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.     

Role of the integrin alphaVbeta3 in mediating increased smooth muscle cell responsiveness to IGF-I in response to hyperglycemic stress.

Under usual conditions, the role of IGF-I in vascular cell types is to maintain cellular protein synthesis and cell size, and even excess IGF-I does not stimulate proliferation. In pathophysiologic states, such as hyperglycemia, smooth muscle cells (SMC) dedifferentiate and change their responsiveness to IGF-I. During hyperglycemia IGF-I stimulates both SMC migration and proliferation. Our laboratory has investigated the molecular mechanism by which this change is mediated. During hyperglycemia SMC secrete increased concentrations of thrombospondin, vitronectin and osteopontin, ligands for the integrin alphaVbeta3. Activation of alphaVbeta3 stimulates recruitment of a tyrosine phosphatase, SHP-2. Exposure of SMC to IGF-I results in phosphorylation of the transmembrane protein, SHPS-1, which provides a docking site for alphaVbeta3-associated SHP-2. After IGF-I stimulation SHP-2 associates with Src kinase, which associates with the signaling protein Shc. Src phosphorylates Shc, resulting in activation of MAP kinases, which are necessary both for stimulation of cell proliferation and migration. Blocking activation of alphaVbeta3 results in an inability of IGF-I to stimulate Shc phosphorylation. Under conditions of normoglycemia, there are insufficient alphaVbeta3 ligands to recruit SHP-2, and no increase in Shc phosphorylation can be demonstrated in SMC. In contrast, if alphaVbeta3 ligands are added to cells in normal glucose, the signaling events that are necessary for Shc phosphorylation can be reconstituted. Therefore when SMC are exposed to normal glucose they are protected from excessive stimulation of mitogenesis by IGF-I. With hyperglycemia there is a marked increased in alphaVbeta3 ligands and Shc phosphorylation in response to IGF-I is sustained. These findings indicate that in SMC hyperglycemic stress leads to altered IGF-I signaling, which allows the cells to undergo a mitogenic response, and which may contribute to the development of atherosclerosis.     

Minireview: Integral membrane proteins that function coordinately with the insulin-like growth factor I receptor to regulate intracellular signaling

Integral membrane proteins that are present on cell surfaces bind to extracellular ligands, and this binding influences multiple cellular processes. Three cell surface proteins, alpha V beta 3 integrin, integrin associated protein, and SHPS-1, have been shown to modulate both IGF-I receptor-linked signaling and cellular growth and migration responses that are stimulated by IGF-I. Ligand occupancy of these three proteins influences the recruitment of the phosphatase SHP-2 to the IGF-I receptor and thereby modulates the duration of IGF-I receptor tyrosine phosphorylation. In addition, changes in ligand occupancy of these three integral membrane proteins can regulate the transfer of SHP-2 phosphatase to downstream signaling molecules, which is also required for stimulation of cell migration and DNA synthesis by IGF-I. Determination of the spectrum of ligands for these three integral membrane proteins and the mechanisms by which each ligand functions to alter IGF-I signaling are important objectives of future research.     

Recruitment of the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase-2 to the p85 subunit of phosphatidylinositol-3 (PI-3) kinase is required for insulin-like growth factor-I-dependent PI-3 kinase activation in smooth muscle cells

IGF-I stimulates smooth muscle cell (SMC) migration and the phosphatidylinositol-3 (PI-3) kinase pathway plays an important role in mediating the IGF-I-induced migratory response. Prior studies have shown that the tyrosine phosphatase Src homology 2 domain tyrosine phosphatase (SHP)-2 is necessary to activate PI-3 kinase in response to growth factors and expression of a phosphatase inactive form of SHP-2 (SHP-2/C459S) impairs IGF-I-stimulated cell migration. However, the mechanism by which SHP-2 phosphatase activity or the recruitment of SHP-2 to other signaling molecules contributes to IGF-I stimulated PI-3 kinase activation has not been determined. SMCs that had stable expression of SHP-2/C459S had reduced cell migration and Akt activation in response to IGF-I, compared with SMC-expressing native SHP-2. Similarly in cells expressing native SHP-2, IGF-I induced SHP-2 binding to p85, whereas in cells expressing SHP-2/C459S, there was no increase. Because the C459S substitution results in loss of the ability of SHP-2 to disassociate from its substrates, making it inaccessible not only to p85 but also the other proteins, a p85 mutant in which tyrosines 528 and 556 were changed to phenylalanines was prepared to determine whether this would disrupt the p85/SHP-2 interaction and whether the loss of this specific interaction would alter IGF-I stimulated the cell migration. Substitution for these tyrosines in p85 resulted in loss of SHP-2 recruitment and was associated with a reduction in association of the p85/p110 complex with insulin receptor substrate-1. Cells stably expressing this p85 mutant also showed a decrease in IGF-I-stimulated PI-3 kinase activity and cell migration. Preincubation of cells with a cell-permeable peptide that contains the tyrosine556 motif of p85 also disrupted SHP-2 binding to p85 and inhibited the IGF-I-induced increase in cell migration. The findings indicate that tyrosines 528 and 556 in p85 are required for SHP-2 association. SHP-2 recruitment to p85 is required for IGF-I-stimulated association of the p85/p110 complex with insulin receptor substrate-1 and for the subsequent activation of the PI-3 kinase pathway leading to increased cell migration.     

Glucose-oxidized low-density lipoproteins enhance insulin-like growth factor I-stimulated smooth muscle cell proliferation by inhibiting integrin-associated protein cleavage

Prior published reports have demonstrated that glucose-oxidized low-density lipoproteins (g-OxLDL) enhance the proliferative response of vascular smooth muscle cells (SMC) to IGF-I. Our previous studies have determined that the regulation of cleavage of integrin-associated protein (IAP) by matrix-metalloprotease-2 (MMP-2) in diabetic mice in response to hyperglycemia is a key regulator of the response of SMC to IGF-I. Because chronic hyperglycemia enhances glucose-induced LDL oxidation, these studies were conducted to determine whether g-OxLDL modulates the response of SMC to IGF-I by regulating MMP-2-mediated cleavage of IAP. We determined that exposure of SMC to g-OxLDL, but not native LDL, was sufficient to facilitate an increase in cell proliferation in response to IGF-I. Exposure to an anti-CD36 antibody, which has been shown to inhibit g-OxLDL-mediated signaling, inhibited the effects of g-OxLDL on IGF-I-stimulated SMC proliferation. The effect of g-OxLDL could be attributed, in part, to an associated decrease in proteolytic cleavage of IAP leading to increase in the basal association between IAP and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1, which is required for IGF-I-stimulated proliferation. The inhibitory effect of g-OxLDL on IAP cleavage appeared to be due to its ability to decrease the amount of activated MMP-2, the protease responsible for IAP cleavage. In conclusion, these data provide a molecular mechanism to explain previous studies that have reported an enhancing effect of g-OxLDL on IGF-I-stimulated SMC proliferation.     

Hyperglycemia alters the responsiveness of smooth muscle cells to insulin-like growth factor-I

IGF-I stimulation of smooth muscle cell (SMC) migration and proliferation requires alphaVbeta3 ligand occupancy. We hypothesized that changes in the levels of extracellular matrix proteins induced by alterations in glucose concentrations may regulate the ability of SMCs to respond to IGF-I. IGF-I stimulated migration and proliferation of SMCs that had been maintained in 25 mM glucose containing media, but it had no stimulatory effect when tested using SMCs that had been grown in 5 mM glucose. IGF-I stimulated an increase in Shc phosphorylation and enhanced activation of the MAPK pathway in SMCs grown in 25 mM glucose, whereas in cells maintained in 5 mM glucose, IGF-I had no effect on Shc phosphorylation, and the MAPK response to IGF-I was markedly reduced. In cells grown in 25 mM glucose, the levels of alphaVbeta3 ligands, e.g. osteopontin, vitronectin, and thrombospondin, were all significantly increased, compared with cells grown in 5 mM glucose. The addition of these alphaVbeta3 ligands to SMCs grown in 5 mM glucose was sufficient to permit IGF-I-stimulated Shc phosphorylation and downstream signaling. Because we have shown previously that alphaVbeta3 ligand occupancy is required for IGF-I-stimulated Shc phosphorylation and stimulation of SMC growth, our data are consistent with a model in which 25 mM glucose stimulates increases in the concentrations of these extracellular matrix proteins, thus enhancing alphaVbeta3 ligand occupancy, which leads to increased Shc phosphorylation and enhanced cell migration and proliferation in response to IGF-I.     

Disruption of the association of integrin-associated protein (IAP) with tyrosine phosphatase non-receptor type substrate-1 (SHPS)-1 inhibits pathophysiological changes in retinal endothelial function in a rat model of diabetes

Aims/hypothesis  

We have previously shown that the association of integrin-associated protein (IAP) with tyrosine phosphatase non-receptor type substrate-1 (SHPS-1) regulates the response of cells, including osteoclasts, osteoblasts, smooth muscle and retinal endothelial cells, to IGF-I. Here we sought to: (1) determine whether the regulation of IGF-I responsiveness by the association of IAP with SHPS-1 is a generalised response of endothelial cells; (2) identify the mechanism by which this association contributes to changes in endothelial cell responses to IGF-I; and (3) determine whether inhibition of this association alters pathophysiological changes occurring in vivo.     

Suppression of AMPK activation via S485 phosphorylation by IGF-I during hyperglycemia is mediated by AKT activation in vascular smooth muscle cells

As a metabolic sensor, the serine/threonine protein kinase AMP-activated protein kinase (AMPK) promotes the adaptation of cells to signals arising from nutrients, hormones, and growth factors. The ability of IGF-I to stimulate protein synthesis is suppressed by AMPK, therefore, these studies were undertaken to determine whether IGF-I modulates AMPK activity. IGF-I dose-dependently suppressed phosphorylation of AMPK T172, and it stimulated AMPK S485 phosphorylation in vascular smooth muscle cells (VSMC). To determine whether stimulation of AMPK S485 phosphorylation was mediating this response, VSMC were transduced with a mutant AMPKα (AMPK S485A). Expression of this altered form inhibited the ability of IGF-I to suppress AMPK T172 activation, which resulted in inhibition of IGF-I-stimulated phosphorylation of P70S6 kinase. In contrast, expression of an AMPK S485D mutant resulted in constitutive suppression of AMPK activity and was associated with increased IGF-I-stimulated P70S6K phosphorylation and protein synthesis. The addition of a specific AKT inhibitor or expression of an AKT1 short hairpin RNA inhibited AMPK S485 phosphorylation, and it attenuated the IGF-I-induced decrease in AMPK T172 phosphorylation. Exposure to high glucose concentrations suppressed AMPK activity and stimulated S485 phosphorylation, and IGF-I stimulated a further increase in S485 phosphorylation and AMPK T172 suppression. We conclude that AMPK S485 phosphorylation negatively regulates AMPK activity by modulating the T172 phosphorylation response to high glucose and IGF-I. IGF-I stimulates S485 phosphorylation through AKT1. The results suggest that AMPK plays an inhibitory role in modulating IGF-I-stimulated protein synthesis and that IGF-I must down-regulate AMPK activity to induce an optimal anabolic response.     

The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.     

C-terminal Src kinase (CSK) modulates insulin-like growth factor-I signaling through Src in 3T3-L1 differentiation

IGF-I stimulates both proliferation and differentiation of adipocyte-precursor cells, preadipocytes in vivo and in vitro. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of MAPK and MAPK activation by IGF-I is mediated through the Src family of nonreceptor tyrosine kinases. In addition, we have shown that when 3T3-L1 cells reach growth arrest and are stimulated to differentiate, IGF-I can no longer activate the MAPK pathway. We hypothesized that the loss of IGF-I signaling to MAPK in differentiating 3T3-L1 cells is due to loss of IGF-I activation of Src family kinases. We measured c-Src kinase activity in cell lysates from proliferating, growth-arrested and differentiating 3T3-L1 cells. Src activity increased 2- to 4-fold in IGF-I-stimulated proliferating cells; however, IGF-I had a marginal affect on Src activity in growth-arrested cells and inhibited Src activity localized at the membrane in differentiating cells. C-terminal Src kinase (CSK), a ubiquitously expressed nonreceptor tyrosine kinase, negatively regulates the Src family kinases by phosphorylation of the Src C-terminal tyrosine. IGF-I decreased phosphorylation of the Src C-terminal tyrosine in proliferating cells and increased phosphorylation of this site in differentiating cells. IGF-I stimulated CSK kinase activity 2-fold in differentiating 3T3-L1 cells. An association between CSK and c-Src was detected by immunoprecipitation following IGF-I stimulation of differentiating but not proliferating 3T3-L1 cells. These results suggest that the loss of IGF-I downstream mitogenic signaling in differentiating 3T3-L1 cells is due to a change in IGF-I activation of c-Src and CSK may mediate the inactivation of c-Src by IGF-I in 3T3-L1 adipogenesis.     

Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation.

Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of alphaIIbbeta3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin alpha2beta1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP-/- mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of alpha2beta1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and alpha2beta1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.     

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.     

Synthetic alphaVbeta3 antagonists inhibit insulin-like growth factor-I-stimulated smooth muscle cell migration and replication.

Porcine aortic smooth cells respond to insulin-like growth factor-I (IGF-I) with increases in DNA synthesis and cell migration. Because ligand occupancy of the alphaVbeta3 integrin has been shown to be necessary for IGF-I to stimulate maximal increases in both processes, we determined whether synthetic alphaVbeta3 antagonists could inhibit IGF-I-stimulated actions on this cell type. Low-molecular-weight compounds that had been selected based on their ability to compete with vitronectin for binding to purified human alphaVbeta3 in vitro were analyzed for their ability to compete with 125I-kistrin (a known ligand for porcine alphaVbeta3) for binding to porcine alphaVbeta3. Nine compounds were screened, and five were found to be potent competitive inhibitors. The most potent compound, SC-69000, resulted in 88% competition at 10(-7) M and was nearly equipotent with echistatin. The compounds that were the most potent inhibitors of kistrin binding were tested for their capacity to inhibit the cell migration response to IGF-I. Three compounds caused between 81-88% inhibition of IGF-I-stimulated migration at 10(-7) M. To determine whether these compounds could inhibit other IGF-I-stimulated actions, their ability to inhibit IGF-I-stimulated [3H]-thymidine incorporation into DNA was analyzed. The four compounds that were the most potent inhibitors of cell migration also inhibited IGF-I-stimulated DNA replication. IGF-I stimulates the synthesis of IGF binding protein-5 by these cells. Preincubation with the four most active compounds also resulted in significant inhibition of the ability of IGF-I to stimulate IGF binding protein-5 synthesis. AlphaVbeta3 occupancy by the ligand vitronectin has been shown to enhance the capacity of IGF-I to activate its receptor tyrosine kinase. The four most active compounds were shown to inhibit IGF-I-stimulated IGF-I receptor autophosphorylation. These findings suggest that blockade of ligand occupancy of the alphaVbeta3 integrin globally inhibits several IGF-I-stimulated biologic actions and that synthetic inhibitors are very active in this regard. Because these compounds can be administered to whole animals, they should be very useful in determining whether blocking alphaVbeta3 occupancy in vivo results in alteration in responsiveness to IGF-I.     

The adapter protein GRB10 is an endogenous negative regulator of insulin-like growth factor signaling

The growth factor IGF-I is critical for normal human somatic growth and development. Growth factor receptor-bound protein (Grb)10 is a protein that interacts with the IGF-I receptor and may thus regulate IGF-I-stimulated growth. However, the role of endogenous Grb10 in regulating IGF-I action is not known. The objective of this study was to determine the function of endogenous Grb10 in IGF signaling responses. Using small interfering RNA, we demonstrate that knockdown of Grb10 enhances IGF-I-mediated phosphorylation of insulin receptor substrate proteins, Akt/protein kinase B, and ERK1/2 and leads to a corresponding increase in DNA synthesis. Although IGF-I receptor autophosphorylation normally correlates with receptor signaling, we demonstrate a decrease in IGF-I-stimulated receptor phosphorylation in Grb10 knockdown cells. Pretreatment of cells with the protein-tyrosine phosphatase inhibitor pervanadate partially reverses this effect of Grb10 knockdown on receptor phosphorylation, indicating that endogenous Grb10 may block phosphatase access to the activated IGF-I receptor. Marked small interfering RNA knockdown of Grb10 does not result in increased or decreased expression of the related proteins Grb7 or Grb14. As further evidence for Grb10 functional specificity, the recently identified Grb10 interacting GYF proteins are shown to interact specifically with Grb10 and not with Grb7 or Grb14, using yeast two-hybrid assays. We conclude that Grb10 functions as a specific endogenous suppressor of IGF-I-stimulated cell signaling and DNA synthesis. Modulation of the Grb10-IGF-I receptor pathway may represent a mechanism that regulates IGF-I-responsive cell and tissue growth.     

Ethyl pyruvate preserves IGF-I sensitivity toward mTOR substrates and protein synthesis in C2C12 myotubes

Bacterial infection decreases skeletal muscle protein synthesis via inhibition of the mammalian target of rapamycin (mTOR), a key regulator of translation initiation. To better define the mechanism by which muscle mTOR activity is decreased, we used an in vitro model of C2C12 myotubes treated with endotoxin [lipopolysaccharide (LPS)]and interferon (IFN)-γ to determine whether stable lipophilic pyruvate derivatives restore mTOR signaling. Myotubes treated with a combination of LPS and IFNγ down-regulated the phosphorylation of the mTOR substrates S6 kinase-1 and 4E binding protein-1. The phosphorylation of ribosomal protein S6 was decreased, whereas phosphorylation of elongation factor-2 was enhanced; all results consistent with defects in both translation initiation and elongation. LPS/IFNγ decreased protein synthesis 60% in myotubes. Treatment with methyl or ethyl pyruvate partially protected against the LPS/IFNγ-induced fall in mTOR signaling. The protective effect of ethyl and methyl pyruvate could not be replicated by an equimolar amount of sodium pyruvate. Although LPS/IFNγ treated myotubes were initially IGF-I responsive, prolonged exposure (≥ 17 h) resulted in IGF-I resistance at the level of mTOR despite normal IGF-I receptor phosphorylation. Ethyl pyruvate treatment restored IGF-I sensitivity as evidenced by the left shift in the IGF-I dose-response curve and maintained IGF-I responsiveness for a prolonged period of time. Ethyl pyruvate also restored IGF-I-stimulated protein synthesis in LPS/IFNγ-treated myotubes. Cotreatment with N-acetyl cysteine or ascorbic acid also preserved IGF-I sensitivity and mTOR activity. The data suggest that the combination of LPS and IFNγ inhibits mTOR activity and that prolonged exposure induces IGF-I resistance in myotubes. Lipophilic pyruvate derivatives and antioxidants show promise at rescuing mTOR activity and muscle protein synthesis by maintaining IGF-I sensitivity in this model.     

Cooperation between low density lipoproteins and IGF-I in the promotion of mitogenesis in vascular smooth muscle cells

Low density lipoproteins (LDL) are an independent risk factor for atherosclerosis and show synergism with some growth factors in vascular smooth muscle cell (VSMC) proliferation. IGF-I has mitogenic actions on VSMC, which, in turn, show enhanced expression of IGF-I and its receptor when exposed to hypercholesterolemic diets in vivo. To investigate the molecular basis of a possible interaction between LDL and the IGF-I signaling system in VSMC, we used A10 cells, where synergism between both factors in DNA synthesis was demonstrated. IGF-I activates phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated MAPK pathways in A10 cells, although insulin receptor substrate-1 (IRS-1)-associated PI3 kinase is more closely linked to IGF-I induced proliferation. LDL, in pathophysiological concentrations, affect the IGF-I signaling pathway at multiple levels: 1) they induce phosphorylation of IGF-I receptor beta and IRS-1 in a time- and dose-dependent manner; 2) they up-regulate IRS-1-associated PI3 kinase/Akt activation in response to IGF-I at early times; and 3) they show additive effects with IGF-I on extracellular signal-regulated MAPK 1/2 phosphorylation. These actions are not present in very low density lipoprotein treatments. Taken together, these results indicate specific cooperation between LDL and the IGF-I signaling pathways and may represent a more general mechanism through which proatherogenic lipoproteins modulate VSMC response to growth factors.     

Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)-1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I-induced increase in IRS-I-associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I-stimulated VSMC proliferation and reduced IGF-I-directed VSMC migration by approximately 60%. These results indicate that activation of PI3 kinase is required for both IGF-I-induced VSMC proliferation and migration.     

Role of insulin receptor substrate-4 in IGF-I-stimulated HEPG2 proliferation

BACKGROUNDS/AIMS: Insulin receptor substrate-4 (IRS-4) is a scaffold protein that mediates the actions of insulin-like growth factor-I (IGF-I). Its expression increases dramatically after partial hepatectomy (a liver regeneration model). Herein, we report IRS-4 expression in a human hepatoblastoma cell line (HepG2) and IGF-I-dependent IRS-4 tyrosine phosphorylation. METHODS: The role of IRS-4 in HepG2 proliferation was established by RNA interference (siRNA). After 72h of transfection with IRS-4 siRNA, we observed a specific reduction in IRS-4 expression. RESULTS: Depletion of IRS-4 levels decreased ERK phosphorylation, p70S6K phosphorylation and IGF-I-stimulated cell proliferation. Changes in ERK phosphorylation in IRS-4-depleted cells were independent of ras/raf/MEK1/2- and PI3K/Akt-cascades. IRS-4 down-regulation abolished IGF-I-, TPA- and IGF-I plus TPA-stimulated ERK and p70S6K activities. Our results suggest that PKC-epsilon mediates the effect of IRS-4 on ERK activity. Moreover, decreased IRS-4 levels diminished FBS- and IGF-I-stimulated HepG2 growth and cause stress fiber disruption in HepG2 cell line. CONCLUSIONS: Collectively, our data suggest that IRS-4 plays an important role in HepG2 proliferation/differentiation and exerts its actions through ERK and p70S6K activation in a ras/raf/MEK1/2- and PI3Kinase/Akt-independent manner and in a PKC-dependent way.