Insulin-like Growth Factor Binding Protein-3 Binding Research Articles

Insulin-Like Growth Factor Binding Protein-3 Binds to Histone 3.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is an essential protein that regulates cellular processes such as cell proliferation, apoptosis, and differentiation. It is known to bind with several proteins to carry out various cellular functions. In this study, we report for the first time that IGFBP-3 is a histone 3 (H3) binding protein. Sub-cellular fractionation was performed to separate into cytosolic fraction, nucleic acid binding protein fraction and insoluble nuclear fraction. Using ligand blot analysis, we identified a ~15 kDa protein that can interact with IGFBP-3 in the insoluble nuclear fraction. The 15 kDa protein was confirmed as histone 3 by far-Western blot analysis and co-immunoprecipitation experiments. A dot-blot experiment further validated the binding of IGFBP-3 with H3. The intensity of IGFBP-3 on dot-blot showed a proportional increase with H3 concentrations between 2.33 pmol–37.42 pmol. Our results support the presence of protein-protein interaction between IGFBP-3 and H3. The physical binding between IGFBP-3 and H3 could indicate its yet another cellular role in regulating the chromatin remodeling for gene transcription.

Biochemical determinants of the IGFBP-3-hyaluronan interaction.

IGFBP-3, the most abundant IGFBP and the main carrier of insulin-like growth factor I (IGF-I) in the circulation, can bind IGF-1 with high affinity, which attenuates IGF/IGF-IR interactions, thereby resulting in antiproliferative effects. The C-terminal domain of insulin-like growth factor-binding protein-3 (IGFBP-3) is known to contain an 18-basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18-amino acid IGFBP-3 peptide is capable of binding either HA or HN with comparable affinities to the full-length IGFBP-3 protein and that IGFBP-3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP-3-HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP-3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP-3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP-3, suggesting that the basic 18-amino acid residue sequence of IGFBP-3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full-length protein. Removing N-linked oligosaccharides from CD44 increased its ability to compete with IGFBP-3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP-3-HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP-3 for binding HA.

New insights on the Intrinsic, Pro-Apoptotic Effect of IGFB3 in Breast Cancer.

New insights on the Intrinsic, Pro-Apoptotic Effect of IGFB3 in Breast Cancer.

Low-density lipoprotein-related receptor protein 1 (LRP-1) is not required for insulin-like growth factor binding protein 3 (IGFBP-3) to suppress L6 myogenic cell proliferation

Insulin-like growth factor binding protein-3 (IGFBP-3) suppresses proliferation of numerous cell types, including myogenic cells, via both insulin-like growth factor (IGF)–dependent and IGF-independent mechanisms; however, the mechanism of IGF-independent suppression of proliferation is not clearly defined. In nonmuscle cells, binding of IGFBP-3 to the low-density lipoprotein receptor-related protein-1 (LRP-1)/activated α 2M receptor is reportedly required for IGFBP-3 to inhibit proliferation. These findings suggest that binding to this receptor also may be required for IGFBP-3 to suppress proliferation of cultured myogenic cells. To investigate the role of the LRP-1 receptor in suppression of myogenic cell proliferation by IGFBP-3, we have examined the effect of receptor-associated protein, an LRP-1 receptor antagonist, on recombinant porcine (rp)IGFBP-3 inhibition of L6 myogenic cell proliferation. Treatment with receptor-associated protein results in a 37% decrease ( P < 0.05) in the ability of rpIGFBP-3 to inhibit L6-cell proliferation. In L6 cells subjected to LRP-1 small interfering RNA treatment for 48 h (LRP-1 silenced), LRP-1 mRNA levels were reduced by greater than 80% compared with control cultures treated with nonsense small interfering RNA (mock silenced). In addition, the 85-kDa transmembrane subunit of LRP-1 was undetectable in Western immunoblots of total protein lysates from LRP-1–silenced cells. Even though LRP-1 mRNA and protein levels were dramatically reduced in LRP-1–silenced L6 cells compared with mock-silenced controls, rpIGFPB-3 suppressed proliferation rate to the same extent in both LRP-1–silenced and mock-silenced cultures. Our results strongly suggest that, in contrast to data obtained for nonmuscle cell lines, the LRP-1 receptor is not required for IGFBP-3 to suppress proliferation of L6 myogenic cells.

GalNAc-T14 may be involved in regulating the apoptotic action of IGFBP-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to induce apoptosis in an insulin-like growth factor (IGF)-dependent and IGF-independent manner, but the mechanism underlying the IGF-independent effects remains unclear. Polypeptide N-acetylgalactosaminyltransferase 14 (GalNAc-T14) is a novel IGFBP-3 binding partner. In this paper, small interference RNA (siRNA) targeting GalNAc-T14 was used to examine whether GalNAc-T14 affects the apoptotic action of IGFBP-3. Using semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot analysis, we determined that GalNAc-T14 expression was downregulated by the siRNA directed against GalNAc-T14. Apoptosis analysis of IGFBP-3-overexpressing cells treated with siRNA against GalNAc-T14 was performed to determine if GalNAc-T14 was specifically involved in IGFBP-3 signalling. The results, as determined by flow cytometric analysis and caspase-3 assay, showed that the extent of apoptosis induced by IGFBP-increased with RNA interference (RNAi) knockdown of GalNAc-T14. Our data suggest that GalNAc-T14 influences the apoptotic action of IGFBP-3 and might mediate the signalling pathway of IGFBP-3. Experiments to determine the role of GalNAc-T14 in the regulation of apoptosis induced by IGFBP-3 are under way.

N-Acetylgalactosaminyltransferase 14, a novel insulin-like growth factor binding protein-3 binding partner

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to inhibit cell proliferation and induce apoptosis in IGF-dependent and IGF-independent manners, but the mechanism underlying IGF-independent effects is not yet clear. In a yeast two-hybrid assay, IGFBP-3 was used as the bait to screen a human fetal liver cDNA library for it interactors that may potentially mediate IGFBP-3-regulated functions. N-Acetylgalactosaminyltransferase 14 (GalNAc-T14), a member of the GalNAc-Tases family, was identified as a novel IGFBP-3 binding partner. This interaction involved the ricin-type beta-trefoil domain of GalNAc-T14. The interaction between IGFBP-3 and GalNAc-T14 was reconfirmed in vitro and in vivo, using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assays. Our findings may provide new clues for further study on the mechanism behind the IGF-independent effects of IGFBP-3 promoting apoptosis. The role of GalNAc-T14 as an intracellular mediator of the effects of IGFBP-3 need to be verified in future studies.

Effects of insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) on endometrial cancer (HHUA) cell apoptosis and EGF stimulated cell proliferation in vitro

Objective IGFBP-3 has been demonstrated to stimulate or inhibit cell proliferation independently of its ability to bind IGF and a specific IGFBP-3 receptor has been proposed. EGF has been implicated in the cancer development and carcinogenesis. Only limited data are available on the crosstalk between IGFBP-3 signaling and EGF induced cell survival and signal transduction. The current studies were undertaken to characterize IGFBP-3 binding to endometrial cancer cells (HHUA) and determine its biological effects, as well as whether IGFBP-3 exposure alters the cell proliferation stimulated by EGF. Methods Cell proliferation and apoptosis were analyzed by ELISA using specific antibodies. The interaction between HHUA cell and IGFBP-3 was analyzed using a biosensor. The phosphorylation abundance of specific proteins and their phosphorylation in response to EGF and IGFBP-3 was analyzed by immunoprecipitation followed by immunoblotting. Results Biosensor analysis showed that IGFBP-3 could bind to HHUA cell surface. IGFBP-3 inhibited BrdU uptake, potentiated ssDNA production and induced p53 in HHUA cells. Although EGF stimulated HHUA cell proliferation and Akt phosphorylation, IGFBP-3 inhibited cell proliferation and Akt phosphorylation that had been stimulated by EGF. However, EGF receptor phosphorylation and expression were not reduced by IGFBP-3. Since HHUA cells lack IGF receptors and do not show biological response to IGF these results suggest that IGFBP-3 can bind to HHUA cells, inhibit cell proliferation and induce apoptosis independently of its ability to bind to IGFs possibly by binding to an IGFBP-3 receptor. Conclusions Taken together these findings demonstrate that IGFBP-3 binds to HHUA cell surface, and inhibits cell division induced by EGF, possibly by modulating the EGF-mediated signal transduction system.

The effects of insulin-like growth factor binding protein-3 (IGFBP-3) on T47D breast cancer cells enriched for IGFBP-3 binding sites

To investigate insulin-like growth factor (IGF)-independent effects of IGF binding protein-3 (IGFBP-3), T47D cells were enriched for a population of cells that expressed binding sites for biotinylated-IGFBP-3 by panning on streptavidin-coated plate. Proliferation of cell enriched for IGFBP-3 binding sites was significantly inhibited by IGFBP-3, whereas IGFBP-3 had no significant effect on the non-enriched cell population. Enriched and non-enriched cells were equally responsive to IGF-I, TGF-beta and EGF. Conditioned medium from enriched cells had less IGFBP-3 than that from non-enriched cells. Cross-linking of biotinylated IGFBP-3 to T47D cell membranes identified complexes with Mr of 32, 80 and 100 kDa. All of these complexes were more abundant in enriched cells compared with the non-enriched cell population. These data demonstrate that despite the anti-proliferative effects of IGFBP-3 it is possible to selectively enriched for cell populations with more abundant IGFBP-3 binding sites. These enriched cells are more responsive to IGFBP-3 and secrete less of this binding protein than non-enriched cells, supporting the concept that IGFBP-3 secretion by human breast cancer cells may function as an autocrine or paracrine modulator of cell proliferation.

Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind 125 I -labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [ 125 I ]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [ 125 I ]bLf to rhIGFBP-3 was shown, but [ 125 I ]bTf did not show binding to rhIGFBP-3. While [ 125 I ]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [ 125 I ]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED 50=3 μg/ml) or displaces (ED 50=1 mg/ml) bound [ 125 I ]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all- trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [ 125 I ]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) binds to fibronectin (FN): demonstration of IGF-I/IGFBP-3/fn ternary complexes in human plasma.

We used a yeast two-hybrid system to identify binding partners for insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3). A partial complementary DNA encoding the carboxyl-terminal of fibronectin (FN), including the cell binding site, the heparin-binding domain, and the fibrin-binding domain, was identified in a screen of a human placental complementary DNA library. The interaction of IGFBP-3 with FN and the 40-kDa heparin-binding carboxyl-terminal fragment of FN was confirmed using Western ligand blotting. Both glycosylated and nonglycosylated IGFBP-3 bound to FN with a K(d) of approximately 0.3 nmol/L. IGF-I and IGFBP-1 had no effect on IGFBP-3 binding to FN. Competitive inhibition of IGFBP-3 binding to FN was observed in the presence of IGFBP-5 and heparin. The binding affinity of the immobilized IGFBP-3/FN complex for [(125)I]IGF-I (K(d) = 0.8 nmol/L) was similar to that of IGFBP-3 alone. The presence of IGF-I/IGFBP-3/FN ternary complexes in human plasma was demonstrated by coimmunoprecipitation of IGFBP-3 and [(125)I]IGF-I with anti-FN monoclonal antibody. These data indicate that FN may have a role in the transportation of IGFBP-3 and IGF-I in the circulation and the sequestration of these proteins in tissues.

Transferrin is an insulin-like growth factor-binding protein-3 binding protein.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) possesses both growth-inhibitory and -potentiating effects on cells that are independent of IGF action and are mediated through specific IGFBP-3 binding proteins/receptors located at the cell membrane, cytosol, or nuclear compartments and in the extracellular matrix. We have here characterized transferrin (Tf) as one of these IGFBP-3 binding proteins. Human serum was fractionated over an IGFBP-3 affinity column, and a 70-kDa protein was eluted, sequenced, and identified (through database searching and Western immunoblot) as human Tf. Tf bound IGFBP-3 but had negligible affinity to the other five IGFBPs, and iron-saturated holo-Tf bound IGFBP-3 more avidly than unsaturated Tf. Biosensor interaction analysis confirmed that this interaction is specific and sensitive, with a high association rate similar to IGF-I, and suggested that binding occurs in the vicinity of the IGFBP-3 nuclear localization site. As an independent confirmation of this interaction, using a yeast two-hybrid system, we cloned Tf from a human liver complementary DNA library as an IGFBP-3 protein partner. Tf treatment blocked IGFBP-3-induced cell proliferation in bladder smooth muscle cells, and IGFBP-3-induced apoptosis in prostate cancer cells. In summary, we have employed a combination of techniques to demonstrate that Tf specifically binds IGFBP-3, and we showed that this interaction has important physiological effects on cellular events.

Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM (125)I-labelled IGFBP-3 to occupy all binding sites present on 100 microg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a K(d) between 2.6x10(-10) and 8.0x10(-10) M and 1.51-4.89x10(11) binding sites/mg of platelet membrane. Kinetic analysis of (125)I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (k(on)) of 8.1x 10(8) per M per min. The reverse rate constant (k(off)) was calculated to be 1.6x10(-1) per min (t(1/2)=4.2 min) and confirmed experimentally to be 3.3x10(-1) per min (t(1/2)=2.1 min). Binding of (125)I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with (125)I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in (125)I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.

Characterization of the inhibition of DNA synthesis in proliferating mink lung epithelial cells by insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.

Characterization of insulin-like growth factor binding protein-3 (IGFBP-3) binding to human breast cancer cells: kinetics of IGFBP-3 binding and identification of receptor binding domain on the IGFBP-3 molecule.

Insulin-like growth factor binding protein-3 (IGFBP-3) binds to specific membrane proteins located on human breast cancer cells, which may be responsible for mediating the IGF-independent growth inhibitory effects of IGFBP-3. In this study, we evaluated IGFBP-3 binding sites on breast cancer cell membranes by competitive binding studies with IGFBP-1 through -6 and various forms of IGFBP-3, including synthetic IGFBP-3 fragments. Scatchard analysis revealed the existence of high-affinity sites for IGFBP-3 in estrogen receptor-negative Hs578T human breast cancer cells (dissociation constant (Kd) = 8.19 +/- 0.97 x 10(-9) M and 4.92 +/- 1.51 x 10(5) binding sites/cell) and 30-fold fewer receptors in estrogen receptor-positive MCF-7 cells (Kd = 8.49 +/- 0.78 x 10(-9) M and 1.72 +/- 0.31 x 10(4) binding sites/cell), using a one-site model. These data demonstrate binding characteristics of typical receptor-ligand interactions, strongly suggesting an IGFBP-3:IGFBP-3 receptor interaction. Among IGFBPs, only IGFBP-5 showed weak competition, indicating that IGFBP-3 binding to breast cancer cell surfaces is specific and cannot be attributed to nonspecific interaction with glycosaminoglycans. This was confirmed by showing that synthetic IGFBP-3 peptides containing IGFBP-3 glycosaminoglycan-binding domains competed only weakly for IGFBP-3 binding to the cell surface. Rat IGFBP-3 was 20-fold less potent in its ability to compete with human IGFBP-3(Echerichia coli), as well as 10- to 20-fold less potent for cell growth inhibition than human IGFBP-3, suggesting the existence of species specificity in the interaction between IGFBP-3 and the IGFBP-3 receptor. When various IGFBP-3 fragments were evaluated for affinity for the IGFBP-3 receptor, only those fragments that contain the midregion of the IGFBP-3 molecule were able to inhibit 125I-IGFBP-3(Escherichia coli) binding, indicating that the midregion of the IGFBP-3 molecule is responsible for binding to its receptor. These observations demonstrate that specific, high-affinity IGFBP-3 receptors are located on breast cancer cell membranes. These receptors have properties that support the notion that they may mediate the IGF-independent inhibitory actions of IGFBP-3 in breast cancer cells.

Characterization of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) Binding to Human Breast Cancer Cells: Kinetics of IGFBP-3 Binding and Identification of Receptor Binding Domain on the IGFBP-3 Molecule

Characterization of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) Binding to Human Breast Cancer Cells: Kinetics of IGFBP-3 Binding and Identification of Receptor Binding Domain on the IGFBP-3 Molecule

The p53/IGF-1 receptor axis in the regulation of programmed cell death.

The loss or functional inactivation of tumor suppressor genes appears to be one of the most fundamental genetic mechanisms of tumorigenesis, and rational insights into the signaling pathways of tumor suppressor genes have emerged as a successful strategy of identifying novel drug discovery targets downstream of the tumor suppressor protein itself. Elucidation of novel pathways downstream of p53 have established a link between this important tumor suppressor gene and the insulin-like growth factor-1 receptor (IGF-1r), either via direct regulation of IGF-1 receptor levels, or modulation of IGFs via transactivation of the insulin-like growth factor-binding protein 3 (IGF-BP3) gene. Binding of IGF-BP3 to IGFs inhibits both their mitogenic and cell survival functions, highlighting a novel pathway whereby p53 may regulate apoptosis in tumor cells.

Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans.

The mitogenic action of insulin-like growth factors (IGFs) on target cells is determined by interaction with signaling IGF-I receptors and modulated by interactions with IGF-binding proteins (IGFBPs). IGFBP-3, an abundant IGFBP that binds IGF-I and IGF-II with high affinity, can form soluble inhibitory complexes with the IGFs that prevent them from binding to IGF-I receptors. Alternatively, IGFBP-3 can bind to the cell surface and possibly potentiate IGF action or act independently of the IGFs. Previous studies showed that heparin inhibited IGFBP-3 binding to the cell surface and increased its accumulation in the medium, suggesting that it might act as a competitive inhibitor of IGFBP-3 binding to structurally similar heparan sulfate proteoglycans on the cell surface. We evaluated this hypothesis by binding 125I-labeled recombinant glycosylated human IGFBP-3 to human fetal skin fibroblasts (GM-10) and to C6 rat glioma cells at 12 C. Heparin inhibited [125I]IGFBP-3 binding more effectively than chondroitin sulfate and dextran sulfate. Complete digestion of cell surface heparan sulfate and chondroitin sulfate glycosaminoglycans using heparitinase and chondroitinase ABC, however, did not significantly decrease IGFBP-3 binding. Quantitative removal was demonstrated by analysis of parallel cultures of cells whose glycosaminoglycans had been biosynthetically labeled using Na2 35SO4. These results suggested that IGFBP-3 did not bind to heparan sulfate glycosaminoglycans on the cell surface, and that the inhibition of IGFBP-3 binding by heparin most likely resulted from its direct interaction with the heparin-binding domains of IGFBP-3. When [125I]IGFBP-3 was incubated with GM-10 fibroblasts or C6 glioma cells at 37 C for 4 h, only 10% of the bound ligand remained associated with the cell surface; approximately 90% of the cell-associated radio-activity was internalized and could be recovered in lysates of acid-washed cells. Incubation with IGF-I or heparin decreased the total cell-associated radioactivity, but did not affect internalization. These results suggest that direct interaction of heparin or IGF-I with IGFBP-3 inhibits its ability to bind to the surface of GM-10 fibroblasts and C6 glioma cells.

Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans

Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action.

In this study, we investigated the effects of various biochemical and pharmacological agents on insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblasts. When cells were preincubated for 48 h with 50 nM recombinant human (rh) IGFBP-3, IGF-I-stimulated [3H]aminoisobutyric acid ([125H]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupting agents during the preincubation with rhIGFBP-3 did not affect IGFBP-3 potentiation of IGF-I action, nor did a variety of serine, aspartate, and metalloproteinase inhibitors. On the other hand, ammonium chloride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Chloroquine and ammonium chloride had no effect alone and did not inhibit IGF-I receptor binding or action in the absence of rhIGFBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen ion pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Competitive [125I]IGF-I binding and affinity cross-linking experiments suggested structure/function changes in cell-bound IGFBP-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 cell adherence, but could not promote dissociation of IGFBP-3 from cells after the 48-h preincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation of IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications that mediate the enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like molecules on the cell surface is not directly involved in this process.