Insulin-like growth factor binding protein expression in human small cell lung cancer cell lines

Insulin-like growth factor binding proteins (IGF-BP) are secreted by several human small cell lung cancer cell lines (SCLC). In order to identify the IGF-BPs from SCLC cell lines the RNA from 10 different SCLC cell lines was analyzed by Northern blot analysis with the probes for three different IGF-BPs, IGFBP-1, IGFBP-2, and IGFBP-3. No hybridization signal could be detected with the probes encoding for IGFBP-1 and IGFBP-3. The hybridization with different IGFBP-2-specific oligodeoxynucleotide probes and with the corresponding full-length cDNA showed that all SCLC cell lines which secreted IGF-BPs express IGFBP-2.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.     

Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.     

Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of insulin-like growth factor-binding proteins in the circulation of four teleost fish species.

Insulin-like growth factor-binding proteins (IGF-BPs) were demonstrated in the circulation of four teleost fish species. In the coho salmon (Oncorhynchus kisutch), serum binding of 125I-labelled human IGF-I (125I-hIGF-I) was competitively inhibited by addition of excess recombinant bovine IGF-I (rbIGF-I) in a manner similar to that when rat serum was used. Western-ligand blot procedure using the same labelled hormone identified at least three major forms of IGF-BPs in the plasma of all four teleost species investigated: coho salmon, striped bass (Morone saxatilis), tilapia (Oreochromis mossambicus), and longjawed mudsucker (Gillichthys mirabilis). The first form is around 40-50 kDa, may be regulated by growth hormone (GH), and seems to be a good candidate for the fish version of mammalian IGF-BP3 (which is in the same size range and is GH-regulated). The second and third forms are 29 kDa and 31 kDa and are good candidates for the fish versions of mammalian IGF-BP1 and IGF-BP2, respectively, as they appear to be regulated by insulin and are in the same size range as their mammalian counterparts. Functionally different classes of circulating IGF-BPs may be conserved between fish and mammal.     

Effects of a single cleavage in insulin-like growth factors I and II on binding to receptors, carrier proteins and antibodies.

Two somatomedin-like peptides were extracted from Cohn fraction IV of human plasma and brought to homogeneity: one focused at pH 7.8 and the other at pH less than 5.6. Each consisted of two peptide chains interlinked by disulphide bonds. The basic peptide was identical to insulin-like growth factor I (IGF-I) and had a single cleavage in the C-domain before Arg37 [IGF-I(Arg36cl)]. The acid peptide showed identity with IGF-II, with a cleavage in the B-domain before Arg30 [IGF-II(Ser29cl)]. The effects of these cleavages on the characteristics of binding to type I and type II receptor sites, to binding proteins and to antibodies was studied. Binding of IGF-I(Arg36cl) to antibodies directed against the B-domain or against the AD-domain of IGF-I was the same as IGF-I binding. Thus the cleavage does not influence these antigenic sites. In contrast, binding of IGF-I(Arg36cl) to the type I receptor on human and bovine placental cell membranes was markedly decreased compared with IGF-I binding. Binding to the insulin receptor on human placental cell membranes was slightly diminished, whereas the interaction with specific type II receptors on bovine placental cell membranes was unaffected. There was only a minor influence of the cleavage on the region involved in binding to binding proteins. The cleavage in IGF-II(Ser29cl) diminished binding to antibodies directed against the C-domain of IGF-II, compared with binding of IGF-II itself. Binding to receptors (type I and type II) was changed less profoundly. With 125I-labelled IGF-II(Ser29cl), less insulin was needed in order to obtain 50% displacement of the tracer compared with displacement of 125I-labelled IGF-II. The cleaved form of IGF-II probably has a greater affinity towards the common receptor population than does native IGF-II. Binding to binding proteins was not affected by the cleavage in IGF-II.     

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of ¹²⁵I-IGF-I and [³H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of ¹²⁵I-IGF-I across HUVE cell monolayers was not significantly different to that of [³H]inulin, a paracellular probe. Moreover, ¹²⁵I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit ¹²⁵I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997. © 1997 Wiley-Liss, Inc.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.     

Estradiol down-regulates the mannose-6-phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: a receptor saturation mechanism to increase the secretion of lysosomal proenzymes.

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.     

Receptor Binding, Endocytosis, and Mitogenesis of Insulin-Like Growth Factors I and II in Fetal Rat Brain Neurons

Cell surface binding, internalization, and biological effects of insulin-like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF-I receptor alpha-subunit (Mr 125,000) binds IGF-I with a KD of 1 nM and IGF-II with 10 times lower affinity. The mannose-6-phosphate (Man-6-P)/IGF-II receptor (Mr 250,000) binds IGF-II with a KD of 0.5 nM and IGF-I with 100 times lower affinity. Surface-bound IGF-I and IGF-II are internalized by their respective receptors binding and internalization of IGF-II but not those of IGF-I. Neuronal synthesis of RNA and DNA is increased twofold by IGF-I with 10 times higher potency than IGF-II. Antibody 3637, which blocks receptor binding of IGF-II, has no effect on the DNA response to IGF-I or IGF-II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that greater than 80% of the bromodeoxyuridine-positive cells become neurofilament positive. It is concluded that IGF-I and IGF-II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF-I and IGF-II. Proliferation of neuronal precursor cells is stimulated by IGF-I and IGF-II via activation of the IGF-I receptor.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

Characterization of insulin-like growth factor binding proteins from human breast cancer cells

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells

The insulin-like growth factor (IGF) system plays an important role in cell proliferation and survival. However, more recently, a small number of studies have shown that IGFs induce apoptosis in some cells. Our initial studies showed this occurred in LIM 1215 colon cancer cells but not RD rhabdomyosarcoma cells. IGFs induced both proliferation and apoptosis in LIM 1215 cells, and the induction of apoptosis was dose-dependent. [R54, R55]IGF-II, which binds to the IGF-I receptor with normal affinity but does not bind to the IGF-II receptor, induced apoptosis to the same extent as IGF-II, whereas [L27]IGF-II, which binds to the IGF-I receptor with 1000-fold reduced affinity, had no effect on apoptosis. These results suggest that the IGF-I receptor is involved in induction of apoptosis. Western blot analyses demonstrated that Akt and Erk1/2 were constitutively activated in RD cells. In contrast, phosphorylation of Akt and Erk1/2 were transient and basal expression of Akt protein was lower in LIM 1215 cells. Analysis of apoptosis-related proteins showed that IGFs decreased pro-caspase-3 levels and increased expression of pro-apoptotic Bad in LIM 1215 cells. IGFs co-activate proliferative and apoptotic pathways in LIM 1215 cells, which may contribute to increased cell turnover. Since high turnover correlates with poor prognosis in colorectal cancer, this study provides further evidence for the role of the IGF system in its progression.     

Urokinase type plasminogen activator receptor is involved in insulin-like growth factor-induced migration of rhabdomyosarcoma cells in vitro

Urokinase-type plasminogen activator (uPA) binds to its receptor, uPAR, on the surface of cancer cells, leading to the formation of plasmin. Rhabdomyosarcoma (RMS) cell lines secrete high levels of insulin-like growth factor II (IGF-II), suggesting autocrine IGFs play a major role in the unregulated growth and metastasis of RMS. In vitro, IGF-II and IGF-I increased migration of RD cells to 124+/-9% (P<0.01) and 131+/-8% (P<0.05) of control, respectively. IGF-II-induced migration was abolished by insulin-like growth factor binding protein-6 (IGFBP-6) (P<0.01), a relatively specific inhibitor of IGF-II, and by plasminogen activator inhibitor type 1 (PAI-1) (P<0.05). Aprotinin, a plasmin inhibitor, and mannosamine, which inhibits the synthesis of glycosylphosphatidylinositol (GPI), thereby preventing anchorage of GPI-linked proteins such as uPAR to the cell membrane, also decreased IGF-II- (P<0.05 for both) but not IGF-I-induced migration. [Arg54,Arg55]IGF-II and [Leu27]IGF-II, which preferentially bind to the IGF-I and IGF-II/mannose-6-phosphate receptors (IGF-II/M6PR), respectively, both induced RD cell migration to 146+/-8% (P<0.01) and 120+/-7% (P<0.05) of control, respectively. An anti-uPAR anti-serum reduced IGF-II- and IGF-I-induced migration (P<0.05 for both). An anti-low density lipoprotein-related protein (LRP) anti-serum reduced IGF-I-induced migration (P<0.05). IGF-I and -II both increased specific 125I-single chain uPA (scuPA) binding to RD cells in a dose-dependent manner (P<0.01). These results suggest involvement of the PA/plasmin system in IGF-induced migration and indicate important roles these systems may have in RMS metastasis.     

Salt stimulation of serum insulin-like growth factor binding protein activity

Insulin-like growth factors (IGF)-I and -II are bound to carrier or binding proteins in serum. There are at least two classes of binding protein: a high molecular weight complex and a low molecular weight species that is relatively unsaturated. Total binding capacity in serum generally is determined by incubating [125I]IGF with protein that has been stripped of IGF by acid gel filtration. We found that addition of NaCl to the assay increased binding to stripped guinea pig binding protein to about two to four times the level measured in the absence of salt. Stimulation by NaCl was optimal between concentrations of 0.6 and 1.4 M and also was observed when fetal calf or human sera were used as sources of stripped binding protein or when IGF-II was the ligand. Using chloride salts, the order of activity with respect to cations was Na+ greater than K+ greater than Li+. Na2HPO4 at 0.6 M was as stimulatory as 1.2 M NaCl but 0.6 M Na2SO4 was less effective. NH4HCO3 was as effective as NaCl at 0.6 M. Scatchard plots of data from competitive dilution experiments with [125I]IGF-I and unlabeled IGF-I showed that binding was heterogeneous in the absence of 0.6 M NaCl but linear in its presence. NaCl did not stimulate binding when whole serum was used, but after gel filtration of serum on Sephacryl 200 at pH 8, which does not dissociate IGFs from binding protein, binding to individual fractions was stimulated three- to fourfold by NaCl. Fractions stimulated included those containing the large complex or the unsaturated binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific Insulin-Like Growth Factor-I Receptors on Circulating Bovine Mononuclear Cells

Abstract

We have examined the presence and properties of specific receptors for IGF-I on bovine mononuclear cells. Competitive binding studies showed that binding of [¹²⁵I]IGF-I to mononuclear cells was inhibited by unlabelled peptides with the rank of IGF-I > IGF-II > insulin. The binding of [¹²⁵I]IGF-I was a function of the cell concentration. Equilibrium dissociation constant and receptor concentration values for the average of 9 adult cows were 1.13 ± 0.11 nM and 108.9 ± 24.1 fMol/10⁷ cells, respectively. Moreover, IGF-I stimulated thymidine incorporation into bovine mononuclear cells in the absence of serum and phytohemagglutinin (PHA). The existence of specific and functional IGF-I receptors on circulating bovine mononuclear cells would provide an easily accessible source for studying IGF-I receptor changes in the bovine, both in physiologic and pathologic states.