The alphaVbeta3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the Src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor

The alphaVbeta3 integrin is an important determinant of IGF-I-stimulated receptor phosphorylation and biological actions. Blocking ligand occupancy of alphaVbeta3 with the distintegrin echistatin reduces IGF-I-stimulated receptor phosphorylation, and it inhibits cellular migration and DNA synthesis responses to IGF-I. We have shown that recruitment of the tyrosine phosphatase Src-homology 2-containing phosphotyrosine phosphatase-2 (SHP-2) to the IGF-I receptor (IGF-IR) is an important determinant of the duration of IGF-IR phosphorylation. These studies were undertaken to determine whether an alteration in the recruitment of SHP-2 to the receptor in the presence of echistatin could account for the decrease in receptor phosphorylation. Following an overnight exposure of smooth muscle cell cultures to echistatin, the addition of IGF-I was accompanied by rapid dephosphorylation of IGF-IR compared with cells exposed to media alone. This was associated with an increase in the rate of SHP-2 recruitment to the IGF-IR. In cells expressing a catalytically inactive form of SHP-2, prior exposure to echistatin had no effect on the rate of receptor dephosphorylation. In contrast to the usual physiologic situation in which following IGF-I exposure SHP-2 is recruited to IGF-IR via SHP-2 substrate-1 (SHPS-1) in the presence of echistatin, SHPS-1 was not used for SHP-2 recruitment. Our findings show that IRS-1 may substitute for SHPS-1 under these conditions. These results demonstrate that the activation state of alphaVbeta3 is an important regulator of the duration of IGF-IR phosphorylation and subsequent downstream signaling and that this regulation is mediated through changes in the subcellular localization of SHP-2.     

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.     

Inhibition of insulin-like growth factor-I signaling by ethanol in neuronal cells

BACKGROUND: Ethanol inhibits insulin-like growth factor-I receptor (IGF-IR) activation. However, the potency of ethanol for inhibition of the IGF-IR and other receptor tyrosine kinases varies considerably among different cell types. We investigated the effect of ethanol on IGF-I signaling in several neuronal cell types. METHODS: IGF-I signaling was examined in SH-SY5Y neuroblastoma cells, primary cultured rat cerebellar granule neurons, and rat NG-108 neuroblastoma x glioma hybrids. The tyrosine phosphorylation of IGF-IR, IRS-2, Shc, and p42/p44 MAP kinase (MAPK), and the association of Grb-2 with Shc, were examined by immunoprecipitations and Western blotting. RESULTS: IGF-I-mediated tyrosine phosphorylation of MAPK was inhibited by ethanol in all cell lines. IGF-IR autophosphorylation was markedly inhibited by ethanol in SH-SY5Y cells, was only mildly inhibited in cerebellar granule neurons, and was unaffected in rat NG-108 cells. In vitro tyrosine autophosphorylation of immunopurified IGF-IR obtained from all cell lines was inhibited by ethanol. There was also differential ethanol sensitivity of IRS-2 and Shc phosphorylation, and the association of Shc with IRS-2, among the different cell types. CONCLUSIONS: The findings demonstrate that IGF-I-mediated MAPK activation is a sensitive target of ethanol in diverse neuronal cell types. The data are consistent with ethanol-induced inhibition of IGF-IR activity, although the extent of IGF-IR tyrosine autophosphorylation per se is a poor marker of the inhibitory action of ethanol on this receptor. Furthermore, despite uniform inhibition of MAPK in the different neuronal cell types, tyrosine phosphorylation of proximal mediators of the IGF-IR are differentially inhibited by ethanol.     

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.     

Essential role of insulin-like growth factor I receptor in insulin-induced fetal brown adipocyte differentiation

To define the specific role of IGF-I receptor (IGF-IR) in adipogenic and thermogenic differentiation of brown adipocytes during late fetal life, we have established immortalized brown adipocyte cell lines from fetuses of IGF-IR-deficient mice (IGF-IR(-/-)) as well as from wild-type mice (IGF-IR(+/+)). IGF-IR(-/-) cells showed an increased insulin sensitivity regarding insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation despite a substantial reduction in IRS-1 protein content. Furthermore, insulin-induced total and IRS-1-associated phosphatidylinositol 3-kinase activities were augmented in IGF-IR-deficient cells compared with wild-type cells. Downstream phosphatidylinositol 3-kinase activation of Akt, but not p70s6 kinase, were elicited at lower doses of insulin in IGF-IR(-/-) brown adipocytes. Activation of protein kinase Czeta by insulin was similar in both cell types as was insulin-induced glucose uptake. Treatment of wild-type brown adipocytes with insulin for 12 h up-regulated fatty acid synthase (FAS) and adipocyte determination and differentiation (ADD1/SREBP) mRNAs; this effect was impaired in the absence of IGF-IR. At the protein level, insulin increased FAS content and the amount of the mature form of adipocyte determination and differentiation (ADD1/SREBP) in the nucleus in wild-type cells, but not in IGF-IR(-/-) cells. Furthermore, 24 h of insulin stimulation induced the expression of both uncoupling protein-1 and CCAAT/enhancer-binding protein alpha (C/EBPalpha) in wild-type brown adipocytes; these effects were abolished in IGF-I-R(-/-) cells. Retrovirus-mediated reexpression of peroxisomal proliferator-activated receptor gamma (PPARgamma) in IGF-IR(-/-) brown adipocytes could overcome FAS mRNA impairment, bypassing insulin signaling. However, insulin further increased FAS mRNA expression in C/EBPalpha-IGF-IR(-/-) cells, but not in PPARgamma-IGF-IR(-/-) cells. In addition, fetal brown adipocytes lacking IGF-IR up-regulated uncoupling protein-1 expression in the absence of insulin when PPARgamma, but not C/EBPalpha, was overexpressed. These data provide strong evidence for a critical role of IGF-IR in the differentiation of the brown adipocyte phenotype in fetal life; this effect is mimicked by PPARgamma in an insulin-independent manner.     

1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.     

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.     

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.     

Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle

Long-term denervation is associated with insulin resistance. To investigate the molecular bases of insulin resistance, the downstream signaling molecules of insulin receptor including insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI 3-K) were examined in skeletal muscle of rats after 7 days of denervation. Long-term denervation attenuated insulin-stimulated activation of the initial steps of the intracellular signaling pathway. Insulin-stimulated tyrosine phosphorylation of insulin receptor was reduced to 36% (P < .005), as was the phosphorylation of IRS-1 to 34% (P < .0001) of control. While insulin receptor protein level was unchanged, the protein expression of IRS-1 was significantly decreased in denervated muscles. Insulin-stimulated percent tyrosine phosphorylation of IRS-1, normalized to the IRS-1 protein expression, was also reduced to 55% (P < .01) of control in denervated muscle. Denervation caused a decline in the insulin-induced binding of p85 regulatory subunit of PI 3-K to IRS-1 to 61% (P < .001) and IRS-1-associated PI 3-K activity to 57% (P < .01). These results provide evidence that long-term denervation results in insulin resistance because of derangements at multiple points, including tyrosine phosphorylation of insulin receptor and its downstream signaling molecule, IRS-1, protein expression of IRS-1, and activation of PI 3-K.     

Association of the p85 regulatory subunit of phosphatidylinositol 3- kinase with an essential erythropoietin receptor subdomain

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.     

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.     

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.     

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.     

Abnormalities of insulin-like growth factor-I signaling and impaired cell proliferation in osteoblasts from subjects with osteoporosis

IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.     

Control of cell size through phosphorylation of upstream binding factor 1 by nuclear phosphatidylinositol 3-kinase

The insulin-like growth factor I/insulin receptor substrate 1 axis controls, in a nonredundant way, ≈50% of cell and body size in animals from Drosophila to mice and in cells in culture. Although other factors may also intervene, cell size is strongly dependent on ribosome biogenesis, which is under the control of RNA polymerase I activity. We have previously shown that insulin receptor substrate 1 (IRS-1) translocates to the nuclei and nucleoli, where it binds to the upstream binding factor (UBF) 1, a regulator of RNA polymerase I activity. Activation of UBF1 requires its phosphorylation. However, IRS-1 is not a kinase, and we searched for an intermediate kinase that can phosphorylate UBF1. We demonstrate here that IRS-1 binds also to the phosphatidylinositol 3-kinase (PI3-K) subunits in nuclear extracts, and that the p110 subunit of PI3-K directly phosphorylates and activates UBF1, an exclusively nucleolar protein. The interaction of IRS-1, PI3-K, and UBF1 in the nucleoli provides one of the mechanisms for the effects of IRS-1 on cell and body size.     

Blocking ligand occupancy of the αVβ3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells

Blocking αVβ3 integrin occupancy results in attenuation of the cellular migration response to insulin-like growth factor I (IGF-I). To determine whether integrin antagonists alter other IGF-I-stimulated biologic actions, quiescent smooth muscle cells (SMCs) were exposed to echistatin and their ability to respond to IGF-I was determined. Echistatin (10⁻⁷ M) inhibited IGF-I-stimulated DNA synthesis by 80%, and the protein synthesis response also was inhibited. Therefore blocking occupancy of αVβ3 inhibited multiple target cell actions of IGF-I. To determine whether blocking αVβ3 occupancy could alter IGF-I receptor-mediated signal transduction, the ability of IGF-I to stimulate phosphorylation of insulin receptor substrate-1 (IRS-1) was analyzed. A 10-min exposure to 100 ng/ml of IGF-I resulted in a substantial increase in phosphorylated IRS-1, and echistatin (10⁻⁷ M) blocked the IGF-I-induced IRS-1 phosphorylation response. Echistatin also attenuated downstream signaling because the capacity of the p85 subunit of phosphatidylinositol-3 kinase (PI-3 kinase) to bind to IRS-1 was blocked. In contrast, exposure of SMCs to vitronectin (1.0 μg/cm²) or thrombospondin (0.25 μg/cm²), two known ligands for αVβ3, resulted in enhancement of the IGF-I-stimulated IRS-1 response. To determine whether these effects were caused by alterations in receptor kinase activity, the IGF-I receptor was immunoprecipitated and then analyzed for phosphotyrosine. Echistatin (10⁻⁷ M) significantly reduced IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor β subunit. We conclude that occupancy of the αVβ3 integrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor and phosphorylate IRS-1. Activation of the αVβ3 receptor results in an interaction with the IGF-I signal transduction pathway, which modulates SMCs responsiveness to IGF-I.     

Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells.

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These findings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (ii) insulin and IGF-I stimulation of hematopoietic cell lines leads to the phosphorylation of a substrate that may be related to but is not identical to IRS-1.     

Role of Akt in growth and survival of PANC-1 pancreatic cancer cells.

INTRODUCTION: Akt is involved in different cellular processes such as cell growth, cell differentiation, and anti-apoptosis. AIMS: To investigate the role of Akt in cell growth and survival in PANC-1 pancreatic cancer cells. METHODOLOGY AND RESULTS: Insulin-like growth factor (IGF)-I induced Akt activation in a dose-dependent manner and stimulated anchorage-dependent and anchorage-independent cell growth of PANC-1 cells. In PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, anchorage-dependent and anchorage-independent cell growth was significantly reduced in the presence or absence of IGF-I compared with cells infected with adenovirus vectors carrying wild-type Akt, although IGF-I significantly stimulated cell growth in both transfected cell lines. Conversely, in PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, typical DNA laddering was undetectable in DNA fragmentation assay, and DNA 3;-OH reactivity was not detected in TUNEL assay. We then examined the role of phosphatidylinositol 3-kinase (PI3-K), an upstream mediator of Akt, on cell survival. In PANC-1 cells infected with adenovirus vector carrying a deletion mutant of the 85-kDa regulatory subunit of PI3-K and in cells treated with PI3-K inhibitor wortmannin, typical DNA laddering was evident in DNA fragmentation assay. In TUNEL assay, nuclear condensation and DNA 3;-OH reactivity was observed in approximately 30% of these cells. CONCLUSION: The present results indicate that Akt is implicated in cell growth, but not in survival in PANC-1 cells. These results suggest that there may be an alternative survival signal cascade from PI3-K in PANC-1 cells.     

Lipid rafts are required for Kit survival and proliferation signals

In addition to its physiologic role as central regulator of the hematopoietic and reproductive systems, the Kit receptor tyrosine kinase (RTK) is pathologically overexpressed in some forms of leukemia and constitutively activated by oncogenic mutations in mast-cell proliferations and gastrointestinal stromal tumors. To gain insight into the general activation and signaling mechanisms of RTKs, we investigated the activation-dependent dynamic membrane distributions of wild-type and oncogenic forms of Kit in hematopoietic cells. Ligand-induced recruitment of wild-type Kit to lipid rafts after stimulation by Kit ligand (KL) and the constitutive localization of oncogenic Kit in lipid rafts are necessary for Kit-mediated proliferation and survival signals. KL-dependent and oncogenic Kit kinase activity resulted in recruitment of the regulatory phosphatidylinositol 3-kinase (PI3-K) subunit p85 to rafts where the catalytical PI3-K subunit p110 constitutively resides. Cholesterol depletion by methyl-beta-cyclodextrin prevented Kit-mediated activation of the PI3-K downstream target Akt and inhibited cellular proliferation by KL-activated or oncogenic Kit, including mutants resistant to the Kit inhibitor imatinib-mesylate. Our data are consistent with the notion that Kit recruitment to lipid rafts is required for efficient activation of the PI3-K/Akt pathway and Kit-mediated proliferation.     

Genistein enhances insulin-like growth factor signaling pathway in human breast cancer (MCF-7) cells

Physiological concentration of genistein, a natural isoflavonoid phytoestrogen, stimulates human breast cancer (MCF-7) cells proliferation. In this study, we hypothesize that low concentration of genistein mimics the action of 17beta-estradiol in stimulation of MCF-7 cell growth by enhancement of IGF-I signaling pathway. Genistein, at 1 microM, stimulated the growth of MCF-7 cells. Cell cycle analysis showed that 1 micro M genistein significantly increased the S phase and decreased the G0G1 phase of MCF-7 cells. The protein and mRNA expression of IGF-I receptor (IGF-IR) and insulin receptor substrate (IRS)-1, but not Src homology/collagen protein, increased in response to 1 microM genistein in a time-dependent manner. These effects could be completely abolished by cotreatment of MCF-7 cells with estrogen antagonist ICI 182780 (1 microM) and tamoxifen (0.1 microM). Our results also showed that genistein induction of IGF-IR and IRS-1 expression resulted in enhanced tyrosine phosphorylation of IGF-IR and IRS-1 on IGF-I stimulation. Taken together, these data provide the first evidence that the IGF-IR pathway is involved in the proliferative effect of low-dose genistein in MCF-7 cells.