Activation of the insulin-like growth factor 1 signaling pathway by the antiapoptotic agents aurintricarboxylic acid and evans blue.

Aurintricarboxylic acid (ATA), an endonuclease inhibitor, prevents the death of a variety of cell types in culture. Previously we have shown that ATA, similar to insulin-like growth factor I (IGF-I), protected MCF-7 cells against apoptotic death induced by the protein synthesis inhibitor cycloheximide. Here we show that ATA and a polysulfonated aromatic compound, Evans blue (EB), similar to IGF-I, promote survival and increase proliferation of MCF-7 cells in serum-free culture medium. This may suggest a common signaling pathway shared by the aromatic polyanions and IGF-I. Therefore, the ability of these aromatic compounds to activate the signal transduction pathway of IGF-I was examined. We found that ATA and EB mimicked the IGF-I effect on tyrosine phosphorylation of the IGF-I receptor (IGF-IR) and its major substrates, insulin receptor substrate-1 (IRS-1) and IRS-2; induced the association of these substrates with phosphatidylinositol 3-kinase and Grb2; and activated Akt kinase and p42/p44 mitogen-activated protein kinases. ATA and EB competed for IGF-I binding to the IGF-IR. ATA was found to be selective for the IGF-IR, whereas EB also activated the insulin receptor. Upon fractionation of commercial ATA by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the survival of MCF-7 cells in the presence of cycloheximide, whereas fractions devoid of IRS phosphorylation activity had no survival ability. Taken together, these results suggest that the survival/proliferation-promoting effects of ATA and EB in MCF-7 cells are transduced via the IGF-IR signaling pathway.     

Activation of the phosphatidylinositol-3' kinase pathway and DNA synthesis by a mutant insulin-like growth factor I receptor lacking the NPXY motif

We have investigated the role of the NPXY motif in the insulin-like growth factor I receptor (IGF-IR) by focusing on the activation of the phosphatidylinositol-3' kinase (PI3-K) pathway and DNA synthesis following IGF-I stimulation. For this purpose, we established stable R-cell lines, which are deficient in endogenous IGF-IR, and express human IGF-IR lacking the whole NPEY(950) sequence (DeltaNPEY). The DeltaNPEY cells showed an apparent autophosphorylation of IGF-IR, albeit with reduced sensitivity to stimulation compared with cells expressing similar levels of wild-type IGF-IR. Activation of insulin receptor substrate (IRS)-1 and IRS-2 was severely impaired in DeltaNPEY cells even at high concentrations of IGF-I. However, recruitment of p85, a regulatory subunit of PI3-K, to activated IRS-2 was similar between the cell lines, but recruitment of p85 to IRS-1 was reduced in DeltaNPEY cells. Essentially similar levels of p85- or phosphotyrosine-associated PI3-K and Akt activities were observed between the cell lines, although the sensitivity to stimulation was reduced in DeltaNPEY cells. Activation of extracellular signal-regulated kinase and DNA synthesis were virtually unaffected by the mutation, in terms of both sensitivity to stimulation and responsiveness. DNA synthesis was completely inhibited by the PI3-K inhibitor, LY294002. These results indicate that the IGF-IR is able to activate the PI3-K pathway and induce DNA synthesis in a normal fashion without the NPXY motif when the receptor is fully activated.     

Inducible expression of dominant negative insulin-like growth factor I receptor in MCF-7 breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) is expressed in many cell types and is critical for normal growth and development. In the healthy mammary gland, the role of IGF-IR is not fully elucidated. However, IGF-IR, which is primarily expressed in the mammary epithelial cells, is known to play an obligatory role in cellular transformation, facilitating the progression to breast cancer. We have utilized the tetracycline regulatory (tet-on) system to generate an in vitro model system to allow us to further investigate IGF-I/IGF-IR function in mammary epithelial cells. A plasmid construct containing a mutant IGF-I receptor (IGF-IR-DN) fused to the tetracycline operator (tetOPh_CMV-IGF-IR-DN) was stably transfected into MCF-7 human breast cancer cells. The conditional regulation of the IGF-IR-DN gene expression was studied in four independent clonal lines. The translated IGF-IR-DN protein was detected only in the stably transfected doxycycline-induced cells, and its expression was up-regulated (three- to sixfold) following induction. IGF-I stimulated cell proliferation diminished (twofold) in doxycycline-induced cells compared to uninduced cells, demonstrating that the transgene construct was functional and ruling out any pleiotropic effect that may be attributed to doxycycline. Interestingly, autophosphorylation of the IGF-IR and phosphorylation of the downstream substrate, insulin receptor substrate-1 (IRS-1), was not inhibited in doxycycline/IGF-I treated cells, suggesting the possibility that activation of downstream substrates other than the IRS-1 may be critical for optimal cell proliferation. This novel in vitro model should allow us to more directly examine the role of IGF-I/IGF-IR signaling and function in mammary epithelial cells.     

Deletion of the pleckstrin and phosphotyrosine binding domains of insulin receptor substrate-2 does not impair its ability to regulate cell proliferation in myeloid cells

32D IGF-I receptor (IR) cells are IL-3-dependent myeloid cells that can be induced to differentiate into granulocytes by IGF-I. Like the parental 32D cells, 32D IGF-IR cells do not express the insulin receptor substrate (IRS)-1 or IRS-2. We investigated the effect of ectopic expression of IRS-2 in 32D IGF-IR cells. Expression in these cells of a wild-type IRS-2 inhibits IGF-I-induced differentiation, and the cells grow indefinitely in the absence of IL-3. We also investigated the effect of a mutant IRS-2 lacking both the pleckstrin (PH) and the phosphotyrosine-binding (PTB) domains, which are known to bind to the IR. The partial differentialPHPTB IRS-2 is fully as capable as the wild-type IRS-2 (and wild-type IRS-1) to stimulate the growth and inhibit the differentiation of 32D IGF-IR cells. In contrast, an IRS-1 protein lacking the same PH and PTB domains is completely inactive in blocking differentiation and stimulating IL-3-independent growth of 32D IGF-IR cells. The partial differentialPHPTB IRS-2 protein is dependent for its effect on an activated IGF-IR, is cytoplasmic, binds to the beta-subunit of the IGF-IR, and requires for its action the presence of phosphatidylinositol 3-kinase binding sequences. These experiments show that the PH and PTB domains of IRS-2 (but not IRS-1) are dispensable for the IGF-I/IRS-2-mediated growth of 32D myeloid cells. Our results also indicate that IRS-2 (either wild type or partial differentialPHPTB) is capable of inhibiting the differentiation of 32D cells.     

Estrogen signaling via a linear pathway involving insulin-like growth factor I receptor, matrix metalloproteinases, and epidermal growth factor receptor to activate mitogen-activated protein kinase in MCF-7 breast cancer cells

We present an integrated model of an extranuclear, estrogen receptor-alpha (ERalpha)-mediated, rapid MAPK activation pathway in breast cancer cells. In noncancer cells, IGF-I initiates a linear process involving activation of the IGF-I receptor (IGF-IR) and matrix metalloproteinases (MMP), release of heparin-binding epidermal growth factor (HB-EGF), and activation of EGF receptor (EGFR)-dependent MAPK. 17beta-Estradiol (E2) rapidly activates IGF-IR in breast cancer cells. We hypothesize that E2 induces a similar linear pathway involving IGF-IR, MMP, HB-EGF, EGFR, and MAPK. Using MCF-7 breast cancer cells, we for the first time demonstrated that a sequential activation of IGF-IR, MMP, and EGFR existed in E2 and IGF-I actions, which was supported by evidence that the selective inhibitors of IGF-IR and MMP or knockdown of IGF-IR all inhibited E2- or IGF-I-induced EGFR phosphorylation. Using the inhibitors and small inhibitory RNA strategies, we also demonstrated that the same sequential activation of the receptors occurred in E2-, IGF-I-, but not EGF-induced MAPK phosphorylation. Additionally, a HB-EGF neutralizing antibody significantly blocked E2-induced MAPK activation, further supporting our hypothesis. The biological effects of sequential activation of IGF-IR and EGFR on E2 stimulation of cell proliferation were also investigated. Knockdown or blockade of IGF-IR significantly inhibited E2- or IGF-I-stimulated but not EGF-induced cell growth. Knockdown or blockade of EGFR abrogated cell growth induced by E2, IGF-I, and EGF, indicating that EGFR is a downstream molecule of IGF-IR in E2 and IGF-I action. Together, our data support the novel view that E2 can activate a linear pathway involving the sequential activation of IGF-IR, MMP, HB-EGF, EGFR, and MAPK.     

Insulin-like growth factor I induces preferential degradation of insulin receptor substrate-2 through the phosphatidylinositol 3-kinase pathway in human neuroblastoma cells

Insulin receptor substrate (IRS) signaling is regulated through serine/threonine phosphorylation, with subsequent IRS degradation. This study examines the differences in IRS-1 and IRS-2 degradation in human neuroblastoma cells. SH-EP cells are glial-like, express low levels of the type I IGF-I receptor (IGF-IR) and IRS-2 and high levels of IRS-1. SH-SY5Y cells are neuroblast-like, with high levels of IGF-IR and IRS-2 but virtually no IRS-1. When stimulated with IGF-I, IRS-1 expression remains constant in SH-EP cells; however, IRS-2 in SH-SY5Y cells shows time- and concentration-dependent degradation, which requires IGF-IR activation. SH-EP cells transfected with IRS-2 and SH-SY5Y cells transfected with IRS-1 show that only IRS-2 is degraded by IGF-I treatment. When SH-EP cells are transfected with IGF-IR or suppressor of cytokine signaling, IRS-1 is degraded by IGF-I treatment. IRS-1 and -2 degradation are almost completely blocked by phosphatidylinositol 3-kinase inhibitors and partially by proteasome inhibitors. In summary, 1) IRS-2 is more sensitive to IGF-I-mediated degradation; 2) IRS degradation is mediated by phosphatidylinositol 3-kinase and proteasome sensitive pathways; and 3) high levels of IGF-IR, and possibly the subsequent increase in Akt phosphorylation, are required for efficient IRS degradation.     

Dysregulation of IGF-I signaling in uterine leiomyoma.

IGF-I expression has been observed in human uterine leiomyomas. To examine whether autocrine IGF-I signaling plays a role in the growth of these tumors, we used an animal model of uterine leiomyoma (the Eker rat) to investigate regulation of IGF-I and the IGF-I receptor (IGF-IR) expression in tumors and normal myometrium. During the normal estrous cycle, myometrial IGF-I expression peaked on the day of proestrus when the rate of proliferation in this tissue is greatest. In leiomyomas, the expression of IGF-I was increased 7.5-fold compared with the age-matched normal tissue. The level of IGF-IR mRNA in both tumor and non-tumor tissues was found to inversely correlate with that of IGF-I. Changes observed in IGF-I signaling components correlated with the activation state of the signal-transducing protein insulin receptor substrate-1 (IRS-1). During diestrus and proestrus when IGF-I levels were increasing, tyrosine phosphorylation of IRS-1 was increased up to 5.7-fold in the normal myometrium relative to estrus, when IGF-I levels were the lowest. Additionally, IRS-1 phosphorylation was 4-fold greater in leiomyomas relative to age-matched normal myometrium. Autocrine stimulation of the IGF-IR may, therefore, play a role in regulating the normal growth of the myometrium, and dysregulation of IGF-I signaling could contribute to the neoplastic growth of uterine leiomyomas.     

Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth

Development of acquired resistance to antiestrogens is a major clinical problem in endocrine treatment of breast cancer patients. The IGF system plays a profound role in many cancer types, including breast cancer. Thus, overexpression and/or constitutive activation of the IGF-I receptor (IGF-IR) or different components of the IGF-IR signaling pathway have been reported to render breast cancer cells less estrogen dependent and capable of sustaining cell proliferation in the presence of antiestrogens. In this study, growth of the antiestrogen-sensitive human breast cancer cell line MCF-7 was inhibited by treatment with IGF-IR-neutralizing antibodies. In contrast, IGF-IR-neutralizing antibodies had no effect on growth of two different antiestrogen-resistant MCF-7 sublines. A panel of antiestrogen-resistant cell lines was investigated for expression of IGF-IR and either undetectable or severely reduced IGF-IR levels were observed. No increase in insulin receptor substrate 1 (IRS-1) or total PKB/Akt (Akt) was detected in the resistant cell lines. However, a significant increase in phosphorylated Akt (pAkt) was found in four of six antiestrogen-resistant cell lines. Overexpression of pAkt was associated with increased Akt kinase activity in both a tamoxifen- and an ICI 182,780-resistant cell line. Inhibition of Akt phosphorylation by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin or the Akt inhibitor SH-6 (structurally modified phosphatidyl inositol ether liquid analog PIA 6) resulted in a more pronounced growth inhibitory effect on the antiestrogen-resistant cells compared with the parental cells, suggesting that signaling via Akt is required for antiestrogen-resistant cell growth in at least a subset of our antiestrogen-resistant cell lines. PTEN expression and activity was not decreased in cell lines overexpressing pAkt. Our data demonstrate that Akt is a target for treatment of antiestrogen-resistant breast cancer cell lines and we suggest that antiestrogen-resistant breast cancer patients may benefit from treatment targeted to inhibit Akt signaling.     

Growth factor-specific regulation of insulin receptor substrate-1 expression in MCF-7 breast carcinoma cells: effects on the insulin-like growth factor signaling pathway

IGFs are potent mitogens that play a crucial role in cell proliferation and/or differentiation and tumorigenesis. Insulin receptor substrate-1 (IRS-1) is a key protein in the IGF signaling pathway in the estrogen-dependent MCF-7 breast carcinoma cell line. In this study, three growth factors [fibroblast growth factor (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)] were tested for their ability to modulate IRS-1 protein expression and the IGF-I signaling pathway. FGF and, to a lesser extent, EGF were found to increase IRS-1 protein, whereas PDGF had no effect. This indicates that growth factors can specifically modulate IRS-1 protein content. The increases provoked by EGF and FGF were dependent on the MAPK signaling pathway but independent of phosphatidylinositol 3-kinase (PI 3-kinase) signaling and required de novo protein synthesis. We noted that the kinetics of MAPK activation was continuous in response to FGF but transient in response to EGF. In addition, transfection of cells with a constitutively active form of MAPK kinase, which results in continuous MAPK activity, increased IRS-1 expression. Taken together, these results suggest that stimulation of IRS-1 expression was therefore stronger when MAPK activity was sustained. Pretreatment of cells with EGF, FGF, or PDGF for 24 h reduced IGF-I-induced tyrosine phosphorylation per molecule of IRS-1. However, IGF-I-induced PI 3-kinase activity was decreased by 24 h of pretreatment with EGF or PDGF but not with FGF. Our results therefore demonstrate that different growth factors are capable of specifically modulating the IGF-I signaling via IRS-1. They further suggest that the FGF-induced increase in IRS-1 counterbalances the inhibition of IRS-1 tyrosine phosphorylation to allow normal stimulation of IGF-I-induced PI 3-kinase activity.     

Polymorphic variants of insulin-like growth factor I (IGF-I) receptor and phosphoinositide 3-kinase genes affect IGF-I plasma levels and human longevity: cues for an evolutionarily conserved mechanism of life span control

Current literature indicates that abrogation of the IGF-I response pathway affects longevity in Caenorhabditis elegans, and that the down-regulation of IGF-I gene expression is associated with an extension of the life span in mice. In this paper we tested the hypothesis that polymorphic variants of IGF-I response pathway genes, namely IGF-IR (IGF-I receptor; G/A, codon 1013), PI3KCB (phosphoinositol 3-kinase; T/C, -359 bp; A/G, -303 bp), IRS-1 (insulin receptor substrate-1; G/A, codon 972), and FOXO1A (T/C, +97347 bp), play a role in systemic IGF-I regulation and human longevity. The major finding of this investigation was that subjects carrying at least an A allele at IGF-IR have low levels of free plasma IGF-I and are more represented among long-lived people. Moreover, genotype combinations at IGF-IR and PI3KCB genes affect free IGF-I plasma levels and longevity. These findings represent the first indication that free IGF-I plasma levels and human longevity are coregulated by an overlapping set of genes, contributing to the hypothesis that the impact of the IGF-I/insulin pathway on longevity is a property that has been evolutionarily conserved throughout the animal kingdom.     

Antiinsulin receptor autoantibodies induce insulin receptors to constitutively associate with insulin receptor substrate-1 and -2 and cause severe cell resistance to both insulin and insulin-like growth factor I.

We report here that antiinsulin receptor (anti-IR) autoantibodies (AIRs) from a newly diagnosed patient with type B syndrome of insulin resistance induced cellular resistance not only to insulin but also to insulin-like growth factor I (IGF-I) for the stimulation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activities and of glycogen and DNA syntheses. The molecular mechanisms of this dual resistance were investigated. Patient AIRs bound the IR at the insulin-binding site and caused insulin resistance at the IR level by inducing a 50% decrease in cell surface IRs and a severe defect in the tyrosine kinase activity of the residual IRs, manifested by a loss of insulin-stimulated IR autophosphorylation and IR substrate-1 (IRS-1)/IRS-2 phosphorylation. In contrast, cell resistance to IGF-I occurred at a step distal to IGF-I receptors (IGF-IRs), as AIRs altered neither IGF-I binding nor IGF-I-induced IGF-IR autophosphorylation, but inhibited the ability of IGF-IRs to mediate tyrosine phosphorylation of IRS-1 and IRS-2 in response to IGF-I. Coimmunoprecipitation assays showed that in AIR-treated cells, IRs, but not IGF-IRs, were constitutively associated with IRS-1 and IRS-2, strongly suggesting that AIR-desensitized IRs impeded IGF-I action by sequestering IRS-1 and IRS-2. Accordingly, AIRs had no effect on the stimulation of mitogen-activated protein kinase activity or DNA synthesis by vanadyl sulfate, FCS, epidermal growth factor, or platelet-derived growth factor, all of which activate signaling pathways independent of IRS-1/IRS-2. Thus, AIRs induced cell resistance to both insulin and IGF-I through a novel mechanism involving a constitutive and stable association of IRS-1 and IRS-2 with the IR.     

Substrate-bound insulin-like growth factor (IGF)-I-IGF binding protein-vitronectin-stimulated breast cell migration is enhanced by coactivation of the phosphatidylinositide 3-Kinase/AKT pathway by alphav-integrins and the IGF-I receptor

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.     

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.     

Insulin-like growth factor-I-dependent signal transduction pathways leading to the induction of cell growth and differentiation of human neuroblastoma cell line SH-SY5Y: the roles of MAP kinase pathway and PI 3-kinase pathway

IGF-I regulates cell growth, differentiation, and survival in many cultured nerve cell lines. The present study was undertaken in the human neuroblastoma cell line, SH-SY5Y, to elucidate whether there are differences in the IGF-dependent signal transduction pathways that stimulate proliferation compared to those that induce differentiation. Quiescent SH-SY5Y cells were treated with IGF-I in the presence or absence of PD98059 (an inhibitor of MEK, a MAP kinase kinase) or LY294002 (an inhibitor of PI 3-kinase). Cell growth was assessed by measuring [3H]thymidine incorporation into DNA and cell number. Cell differentiation was assessed by measuring mRNA levels of NPY and neurite outgrowth. IGF-I both induced cell proliferation and differentiation. It stimulated tyrosine phosphorylation of the type I IGF receptor (IGF-IR) beta-subunit, IRS-I, IRS-2, and Shc, and these changes were associated with activation of Erk and Akt. PD98059 inhibited activation of Erk and LY294002 repressed activation of Akt in response to IGF-I, but did not affect tyrosine phosphorylation of the IGF-IR, IRS-1, IRS-2, or Shc. Each PD98059 and LY294002 inhibited IGF-I-dependent cell proliferation in a concentration-dependent manner. In contrast, each of these inhibitors only partially depressed NPY gene expression induced by IGF-I and slightly inhibited IGF-I-mediated neurite outgrowth; however, when both PD98059 and LY294002 were present, IGF-I-dependent NPY gene expression and neurite outgrowth were abolished completely. These results suggest that in these nerve cells, 1) the IGF-I signals through the MAP kinase pathway and PI-3 kinase pathway are independently essential to induce IGF-I-dependent growth, and 2) alternate activation of the MAP kinase pathway and PI 3-kinase pathway is sufficient for the cells to undergo IGF-I-dependent differentiation.     

Epidermal growth factor-induced activation of the insulin-like growth factor I receptor in rat hepatocytes

Insulin-like growth factor I (IGF-I) plays a critical role in the induction of cell cycle progression and survival in many cell types. However, there is minimal IGF-I binding to hepatocytes, and a role for IGF-I in hepatocyte signaling has not been elucidated. The dynamics of IGF-I receptor (IGF-IR) activation were examined in freshly isolated rat hepatocytes. IGF-I did not activate the IGF-IR. However, des(1-3)IGF-I, which weakly binds IGF binding protein-3 (IGFBP-3), induced IGF-IR phosphorylation. IGFBP-3 surface coating was identified by confocal immunofluorescence microscopy. In contrast with the inactivity of IGF-I, epidermal growth factor (EGF) induced the tyrosine phosphorylation of the IGF-IR in parallel with EGF receptor phosphorylation. Transactivation of the IGF-IR by EGF was inhibited by tyrphostin I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR. Src kinase inhibitors pyrazolopyrimidine PP-1 and PP-2 inhibited transactivation of the IGF-IR by EGF. EGF stimulated the tyrosine phosphorylation of Src, and induced its association with the IGF-IR. EGF-induced phosphorylations of insulin-related substrate (IRS)-1, IRS-2, Akt, and p42/44 mitogen-activated protein kinases (MAPKs) were inhibited variably by I-Ome-AG538. In conclusion, the data show an EGF- and Src-mediated transactivation pathway for IGF-IR activation in hepatocytes, and indicate a role for the IGF-IR in hepatocyte intracellular signaling. The findings also show a role for IGFBP-3 in the inhibition of IGF-I signaling in hepatocytes.     

Insulin-like growth factor (IGF) binding protein-3 inhibits type 1 IGF receptor activation independently of its IGF binding affinity

Insulin-like growth factor binding proteins (IGFBPs) regulate the cellular actions of the IGFs owing to their strong affinities, which are equal to or stronger than the affinity of the type 1 IGF receptor (IGF-IR), the mediator of IGF signal transduction. We recently found that IGFBP-3 modulates IGF-I binding to its receptor via a different mechanism possibly involving conformational alteration of the receptor. We have now investigated the effects of IGFBP-3 on the initial steps in the IGF signaling pathway. MCF-7 breast carcinoma cells were preincubated with increasing concentrations of IGFBP-3 and then stimulated with IGF-I, des(1-3)IGF-I, or [Q(3)A(4)Y(15)L(16)]-IGF-I, the latter two being IGF-I analogs with intact affinity for the type 1 IGF receptor, but weak or virtually no affinity for IGFBPs. Stimulation of autophosphorylation of the receptor and its tyrosine kinase activity was dose-dependently depressed. At 2.5 nM, IGFBP-3 provoked more than 50% inhibition of the stimulation induced by 3 nM des(1-3)IGF-1 and, at 10 nM, more than 80% inhibition. Similar results were obtained with [Q(3)A(4)Y(15)L(16)]-IGF-I. Cross-linking experiments using iodinated or unlabeled IGFBP-3 and anti-IGF-IR antibodies indicated that the inhibitory effects do not involve direct interaction between IGFBP-3 and IGF-IR. The inhibition appeared to be specific to IGFBP-3, because IGFBP-1 and IGFBP-5 at 10 nM had no significant effect. Also, inhibition was restricted to the IGF receptor, because IGFBP-3 failed to inhibit the tyrosine kinase activity of the insulin receptor stimulated by physiological concentrations of insulin. Our results provide the first demonstration that IGFBP-3 can specifically modulate the IGF-I signaling pathway independently of its IGF-I-binding ability. They also reveal a regulatory mechanism specific to the type 1 IGF receptor, with no effect on insulin receptor activation.     

Interactions between IGF-I, estrogen receptor-α (ERα), and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

Understanding of the interactions between estradiol (E?) and IGF-I is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating noninterfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions, and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human GH plus epidermal growth factor, but E? did not cause an increase in the number of the IGF-IR.low cells compared to controls. The proliferation rate of IGF-IR.low cells was only reduced in response to E? compared to controls, whereas their basal and hormone-stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E?, without affecting control cells. Furthermore, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. In conclusion, suppressing IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK, which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.     

Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor-alpha

Recent data indicate that insulin-like growth factor I (IGF-I) may have a function in mediating the mitogenic effects of 17beta-estradiol (E2) in the uterus and in regulating the growth of uterine neoplasms. This study was designed to determine whether synthetic and plant-derived chemicals that interact with estrogen receptor-alpha (ERalpha) and elicit estrogenic responses also mimic E2 by activating the uterine IGF-I signaling pathway. Ovariectomized adult female mice were treated with both environmental and clinically relevant chemicals previously reported to display estrogenic and/or antiestrogenic properties, and their uteri were evaluated for an activated IGF-I signaling pathway. Diethylstilbestrol, 4-hydroxytamoxifen, the raloxifene analog LY353381, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), bisphenol A, and genistein were shown to mimic E2 in the uterus by increasing the level of IGF-I messenger RNA, inducing IGF-I receptor (IGF-IR) tyrosine phosphorylation, stimulating the formation of IGF-IR signaling complexes, and increasing both proliferating cell nuclear antigen expression and the number of mitotic cells in the epithelium. The dose of chemical necessary to activate IGF-I signaling varied, with the order of potency: E2 = diethylstilbestrol > LY353381 > 4-hydroxytamoxifen > genistein > HPTE > bisphenol A. Administration of the chemicals to ERalpha knockout mice did not activate IGF-IR, indicating that ERalpha is required for activation of uterine IGF-IR by these diverse chemicals. This study demonstrates that several chemicals shown previously to display estrogenic activities also mimic E2 by activating uterine IGF-I signaling.     

Effects of genistein and synergistic action in combination with eicosapentaenoic acid on the growth of breast cancer cell lines

Genistein, a prominent isoflavone in soy products, produced dose- and time-dependent in vitro growth inhibition at high concentrations (at least 185 microM) with an IC50 of 7.0-274.2 microM after 72 h incubation in four breast cancer cell lines (DD-762, Sm-MT, MCF-7 and MDA-MB-231) and one breast epithelial cell line (HBL- 100) of human and animal origin; it stimulated estrogen-receptor-positive MCF-7 cells at low concentrations (3.7 nM-37 microM). Genistein-exposed cells underwent apoptosis, confirmed by G2/M arrest followed by the appearance of a sub-G1 fraction in cell-cycle progression, and by a characteristic cell ultrastructure. The apoptosis cascade was due to up-regulation of Bax protein, down-regulation of Bcl-XL protein, and activation of caspase-3. Genistein acted in synergism with eicosapentaenoic acid (EPA), a fish oil component, on human breast cancer MCF-7 cells (genistein > 93.2 microM and EPA > 210.9 microM) and on MDA-MB-231 cells (genistein > 176.1 microM and EPA > 609.3 microM). Dietary intake of genistein in combination with EPA may be beneficial for breast cancer control.