransferase CBP/p300, binds to full length FKHR and increases FKHR-stimulated transcription. We are determining whether CBP/p300 is involved in insulin regulation of transcription by full-length mFKHR and the C-terminal fragments. (ii) IGFBP-3, the most abundant IGFBP in serum, inhibits cell proliferation and stimulates apoptosis. It has been proposed that IGFBP-3 mediates the growth inhibitory effects of potent anti-proliferative agents such as TGF-beta, retinoids, antiestrogens, vitamin D3 and p53, and that high plasma IGFBP-3 is a negative risk factor for several common cancers. It has been assumed that IGFBP-3 acts by inhibiting IGF stimulation of cell survival and proliferation. IGFBP-3, however, also can stimulate apoptosis and inhibit cell proliferation directly, but these IGF-independent actions only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. (a) To assess the general importance of IGF-independent mechanisms, we generated a human IGFBP-3 mutant that can not bind IGF-I or IGF-II by substituting alanine for six residues in the proposed IGF binding site, I56/Y57/R75/L77/L80/L81. Binding of both IGF-I and IGF-II to 6m-hIGFBP-3 under non-denaturing conditions was reduced >80-fold. The non-binding 6m-hIGFBP-3 mutant only can act by IGF-independent mechanisms since it can not prevent IGF-I and IGF-II from activating the IGF-I receptor. The 6m-hIGFBP-3 mutant inhibited DNA synthesis in Mv1 mink lung epithelial cells, confirming our previous conclusion that wild-type hIGFBP-3 inhibited Mv1 cell DNA synthesis entirely by IGF-independent mechanisms. Wild-type and 6m-hIGFBP-3 also stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence in serum-deprived PC-3 human prostate cancer cells which synthesize and respond to IGF-II so that IGFBP-3 could have acted by either IGF-dependent or IGF-independent mechanisms. These results demonstrate that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells, and suggest that they may play a wider role in the anti-proliferative and anti-tumorigenic actions of IGFBP-3. (b) We also investigated whether IGFBPs synthesized by Mv1 cells are involved in the inhibition of DNA synthesis by TGF-beta. IGFBP-2 is the only IGFBP synthesized and secreted by Mv1 cells. Recombinant bovine IGFBP-2, like exogenous IGFBP-3, inhibits Mv1 cell DNA synthesis in an IGF-independent manner. Coincubation with an IGF-I analogue that has high affinity for IGFBP-2 and low affinity for IGF-I receptors, Leu60-IGF-I, induces a conformational change in IGFBP-2 that blocks it from inhibiting DNA synthesis. Coincubation with Leu60-IGF-I also decreased the inhibition of Mv1 DNA synthesis by TGF-beta by up to 70%, whereas coincubation with Long-R3-IGF-I, an IGF-I analogue with high affinity for the IGF-I receptor and low affinity for IGFBP-2, had no effect. These results indicate that Leu60-IGF-I decreases TGF-beta inhibition of Mv1 DNA synthesis by binding to IGFBP-2, not by activating IGF-I receptors. bIGFBP-2 binds to Mv1 plasma membranes at 0 C; binding is blocked by coincubation with Leu60-IGF-I. We propose that secreted IGFBP-2 must reassociate with Mv1 cells to inhibit DNA synthesis; Leu60-IGF-I prevents this reassociation. TGF-beta sensitizes Mv1 cells to the latent growth inhibitory effects of endogenous IGFBP-2 by promoting the binding or uptake of IGFBP-2, or potentiating an IGFBP-2 signaling pathway.