Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

Secretion and biological actions of insulin-like growth factor binding proteins in two human tumor-derived cell lines in vitro.

The insulin-like growth factors (IGFs) I and II are present in extracellular fluids associated with specific binding proteins (IGFBPs) that can modify their biologic actions. These studies were undertaken to determine which forms of IGFBP are secreted by endometrial carcinoma (HEC-1B) and breast carcinoma (MDA-231) cells, to characterize variables that control IGFBP secretion, and to study the effect of IGFBP-1 and IGFBP-2 on IGF-I stimulated cell proliferation. Secreted IGFBPs were identified by ligand blotting and IGFBP-1 was quantified using a specific radioimmunoassay (RIA). MDA-231 cell conditioned media (CM) contained four (43,000, 39,000, 30,000 and 24,000 Mr) forms of IGFBP, and HEC-1B cell CM contained three forms (39,000, 34,000 and 30,000 Mr). Immunoblotting showed that the 30,000 Mr form secreted by both cell types was IGFBP-1. Likewise the 34,000 Mr band in HEC-1B media reacted with IGFBP-2 antiserum and the 39,000 and 43,000 Mr bands reacted with IGFBP-3 antiserum. IGF-I stimulated the secretion of IGFBP-3 from both cell types and IGFBP-2 from HEC-1B cells but either decreased or caused no change in secretion of IGFBP-1 and a 24,000 Mr form. In contrast, insulin inhibited the secretion of IGFBP-1 but increased the secretion of the 24,000 Mr form. Compounds that elevate intracellular cAMP levels increased the secretion of IGFBP-3, IGFBP-1, and the 24,000 Mr form from both MDA-231 and HEC-1B cells. When sparse cultures of MDA-231 cells were used, addition of IGF-I caused a 24% increase in cell number after 48 hr. This mitogenic response was enhanced by the presence of recombinant human IGFBP-1 (45% increase in cell number, P less than 0.001). Bovine IGFBP-2 did not potentiate IGF-I stimulated cell proliferation. These findings show that two tumor cell lines secrete distinct forms of IGFBPs and that there is differential regulation of IGFBP secretion. At least one form secreted by both tumors may act as a positive autocrine modulator of IGF-I's growth stimulating actions.     

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β (TGF-β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-β stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-β differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I. J. Cell. Physiol. 174:240–250, 1998. © 1998 Wiley-Liss, Inc.     

Production of Insulin-like Growth Factors and Their Binding Proteins in Primary Cultures of Rat Liver Parenchymal and Nonparenchymal Cells

Regulation of the production of insulin-like growth factor (IGF)-I, IGF-II, IGF binding proteins (IGFBPs), and their related proteins by various hormones was investigated in primary cultures of rat liver parenchymal and nonparenchymal cells.

Freshly isolated parenchymal cells contained mRNAs of IGF-I, IGF-II, IGFBP-1, IGFBP-4, growth hormone (GH) receptor, and the acid-labile subunit (ALS), which forms a ternary complex with IGF-I and IGFBP-3; however, parenchymal cells did not express the IGFBP-3 gene. In contrast, nonparenchymal cells contained IGFBP-3 mRNA exclusively, as we reported previously [Takenaka et al. Agric. Biol. Chem., 55, 1191–1193 (1991)]. Cultured rat parenchymal cells produced IGF-I, IGFBP-1, and IGFBP-4 prominently. In these cells, secretion of IGF-I and the content of IGF-I mRNA was greatly increased in the presence of GH in the medium. Insulin also increased the production of IGF-I. Secretion of IGFBP-l into the medium was enhanced by treatment with glucagon, dibutyrylcyclic AMP (Bu₂cAMP), and dexamethasone (Dex) and these enhancements with glucagon and Dex reflected the increase in its mRNA content. Insulin depressed the secretion of IGFBP-l. The content of IGFBP-4 in the parenchymal cells was increased by insulin, Bu₂cAMP, and triiodothyronine (T₃), thereby enhancing the production of IGFBP-4 and secretion into the medium. Cultured liver nonparenchymal cells of rats produced IGFBP-1, IGFBP-3, and IGFBP-4. Secretion of IGFBP-l was increased by Bu₂cAMP in the medium, that of IGFBP-3 by IGF-I, and that of IGFBP-4 by both IGF-I and Bu2cAMP. Regulation of the production of IGFBP-3 by IGF-I was demonstrated in these investigations.

These results suggest that GH increases production of IGF-I in the parenchymal cells and this IGF-I, in turn, increases the production of IGFBP-3 in nonparenchymal cells. As we found GH also increases ALS production in parenchymal cells, by these mechanisms, GH increases the formation of the ternary complex of IGF-I, IGFBP-3, and ALS. This study clearly demonstrates the interrelationship between parenchymal and nonparenchymal cells in the production of IGF-I and IGFBPs in the liver.     

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12–O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt₂cAMP) augmented the secretion In contrast, TPA increased the level of type IIGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt₂cAMP affected neither IGF-l receptor number nor the IGFBP secretion. These results indicate that the production of IGF-l and the expression of IGF-l receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells. © 1994 Willey-Liss, Inc.     

Resection upregulates the IGF-I system of parenterally fed rats with jejunocolic anastomosis

Rats maintained with parenteral nutrition following 60% jejunoileal resection plus cecectomy exhibit minimal adaptive growth in the residual jejunum but a dramatic adaptive growth in the residual colon. Coinfusion of insulin-like growth factor I (IGF-I) with parenteral nutrition induces jejunal growth but has minimal effects in the colon. Our objective was to study the role of the endogenous IGF-I system in the differential responses of jejunum and colon to resection and/or IGF-I during parenteral nutrition. We measured concentrations of immunoreactive IGF-I in plasma, jejunum, and colon, IGF-I receptor binding, and levels of IGF receptor, IGF-I, IGF binding protein (IGFBP)-3 and IGFBP-5 mRNA in residual jejunum and colon 7 days after resection and/or IGF-I treatment. IGF-I receptor number was increased (74-99%) in jejunum and colon due to resection; IGF-I mRNA was increased 5-fold in jejunum and 15-fold in colon due to resection. Resection increased circulating IGFBPs but did not alter plasma IGF-I concentration. Resection induced colonic growth in association with significantly greater colonic IGFBP-5 mRNA and significantly lower colonic immunoreactive IGF-I. IGF-I treatment had no significant effect on IGF-I mRNA or IGF-I receptor number. Concentrations of plasma and jejunal immunoreactive IGF-I were significantly increased in rats given IGF-I in association with jejunal growth. IGF-I treatment significantly increased IGFBP-5 mRNA in the jejunum, which also correlated with jejunal growth. Thus resection upregulated IGF-I receptor number and IGF-I mRNA in residual jejunum and colon, but differential adaptation of these segments correlated with differential regulation of IGFBP-5 mRNA.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.     

Insulin-like growth factor-I is a serum component stimulating growth of human neuroblastoma

Summary Human non-autocrine neuroblastoma cells SK-N-SH and LF require serum for proliferation in vitro. We wished to determine the role of serum-borne insulin-like growth factor I (IGF-I) as mitogen for these cells. Introduction of the monoclonal antibodyα-IR₃ against human IGF-I receptor reduced proliferation in the presence of fetal bovine serum (FBS). IGF-I (5 nM) was as effective as FBS (10%) in stimulating proliferation. Porcine insulin mimicked the effects of IGF-I, but at a 1000-fold higher concentration. The antibodyα-IR₃ reduced growth stimulated by IGF-I more effectively than growth stimulated by insulin. Thus, proliferation of human non-autocrine neuroblastoma cells can be effectively manipulated by exogenous IGF-I.     

Effects of aging and caloric restriction on IGF-I, IGF-I receptor, IGFBP-3 and IGFBP-4 gene expression in the rat stomach and colon

The purpose of this study was to determine the effects of aging and caloric restriction (CR) on insulin-like growth factor-I (IGF-I), IGF-I receptor (IGF-IR), IGF-binding protein-3 (IGFBP-3) and IGFBP-4 expression in the stomach and colon of male Fischer 344 rats. Stomach and colonic RNA were prepared from ad libitum (AL) fed or long-term CR rats. Stomach IGF-I, IGFBP-3 and IGFBP-4 mRNA levels increased significantly (P相似文献   

Differential regulation of insulin-like growth factor-(IGF) I and IGF-binding protein (IGFBP) secretion by human peripheral blood mononuclear cells

BACKGROUND: Recent studies have shown that immunocompetent cells synthesize and express growth hormone (GH), growth hormone receptors (GH-R), insulin-like growth factor I (IGF-I), IGF-I receptors (IGF-I-R) and different insulin-like growth factor binding proteins (IGFBPs). The aim of the current study was to evaluate the regulation of IGFBP and IGF-I secretion from immunocompetent cells by different mitogens. METHODS/RESULTS: We studied the in vitro secretion pattern of IGFBPs and IGF-I from human peripheral blood mononuclear cells (PBMC), derived from 10 normal adults and 8 GH-deficient patients with adult onset. In serum-free conditioned medium of unstimulated PBMC, derived from normal adults, Western ligand blotting (1D-WLB) revealed a 24-kD, a 34-kD and a 39/43-kD doublet band to be most prominent. According to their molecular weight and two-dimensional Western ligand blot analysis (2D-WLB), these bands are deglycosylated IGFBP-4, IGFBP-2 and IGFBP-3, respectively. When the cells were treated with the T-cell mitogen phytohemagglutinin (PHA) (10 microg/ml), a differential stimulation of IGFBPs was found with a 2.57 +/- 0.48-fold increase of IGFBP-4 (p < 0.01), a 1.55 +/- 0.13-fold increase of IGFBP-2 (p < 0.01), and a 1.35 +/- 0.19-fold increase of IGFBP-3 (n.s.). In contrast, treatment with the B-cell mitogen pokeweed mitogen (PWM) (10 microg/ml) caused only a modest 1.40 +/- 0.07-fold increase of IGFBP-4 (p < 0.01). Treatment with rhGH (100 ng/ml) or rhIGF-I (200 ng/ml) caused no significant induction of any specific band, respectively. In contrast to the secretion pattern of IGFBPs, IGF-I secretion of the PBMC was not stimulated by either PHA or PWM, but showed a significant increase after GH incubation (p < 0.01). A similar differentiated secretion pattern of IGFBPs and IGF-I was also observed in the conditioned medium of PBMC, derived from GH-deficient patients. CONCLUSION: In summary, at least three different IGFBPs are secreted by human PBMC. Secretion of IGFBPs by PBMC is differentially regulated by different lymphocyte mitogens. Secretion of IGFBPs by PBMC is independent of GH or IGF-I, whereas the secretion of IGF-I is stimulated by GH. PBMC derived from normal adults and GH-deficient patients show similar patterns of IGF-I and IGFBPs secretion, thus indicating that the paracrine/autocrine IGF-I-IGFBPs interactions of the PBMC are not altered by pituitary GH deficiency.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.     

Insulin-like growth factor-binding protein enhancement of insulin-like growth factor-i (IGF-I)–mediated DNA synthesis and IGF-I binding in a human breast carcinoma cell line

The insulin-like growth factors (IGFs) are potent mitogens for malignant cell proliferation. The majority of secreted IGFs are bound to specific IGF-binding proteins (IGFBPs) that are secreted by a large number of cells. These proteins may either inhibit or enhance IGF actions. Breast carcinoma cells secrete a variety of IGFBPs. We have previously demonstrated that retinoic acid (RA) inhibition of IGF-l– stimulated MCF-7 cell proliferation is associated with increased IGFBP-3 levels in the conditioned media. We therefore investigated the effect of recombinant IGFBP-3 as well as IGFBP-2, -4 and -5 on IGF-l stimulation of DNA synthesis and IGF-I binding in the MCF-7 human breast carcinoma cell line. IGFBP-2 and -3 enhanced IGF-l stimulation of DNA synthesis in MCF-7 cells while IGFBP-4 and -5 had no effect. Transfection of MCF-7 cells with an IGFBP-3 expression vector resulted in the enhanced secretion of IGFBP-3 with an accompanying increase in IGF-l binding as well as increased cell proliferation upon treatment of the cells with IGF-l. IGF-l preincubation of MCF-7 cells transfected with control pSVneo plasmids results in cells refractory to further IGF-l stimulation of thymidine incorporation while IGF-l continues to stimulate [³H]-thymidine incorporation in IGFBP-3–transfected MCF-7 cells, suggesting that IGFBP-3 protects the cells from IGF-l–mediated down regulation of its receptor. Therefore, IGFBP-3 secreted by MCF-7 cells can enhance IGF-l stimulation of DNA synthesis, increase IGF-l binding to these cells, and prevent IGF-l–induced desensitization of its own receptor, suggesting that IGFBP-3 plays a significant role in IGF-l–mediated breast carcinoma proliferation. © 1994 Wiley-Liss, Inc.