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.     

Overexpression of ovine insulin-like growth factor-I stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells.

To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-I under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I peptides.     

Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.     

Cell-associated insulin-like growth factor-binding proteins inhibit insulin-like growth factor-I-induced endometrial cancer cell proliferation.

Insulin-like growth factor I (IGF-I) is a peptidic growth factor implicated in the proliferation of a wide variety of cell types, and especially endometrial epithelial cells. Its action is modulated by the presence of IGF-binding proteins (IGFBPs) which are secreted by IGF-I target cells. The partition of IGFBPs between cell-associated and soluble form determines the potentiation or the inhibition of IGF-I action. It is commonly accepted that cell-associated IGFBPs potentiate the IGF-I action while the soluble form of IGFBPs has an inhibitory effect. In endometrial adenocarcinoma, IGF-I is involved in tumoral progression and IGFBPs may be key modulators of the IGF-I-induced cell proliferation. Here we showed that the responsiveness of human endometrial adenocarcinoma cells (HEC-IA cell line) to the mitogenic activity of IGF-I was dependent on the pre-incubation conditions. This responsiveness to IGF-I was conditioned by a differential expression of the IGF system components (IGFBPs and IGF-I receptor) and particularly of the IGFBPs. Indeed, the IGF-I-induced proliferation of the HEC-1A cells was attenuated by the presence of cell-associated IGFBPs. Moreover, the IGF-I incubation induced a release of IGFBP-3 in the culture media as the consequence of an interaction between IGF-I and the cell-associated IGFBP-3. This effect was dose-dependent and was associated with the attenuation of the IGF-I action on cellular proliferation. Thus, IGFBP-3 might be initially expressed as a cell-associated form and then released in the interstitial fluid after a direct interaction with IGF-I. Therefore, in HEC-IA endometrial adenocarcinoma cells responsive to IGF-I, the IGFBP-3 is the main binding protein expressed and both soluble and cell-associated forms act as inhibitors of IGF-I-induced cellular proliferation.     

Protective role of retinoic acid from antiproliferative action of TNF-alpha on lung epithelial cells

Tumor necrosis factor (TNF)-alpha is a key molecule in lung inflammation. We have established the insulin-like growth factor binding protein 2 (IGFBP-2) as a marker associated with the growth arrest of lung alveolar epithelial cells (AEC). Here, we studied the effects of TNF-alpha on AEC proliferation and the putative protective role of retinoic acid (RA). We documented an antiproliferative action of TNF-alpha that was reversible only at 24 h and then became irreversible with induction of apoptosis. TNF-alpha treatment was associated with a dramatic induction of IGFBP-2. To discover the mechanism of action of IGFBP-2, we further tested the mitogenic potential of IGF-I to counteract TNF-alpha inhibition. Addition of IGF-I to the TNF-alpha containing medium did not stimulate proliferation, whereas des(1-3)IGF-I, an analog of IGF-I that bears low affinity for IGFBPs, was able to restore cell growth. Interestingly, we observed that RA abrogated TNF-alpha-induced growth arrest and that this effect was associated with a dramatic decrease in IGFBP-2 expression. These results suggest a protective role of RA from TNF-alpha antiproliferative action, through mechanisms involving modulation of IGFBP-2 production.     

Insulin-like growth factors modulate the growth inhibitory effects of retinoic acid on MCF-7 breast cancer cells

Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I,IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (≤10^?6M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10^?7 M RA, and that 10^?9–10^?7 M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10^?9–10^?7 M RA. Treatment with the IGF-I receptor blocking antibody, αIR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10^?9–10^?7 M RA enhancing cell proliferation and ≥ 10^?6M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies. © 1995 Wiley-Liss Inc.     

High retinoid X receptor expression in JEG-3 choriocarcinoma cells: Involvement in cell function modulation by retinoids

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RARα and RXRα, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RARα and RXRα. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 ± 2%) being observed after 4 days of treatment with Ro 25, a RXRα specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 ± 10%) occurred at 48 h with the RXRα-specific ligand. The RARα-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXRα in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA. J. Cell. Physiol. 176:595–601, 1998. © 1998 Wiley-Liss, Inc.     

Autocrine Production of Insulin-like Growth Factor-I (IGF-I) Affects Paracellular Transport Across Epithelial Cells In Vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.     

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.     

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.     

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.     

Interaction of retinoids and tamoxifen on the inhibition of human mammary carcinoma cell proliferation

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).     

Expression of Insulin-like Growth Factor Binding Protein-3 and Regulation of the Insulin-like Growth Factor-I Axis in Pig Testis

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs.     

Vitamin D regulates human ectocervical epithelial cell proliferation and insulin-like growth factor-binding protein-3 level

The differentiation status of the cervical epithelial cell has an important influence on responsiveness to estrogens and progestins. Several agents, including glucocorticoids and retinoids, are known to influence cervical cell differentiation. However, the effects of vitamin D have not been examined. Vitamin D is known to regulate cell proliferation and gene expression in a variety of epithelial cells. In the present study we investigated the ability of 1alpha25-dihydroxyvitamin D3 (D3) to regulate cell proliferation and expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in human ectocervical epithelial cells. ECE16-1, a non-tumorigenic cervical cell line, was growth inhibited by D3 with maximal inhibition at 1000 nM. IGFBP-3 levels increased in parallel with the growth inhibition. IGFBP-3 levels were half-maximally increased at approximately 10-100 nM and maximally increased (10- to 30-fold) at 1000 nM D3. These studies show that vitamin D regulates cervical epithelial cell gene regulation and cell proliferation and that IGFBP-3 may be an in vivo marker of vitamin D action in the cervix.     

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins.

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.     

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.     

Autocrine production of insulin-like growth factor-I (IGF-I) affects paracellular transport across epithelial cells in vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.