TGF-ß-induced expression of IGFBP-3 regulates IGF1R signaling in human osteosarcoma cells.

Signaling pathways initiated by transforming growth factor-ß (TGF-ß) and insulin-like growth factors (IGFs) are important in osteosarcoma cell growth. We have investigated a role for endogenous IGF binding protein-3 (IGFBP-3) in mediating cross-talk between TGF-ß receptor and type I IGF receptor (IGF1R) signaling pathways in MG-63 osteosarcoma cells. TGF-ß1 indirectly activated the Ras/Raf/MAPK pathway and induced the expression of IGFBP-3, an important regulator of IGF1R activity. IGFBP-3 attenuated TGF-ß1 activation of ERK1/2 and Akt in MG-63 cells, and inhibited TGF-ß1-induced cell cycle progression and proliferation. This effect of IGFBP-3 was blocked by inhibiting IGF1R signaling. TGF-ß1 phosphorylated Smad2 on the non-receptor substrate sites (Ser245/250/255). Blocking the TGF-ß1-induced expression of IGFBP-3 enhanced pSmad2(Ser245/250/255) and increased its nuclear accumulation. These results suggest an important role for TGF-ß1 in osteosarcoma cell growth, with the induction of IGFBP-3 by TGF-ß1 serving in a negative-feedback loop to control cell growth by preventing activation of the IGF1R.

Effects of endogenous insulin-like growth factor binding protein-3 on cell cycle regulation in breast cancer cells.

High tissue insulin-like growth factor binding protein-3 (IGFBP-3) expression in breast cancers is associated in some studies with rapid growth and poor outcome. This study examined the effects of endogenous IGFBP-3 in Hs578T breast cancer cells, which are IGF-unresponsive and grow aggressively despite relatively high IGFBP-3 expression. IGFBP-3 downregulation using siRNA was associated with increases in DNA synthesis, the percentage of cells in S phase and viable cell numbers, accompanied by increases in cyclins A and D1, a decrease in p27 expression, and increased phosphorylation of retinoblastoma (Rb) on Ser795. Downregulation of IGFBP-3 inhibited extracellular signal-regulated kinase (ERK) activation and cell migration in a monolayer wound healing assay. These results indicate that endogenous IGFBP-3 is anti-proliferative and pro-migratory in Hs578T cells and that these effects are IGF-independent. Poor outcome in breast tumours expressing high levels of IGFBP-3 may be due to the effects of IGFBP-3 on cell migration rather than cell growth.

Inhibition of adipocyte differentiation by insulin-like growth factor-binding protein-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) interacts with the type II nuclear receptors retinoid X receptor (RXR)alpha and retinoic acid receptor-alpha and modulates their transcriptional activity. Peroxisome proliferator-activated receptor (PPAR)gamma, a related nuclear receptor that dimerizes with RXRalpha, plays an important role in adipocyte differentiation. IGFBP-3 is regulated during adipocyte differentiation, but its role in this process is unknown. We demonstrate that IGFBP-3 interferes with the PPARgamma-dependent processes of adipocyte differentiation and maintenance of the gene expression characteristic of mature adipocytes. Treatment of adipocytes with exogenous IGFBP-3, but not an IGFBP-3 mutant that does not bind RXRalpha or PPARgamma, decreased markers of adipocyte differentiation, PPARgamma, and resistin but increased the preadipocyte marker plasminogen activator inhibitor-1. Furthermore, expression of human IGFBP-3, but not the IGFBP-3 mutant, by preadipocytes inhibited preadipocyte differentiation as determined by gene markers and lipid accumulation. IGFBP-3 interacted with PPARgamma in vitro and in 3T3-L1 adipocyte lysates and inhibited PPARgamma heterodimerization with RXRalpha in vitro. Wild-type IGFBP-3, but not mutant IGFBP-3, blocked ligand-induced transactivation of PPAR response element in 3T3-L1 cells. The observation that IGFBP-3 inhibits adipocyte differentiation and impacts on the PPARgamma system suggests a role for IGFBP-3 in the pathogenesis of obesity and insulin resistance.

Molecular basis of the interaction between IGFBP-3 and retinoid X receptor: role in modulation of RAR-signaling.

IGFBP-3 interacts with the retinoid X receptor-alpha (RXRalpha) and retinoic acid receptor-alpha (RARalpha) and thereby interferes with the formation of RXR:RAR heterodimers. Here we identify the domains in RXRalpha and IGFBP-3 that participate in this interaction. When different regions of RXRalpha were expressed independently, we found that only the DNA-binding domain (C-domain) bound IGFBP-3. Residues in the second Zn-finger loop (Gln49, Arg52), which contribute to C-domain dimerization on DR1 response elements, proved essential to IGFBP-3 binding. In complementary studies, we found that residues within the N-terminal domain of IGFBP-3 (Thr58, Arg60) and motifs in its C-terminal domain ((220)LysLysLys, (228)LysGlyArgLysArg) were required for interaction with RXRalpha and RARalpha. Unlike wild-type IGFBP-3, the non-retinoid receptor-binding mutants of IGFBP-3 were unable to attenuate all-trans-retinoic acid-induced transactivation of the RAR response element by RXR:RAR heterodimers. We conclude that residues in both the N- and C-terminal domains of IGFBP-3 are involved in binding the retinoid receptors, and that this interaction is essential to the modulation of RAR-signaling by IGFBP-3.

Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts.

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen alpha1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.

Insulin-like growth factor binding protein-3 prevents retinoid receptor heterodimerization: implications for retinoic acid-sensitivity in human breast cancer cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) has both IGF-dependent and -independent effects on cell growth, which are frequently growth-inhibitory. Interestingly, the development of a more aggressive phenotype in breast cancer cells (BCCs) correlates positively with elevated expression of IGFBP-3 and is often associated with all-trans-retinoic acid (atRA)-resistance. IGFBP-3 was previously demonstrated to interact directly with retinoid X receptor (RXR). In this study we have shown that IGFBP-5 also interacts with RXR and that both IGFBPs interact with retinoic acid receptor (RAR). To investigate whether the presence of IGFBP-3 regulates breast cancer cell responsiveness to atRA, we immuno-neutralized the IGFBP-3 expressed by the atRA-resistant Hs578T and MDA-MB-231 BCCs (which express IGFBP-3 constitutively) and showed that they become more sensitive to the growth-inhibitory effects of atRA. Similarly, in Hs578T cells expressing a reporter gene under the control of an RAR response element (RARE), depletion of IGFBP-3 resulted in the induction of reporter gene expression in response to atRA. In investigating possible mechanisms for IGFBP-3 regulation of atRA-sensitivity, we found that IGFBP-3 blocked the formation of RAR:RXR heterodimers and disrupted the ligand-inducible receptor complex. Thus, IGFBP-3 has the potential to reduce the RARE-mediated transactivation of target genes and modulate the atRA-response in BCCs.

Phosphorylation of insulin-like growth factor binding protein-3 by deoxyribonucleic acid-dependent protein kinase reduces ligand binding and enhances nuclear accumulation.

The IGF binding proteins (IGFBPs) regulate the mitogenic effects of IGFs in the extracellular environment. Several members of this family, including IGFBP-3, also appear to have IGF-independent effects on cell function. For IGFBP-3 and IGFBP-5, both of which are translocated to the cell nuclei, these effects may be related to their putative nuclear actions. Because reversible phosphorylation is an important mechanism for controlling nuclear protein import, we have examined the effect of phosphorylating IGFBP-3 with a number of serine/threonine protein kinases on its nuclear import. Phosphorylation of IGFBP-3 by the double-stranded DNA-dependent protein kinase (DNA-PK) increased both the nuclear import of IGFBP-3 and the binding of IGFBP-3 to components within the nucleus compared with nonphosphorylated IGFBP-3. However, there was no difference in the binding of the nuclear transport factor, importin beta, to nonphosphorylated and phosphorylated IGFBP-3. The ability of the DNA-PK phosphoform of IGFBP-3 to bind IGFs was severely attenuated, and in contrast to nonphosphorylated IGFBP-3, the DNA-PK phosphoform was unable to transport IGF-I to the nucleus. Furthermore, IGFBP-3 was phosphorylated by DNA-PK when complexed to IGF-I causing the phosphoform to release IGF-I. Together, these results suggest that when IGF-I is cotransported into the nucleus by IGFBP-3, phosphorylation of IGFBP-3 by nuclear DNA-PK provides a means for releasing bound IGF-I and creating a phosphoform of IGFBP-3 with increased affinity for nuclear components.

Role of insulin-like growth factor binding protein-3 in breast cancer cell growth.

The mitogenic effects of insulin-like growth factors (IGFs) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs). One member of this family, IGFBP-3, mediates the growth-inhibitory and apoptosis-inducing effects of a number of growth factors and hormones such as transforming growth factor-beta, retinoic acid, and 1,25-dihydroxyvitamin D3. IGFBP-3 may act in an IGF-dependent manner by attenuating the interaction of pericellular IGFs with the type-I IGF receptor. It may also act in an IGF-independent manner by initiating intracellular signaling from a cell surface receptor, or by direct nuclear action, or both. The possibility of a membrane-bound receptor is strengthened by recent studies which have identified members of the transforming growth factor-beta receptor family as having a role, either directly or indirectly, in signaling from the cell surface by IGFBP-3. A number of growth factors and hormones stimulate the expression and secretion of cellular IGFBP-3, which then signals from the cell surface to bring about some of the effects attributed to the primary agents. Within the cell, the apoptosis-inducing tumor suppressor, p53, can also induce IGFBP-3 expression and secretion. Since IGFBP-3 upregulates the cell cycle inhibitor, p21(Waf1), and increases the ratio of proapoptotic to antiapoptotic members of the Bcl family, it appears to exert the same effects on major downstream targets of cell signaling as p53 does. The nuclear localization of IGFBP-3 has been described in a number of cell types. IGFBP-3 may act to import IGFs or other nuclear localization signal-deficient signaling molecules into the nucleus. It may also act directly in the nucleus by enhancing the activity of retinoid X receptor-alpha and thereby promote apoptosis. All of the above phenomena will be discussed with particular emphasis on the growth of breast cancer cells.