Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1.

The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, except that bovine IGF-1 showed a severalfold greater affinity for the monoclonal antibody than for either polyclonal antiserum. Membranes isolated from human placenta, sheep placenta and foetal-human liver were used as sources of cellular receptors. In human placental membranes, most of the binding of IGF-1 tracers could be attributed to a type-1 receptor, because insulin inhibited up to 65% of tracer binding. The other two tissues apparently contain only type-2 receptors, as evidenced by the very low potency of bovine or human IGF-1 in competing for binding with IGF-2 tracers and the absence of any competition by insulin. In competition for binding with labelled bovine or human IGF-1 to human placental membranes, bovine IGF-1 had a similar potency to human IGF-1, whereas bovine IGF-1 was more potent in binding studies with tissues rich in type-2 receptors. Rat IGF-2 was considerably less effective than human IGF-2 in competition for receptors on any of the membrane preparations.     

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.     

Hsp10 and Hsp60 suppress ubiquitination of insulin-like growth factor-1 receptor and augment insulin-like growth factor-1 receptor signaling in cardiac muscle: implications on decreased myocardial protection in diabetic cardiomyopathy

We have investigated the effects of two heat shock proteins, Hsp10 and Hsp60, on insulin-like growth factor-1 receptor (IGF-1R) signaling in cardiac muscle cells. Neonatal cardiomyocytes were transduced with Hsp10 or Hsp60 via adenoviral vector. Compared with the cells transduced with a control vector, overexpression of Hsp10 or Hsp60 increased the abundance of IGF-1R and IGF-1-stimulated receptor autophosphorylation. Thus, Hsp10 and Hsp60 overexpression increased the number of functioning receptors and amplified activation of IGF-1R signaling. IGF-1 stimulation of MEK, Erk, p90Rsk, and Akt were accordingly augmented. Transducing cardiomyocytes with antisense Hsp60 oligonucleotides reduced Hsp60 expression, decreased the abundance of IGF-1R, attenuated IGF-1R autophosphorylation, and suppressed the pro-survival action of IGF-1 in cardiomyocytes. Using cycloheximide to inhibit protein synthesis did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60. Additional experiments showed that Hsp10 and Hsp60 suppressed polyubiquitination of IGF-1 receptor. These data indicate that Hsp10 and Hsp60 can modulate IGF-1R signaling through post-translational modification. In animal models of diabetes, diabetic myocardium is associated with decreased abundance of Hsp60, increased ubiquitination of IGF-1R, and lower level of IGF-1R protein. Declined myocardial protection is a major feature of diabetic cardiomyopathy. These data suggest that decreased Hsp60 expression and subsequent decline of IGF-1R signaling may be a fundamental mechanism underlying the development of diabetic cardiomyopathy.     

EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells

The IGF/IGF-1R system, which includes the IGF, IGF-1R, and IGFBPs proteins, plays an important role in the development and growth of colorectal cancer. We previously reported that in the HT29 human colon cancer cell line EGCG, the major biologically active component of green tea, inhibits activation of the RTKs EGFR, HER2, and HER3, and that this is associated with inhibition of multiple downstream signaling pathways. Since IGF-1R is also a RTK, in this study we examined the effects of EGCG on the activity of IGF/IGF-1R system in human colon cancer cells. We found that the colon cancer cell lines Caco2, HT29, SW837, and SW480 express high levels of the IGF-1R receptor, and that both SW837 and SW480 cells display constitutive activation of this receptor. Treatment of SW837 cells with 20 microg/ml of EGCG (the IC50 concentration for growth inhibition) caused within 6 h a decrease in the phosphorylated (i.e., activated) form of the IGF-1R protein. At 12 h, there was a decrease in the levels of both IGF-1 protein and mRNA and within 3-6 h there was an increase in the levels of both IGFBP-3 protein and mRNA. The increased expression of the latter protein was sustained for at least 48 h. When SW837 cells were treated with EGCG for a longer time, i.e., 96 h, a very low concentration (1.0 microg/ml) of EGCG also caused inhibition of activation of IGF-1R, a decrease in the IGF-1 protein, and an increase in the IGFBP-3 protein. EGCG also caused a decrease in the levels of mRNAs that encode MMPs-7 and -9, proteins that proteolyze IGFBP-3. In addition, treatment with EGCG caused a transient increase in the expression of TGF-beta2, an inducer of IGFBP-3 expression. These findings expand the roles of EGCG as an inhibitor of critical RTKs involved in cell proliferation, providing further evidence that EGCG and related compounds may be useful in the chemoprevention or treatment of colorectal cancer.     

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.     

Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells.

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific (125)I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rbeta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Crystal structure of the Apo,unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.     

Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.     

Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.     

Growth regulation of human glioblastoma t98g cells by insulin-like growth factor-1 and its receptor

The interaction of insulin-like growth factors (IGFs) with the IGF-1 receptor is an important step in the control of cell proliferation and development. In particular, IGF-1 and IGF-2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum-free medium at 34°C, despite their secretion of substantial amounts of bioactive IGF-1. To be stimulated to divide, growth-arrested cells required the addition of platelet-derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum-free medium could also be obtained by shifting the cells to a temperature of 39.6°C. Treatment of growth-arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF-1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF-1 receptor allowed autonomous growth in serum-free medium at 34°C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF-1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF-1 receptor mRNA. These results demonstrate that the IGF-1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF-1 with its receptor regulates both autonomous and anchorage-independent growth of these cells. © 1994 wiley-Liss, Inc.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics

Because insulin-like growth factor-1 (IGF-1) and its receptor play a pivotal role in many cancers, it is an attractive target for the design of inhibitors. Here we present a new family of bioisostere inhibitors, based on the structure of AG 538. AG 538 is a substrate-competitive inhibitor of the IGF-1 receptor (IGF-1R), with an IC50 = 61 nM in a cell-free kinase assay (Blum, G., Gazit, A., and Levitzki, A. (2000) Biochemistry 39, 15705-15712). AG 538 is a low molecular weight compound containing two catechol rings, which are sensitive to oxidation in cells. We have therefore prepared and examined catechol bioisosteres of AG 538. These AG 538 bioisosteres possess similar biological properties to AG 538; they inhibit IGF-1R by a substrate-competitive mechanism and are non-competitive vis à vis ATP. They inhibit IGF-1R kinase activity in the sub-micromolar concentration range in cell-free assays. IGF-1 induced IGF-1R autophosphorylation; IRS-1 phosphorylation and protein kinase B activation are inhibited at a low micromolar concentration range when applied to intact cells. These inhibitors also block the formation of colonies in soft agar by prostate and breast cancer cells. The ability to replace catechol groups with a moiety that is more stable in cells may aid in developing non-catechol-containing substrate-competitive inhibitors targeted toward IGF-1R and possibly against other protein-tyrosine kinases.     

Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p<0.05 vs. controls), whereas it had no effect of PDGF-stimulated signaling pathways or cell growth. Both, IGF-1 and PDGF-BB, induced MT1-MMP expression, but only IGF-1-mediated MT1-MMP induction was inhibited by dec-CMK. Induction of MMP-2 by IGF-1 was inhibited by the PI3-kinase inhibitor wortmannin, but not by the MEK-inhibitor PD98059. Dec-CMK inhibited VSMC chemotaxis comparable to the effects of the MMP-inhibitor GM6001 (both p<0.05 vs. controls), supporting that MMPs are involved. In conclusion, this study demonstrates that targeting furin-like PCs and thus inhibiting IGF-1R activation is a novel target to inhibit IGF-1-mediated signaling and cell functions, such as IGF-1-induced MT1-MMP/MMP-2 in VSMCs.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.