IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100% inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60% inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.     

Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v-src and various mitogens.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.     

Insulin-like growth factor binding protein (IGFBP-3), an inhibitor of serum growth factors other than IGF-I and -II.

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.     

An IGF binding protein is an inhibitor of FGF stimulation.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.     

Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.     

Isolation and amino-terminal sequence of a novel cellular growth inhibitor (inhibitory diffusible factor 45) secreted by 3T3 fibroblasts

A growth inhibitory protein named inhibitory diffusible factor 45 (IDF45) has been purified to homogeneity from medium conditioned by dense cultures of mouse 3T3 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fast protein liquid chromatography-purified bioactive material showed a single band corresponding to a protein of 45 kDa. The molecule is a monomer. The pI of the molecule, as determined by isoelectric focusing, is about 6.5. The amino-terminal sequence of the protein was established as Ser-Ala-Gly-Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. The protein inhibits DNA synthesis in chicken embryo fibroblasts in a dose-dependent manner (ED50: 40 ng/ml, approximately 1 nM). The chemical properties of IDF45, i.e. molecular weight and amino-terminal sequence, clearly distinguish it from other known growth inhibitory proteins.     

Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells.

Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media. The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE. The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein. When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed. This expressed hIGFBP-3 protein maintains its ability to bind IGF-I. N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E. coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa). Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages. These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells.     

Presence of IDF45 (mlGFBP-3) binding sites on chick embryo fibroblasts.

IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn2+ (10(-3)M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10(-8)M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface.     

High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.     

Insulin-like growth factor I receptors in retinal rod outer segments

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.     

Inhibitory diffusible factor IDF45, a G1 phase inhibitor

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio-Gel P150 chromatography and 2 reverse-phase FPLC. After the final step of purification, 60 ng/ml of IDF45 inhibited 50% of alpha-globulin-stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by alpha-globulin.     

Purification and properties of an endothelial cell growth factor from human platelets

An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin.     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

Insulin-like growth factor binding proteins of equine serum.

Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum binds IGF-I and IGF-II specifically and appears to be unique to this species. The nature of this protein is at present unknown.     

Identification of L-cell growth stimulating antibody as anti-actin

Anti-L-cell antisera having potent cell growth stimulatory properties were shown by Western blotting to have predominant specificity toward a protein with a molecular weight of 42K which we identified as actin. Extractions of L cells, based upon the known insolubility of cytoskeletal proteins (including actin) in Triton X-100 and the solubility of actin in low ionic strength Ca2+ and ATP-containing buffer, led to actin-enriched preparations that retained immunoreactivity with the anti-L-cell antisera. The 42-kDa antigen binds to deoxyribonuclease I, has a pI = 5.2-5.4, and has an amino acid composition, including the presence of 3-methylhistidine, compatible with compositions determined for actins from other sources. Rabbit antiserum specific for this 42-kDa protein, isolated by SDS-PAGE, reproduced the cell growth stimulation by the anti-L-cell antisera and absorption of the antiserum with purified L-cell actin eliminated this stimulation. Moreover, these antibodies bind to the microfilaments of 3T3 fibroblasts. When purified actins were used as soluble antigen inhibitors of the immune reactivity of antiserum to 42-kDa protein with intact L cells, rabbit thymus actin competed with the surface molecules on L cells and reduced the stimulatory effect of the antiserum by 80% at an actin concentration of 150 micrograms/ml. Chicken muscle actin reduced the antibody stimulation effect by only 24% at the same protein concentration, and mouse muscle actin was ineffective as an inhibitor. The F(ab')2 fraction of anti-42K IgG was effective in stimulating L cells, thus documenting the immune nature of the actin-anti-42K interaction. We conclude that anti-actin antibodies, upon binding to actin-like cell surface determinants on L cells, stimulate cellular metabolism.     

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.     

Stimulation by TGF beta of chick embryo fibroblasts--inhibition by an IGFBP-3.

The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).     

Porcine insulin-like growth factor (IGF) binding protein blocks IGF-I action on porcine adipose tissue

A growth hormone-dependent insulin-like growth factor (IGF) binding protein (IGFBP) purified from porcine serum specifically blocked the acute insulin-like effects of IGF-I on lipogenesis and glucose oxidation in porcine adipose tissue. This inhibition was dose dependent with half-maximal effective concentrations of IGFBP of 530 ng/ml for lipogenesis and 590 ng/ml for glucose oxidation in the presence of 10(-8) M IGF-I. The IGFBP also caused decreased rates of lipogenesis following a 1-hr preincubation of tissue with IGF-I (10(-8) M). The IGFBP had no effect on insulin action on porcine adipose tissue. These findings demonstrate the inhibitory effects of a highly purified porcine serum IGFBP on the biologic effects of IGF-I in vitro, and provide evidence that the growth hormone-dependent IGFBP blocks the acute insulin-like actions of IGF-I in vivo.     

Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa)

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.