Regulation of insulin-like growth factor (IGF)-binding protein expression by growth factors and cytokines alters IGF-mediated proliferation of postnatal lung fibroblasts

Postnatal day 5 is the beginning of septation and the peak of postnatal fibroblast proliferation. The author and colleagues studied fibroblasts from this developmental time period to determine factors that regulate cell proliferation. Exposure of cells to insulin-like growth factor (IGF)-I for 48 hours increased cell number whereas exposure to epithelial growth factor (EGF), platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-7, FGF-2, tumor necrosis factor-alpha (TNF-alpha), or interleukin (L)-1beta did not alter cell number. Long[R3]IGF-I (a synthetic IGF analog with reduced affinity for IGF-binding proteins [IGFBPs]) was more potent than IGF-I, with half-maximal stimulation at a dose of 0.6 nM for long[R3]IGF-I compared to 1.5 nM for IGF-I, suggesting that IGFBPs in the conditioned medium (CM) inhibit IGF activity. Addition of exogenous IGFBP-3 inhibited the IGF-stimulated increase in cell number. Addition of IGFBP-4 did not alter IGF activity because IGF-I stimulated proteolysis of IGFBP-4. The expression of mRNA for PAPP-A (a known IGFBP-4 protease) suggests that the clearance of IGFBP-4 is mediated by pregnancy-associated plasma protein (PAPP)-A. Exposure of cells to TNF-alpha or IL-1beta increased IGFBP-3 mRNA abundance and IGFBP-3 protein in CM. PDGF-BB and IL-1beta increased IGFBP-4 protein abundance and PDGF-BB and dibutyryl cAMP increased IGFBP-4 mRNA. The increase in CM IGFBP-3 following TNF-alpha exposure blocked IGF-mediated cell proliferation, suggesting that the growth factor- and cytokine-mediated changes in IGFBP abundance regulate postnatal fibroblast cell proliferation.     

Basic fibroblast growth factor induces proteolysis of secreted and cell membrane-associated insulin-like growth factor binding protein-2 in human neuroblastoma cells.

Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.     

Human glomerular endothelial cells IGFBPs are regulated by IGF-I and TGF-beta1.

The release of insulin-like growth factor binding proteins (IGFBPs) and their regulation in human glomerular endothelial cells (GENC) was characterised. GENC produce IGFBP-4, IGFBP-2 and IGFBP-3 and express mRNA for IGFBP-2 to IGFBP-5. Due to the fact that IGF-I and TGF-beta1 modulate glomerular hypertrophy, their action on IGFBP release and GENC growth was studied. IGF-I increased IGFBP-3, IGFBP-2 and decreased IGFBP-4, while TGF-beta1 decreased IGFBP-3 and apparently increased IGFBP-4. All of the IGFBPs, except the TGF-beta1-regulated IGFBP-4, were modulated at mRNA level. IGF-I stimulated GENC proliferation, while TGF-beta1 inhibited their growth. It was demonstrated that an IGFBP-3 antibody reduced GENC proliferation. However, rhIGFBP-3 alone had no effect on GENC, but after 48 h pre-incubation the IGF-I stimulated GENC growth was increased, suggesting that IGFBP-3 could modulate the IGF-I induced GENC proliferation. It was concluded that the stimulatory IGFBP-3 and the inhibitory IGFBP-4 could regulate GENC growth, although the IGFBP-3 seems to have a predominant effect in this control.     

Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6.

BACKGROUND/AIMS: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1beta and tumor necrosis factor alpha (TNFalpha) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. METHODS: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [(125)I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. RESULTS: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1beta or TNFalpha were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. CONCLUSIONS: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.     

Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4.

The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testosterone and insulin-like growth factor (IGF) I interact in controlling IGF-binding protein production in androgen-responsive foreskin fibroblasts

The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.     

Role of insulin-like growth factor binding proteins in controlling IGF actions

The insulin-like growth factors (IGF) stimulate growth in multiple connective tissue cell types. The capacity of IGF-I and -II to access cell surface receptors is controlled by insulin-like growth factor binding proteins (IGFBPs). Connective tissue cells synthesize four of the IGFBPs (IGFBP-2 through -5). Synthesis is controlled by growth hormone and several other growth factors. In addition to regulating synthesis, other variables regulate the abundance of the IGFBPs including specific serine proteases that are produced for each form of IGFBP. Following cleavage, the IGFBPs have reduced affinity for IGF-I and -II, thus allowing release to receptors. Variables that regulate the amount of proteolysis have been shown to regulate IGF action. In addition to being proteolytically cleaved, three forms of IGFBPs (IGFBP-2, -3 and -5) can associate with extracellular matrix (ECM). In the case of IGFBP-5 binding to ECM, its affinity is lowered substantially allowing IGF to better equilibrate with the receptors. This event results in a potentiation of IGF-I action on fibroblasts and smooth muscle cells (SMC). In summary, IGFBPs are important molecules for regulating the bioavailability of IGF-I and -II to receptors. Understanding the variables that regulate their abundance may lead to a better understanding of the factors that regulate IGF action in skeletal tissues.     

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.     

Non-receptor mediated, post-transcriptional regulation of insulin-like growth factor binding protein (IGFBP)-3 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells secrete insulin-like growth factor binding protein 3 (IGFBP-3) as the major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by the use of cell monolayer affinity cross-linking or immunoperoxidase staining of the cell surface with a specific polyclonal anti-human IGFBP-3 antibody (alpha IGFBP-3 gamma 1). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media (CM) following addition of IGF-I and -II (maximum 13 fold increase at 100 ng/ml), but not by insulin up to 1 mg/ml; 2) no change in CM IGFBP-3 level by [Gln3,Ala4,Tyr15,Leu16] IGF-I, which has decreased affinity for IGFBPs; 3) no change in IGFBP-3 mRNA following addition of IGFs; 4) release of cell surface-associated IGFBP-3 into CM by the addition of IGFs, but not by [Gln3,Ala4,Tyr15,Leu16]IGF-I. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated dissociation of cell surface-associated IGFBP-3.     

Post-transcriptional and post-translational regulation of insulin-like growth factor binding protein-3 and -4 by insulin-like growth factor-I in uterine myometrial cells.

Involution of the uterus induced by oestrogen depletion is associated with a decrease in uterine insulin-like growth factor (IGF)-I and an increase in IGF binding protein (IGFBP) gene expression. We examined the effects of IGF-I on primary uterine myometrial cell proliferation, and on IGFBP-3 and IGFBP-4 gene expression. IGF-I enhanced DNA synthesis in these cells. In conditioned media, IGF-I increased IGFBP-3 accumulation by release of cell associated IGFBP-3. A low dose of IGF-I increased IGFBP-4 accumulation, and a high dose caused IGFBP-4 to disappear. In cell-free conditioned media IGF-I protected IGFBP-3 and enhanced IGFBP-4 proteolysis. Co-incubation of [(125)I]-IGFBP-4 with cell-free conditioned media cleaved IGFBP-4 into 18 and 12 kDa fragments. Northern blot analysis indicated that IGF-I increased IGFBP-4 mRNA accumulation by stabilizing the mRNA while IGFBP-3 gene expression was slightly decreased. The results demonstrate that IGF-I regulates IGFBP-4 post-trancriptionally and post-translationally, whereas IGFBP-3 is only affected post-translationally. By enhancing IGFBP-4 proteolysis, increasing cell-associated IGFBP-3 and stabilizing IGFBP-3, IGF-I may initiate a mitogenic response.     

Prolonged hypoxia induced by the reduction of maternal uterine blood flow alters insulin-like growth factor-binding protein-1 (IGFBP-1) and IGFBP-2 gene expression in the ovine fetus.

Insulin-like growth factors (IGF-I and IGF-II) are potent mitogenic and differentiating peptides which are synthesized by many fetal tissues. In the circulation and tissue fluids, IGFs are bound to binding proteins (BPs) which not only function as carrier proteins, but also inhibit or modulate the biological actions of IGFs. We have previously shown that prolonged hypoxia in the ovine fetus induced by the reduction of maternal uterine blood flow for 24 h causes a reduction in the DNA synthesis rate in selected fetal tissues. To determine if this effect is due to alterations in the local synthesis of tissue IGFs and their binding proteins or to changes in systemic concentrations of IGFs and IGFBPs, we have investigated the abundance of mRNAs encoding IGFs and IGFBPs in selected tissues and changes in plasma IGFs and IGFBPs. Ovine fetuses (115-120 days gestation; n = 6) underwent 24 h of hypoxia by the reduction of maternal uterine blood flow (RUBF). Controls (n = 6) underwent the same surgical procedure without RUBF. Serial plasma samples were collected before, during, and after the experiment, and tissues were collected at the end of 24 h. Mean plasma IGF-I and IGF-II concentrations tended to be lower in hypoxic fetuses than in controls during the course of hypoxia, but these differences were not statistically significant. Tissue mRNA levels for IGF-I and IGF-II in lung, muscle, thymus, and kidney were similar in control and hypoxic fetuses after 24 h of hypoxia. The relative abundance of liver IGF-I and IGF-II mRNAs was lower in hypoxic fetuses, but only IGF-I mRNA levels were significantly different from the control values (P < 0.05). Compared to control fetuses, IGFBP-1 mRNA levels in the liver of hypoxic fetuses were increased 3- to 7-fold, and IGFBP-1 mRNA expression was induced in kidneys of some hypoxic fetuses (two of six). In addition, IGFBP-2 mRNA levels were decreased in the liver (50%) and kidney (30%) of hypoxic fetuses. The increase in liver IGFBP-1 mRNA abundance and the decrease in liver and kidney IGFBP-2 mRNA abundance were accompanied by an increase in IGFBP-1 levels and a decrease in IGFBP-2 levels in fetal plasma. No changes were observed in either plasma levels or tissue mRNA abundance for IGFBP-3. Analysis of the time course of changes in plasma revealed that the changes in IGFBP-1 and IGFBP-2 occurred within 4 h of hypoxia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transfection of human insulin-like growth factor-binding protein 3 gene inhibits cell growth and tumorigenicity: a cell culture model for lung cancer

IGF-I and IGF-II are potent mitogens, postulated to exert autocrine/paracrine effects on growth regulation in human lung cancer. Their proliferative effects are modulated by IGF-binding proteins (IGFBPs), which are found in conditioned medium (CM) of lung cancer cell lines. The biological role of the IGFBPs, which are ontogenetically and hormonally regulated, is not fully understood. Both inhibitory and stimulatory effects on cell growth have been demonstrated. Exogenous IGFBP-3 has been consistently shown to block IGF action, inhibiting cell growth in vitro. In order to evaluate the action of endogenously produced IGFBP-3 on cell growth in lung cancer, we stably transfected the non-small cell lung cancer cell line NCI-H23 with human IGFBP-3 cDNA (resulting in NCI-H23 pOPI3/BP-3) or with the vector pOPI3CAT as control (resulting in NCI-H23 pOPI3CAT). RT-PCR confirmed expression of IGFBP-3-specific mRNA in NCI-H23 pOPI3/BP-3, but not in N! CI-H23 or NCI-H23 pOPI3CAT. Western ligand blot and Western immunoblot analysis of CMs yielded strong signals of the characteristic 40-44 kDa human IGFBP-3 protein in NCI-H23 pOPI3/BP-3. An IGFBP-3 ELISA demonstrated a 20-fold increase in IGFBP-3 protein expression in NCI-H23 pOPI3/BP-3 as compared with NCI-H23. The growth of NCI-H23 pOPI3/BP-3 in serum-containing medium was significantly slower (1.7-fold) than that of NCI-H23 or the vector-transfected control NCI-H23 pOPI3CAT. While the proliferation rate of parental and vector-transfected cells could be stimulated by IGF-I, IGF-II, IGF-I analog Long R(3) IGF-I or insulin, that of NCI-H23 pOPI3/BP-3 could not. Xenotransplantation in nude mice resulted in marked tumor growth after the injection of NCI-H23 or NCI-H23 pOPI3CAT, but absent or minimal growth for the IGFBP-3-transfected cell line. These data suggest that IGFBP-3 is a potent inhibitor of cell growth in human lung cancer c! ell lines and may impair tumorigenicity in vivo.     

Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.     

Coordinated control of fetal gastric epithelial functions by insulin-like growth factors and their binding proteins

The influence of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) on human gastric functions are unknown. This study was undertaken to evaluate the ability of fetal gastric mucosa to produce IGFBPs and to test the effects of IGF-I, IGF-II, and synthetic truncated IGFs that do not interact with IGFBPs on epithelial cell proliferation and glandular zymogenic function. Western blots, Far Western blots, and immunohistochemistry were performed to characterize the expression of IGFBP-1 to -6 and IGF-I receptor. The effects of growth factors on DNA synthesis and lipase and pepsin activities were determined in gastric explants maintained in serum-free organ culture. All gastric epithelial cells expressed the IGF-I receptor. IGFBP-2 to -6 were produced endogenously, and they were differentially localized along the foveolus-gland axis and modulated in culture. Exogenous IGF-I and IGF-II were able to reduce lipase activity without affecting pepsin, whereas they exerted different effects on cellular proliferation: IGF-I was stimulatory and IGF-II had no influence. Illustrating the complex regulatory effect that IGFBPs exert on IGF bioactivity, both truncated IGF-I and IGF-II stimulated DNA synthesis more than IGF-I. Moreover, the striking difference in mitogenic activity between truncated and native forms of IGF-II probably reflects the abundance of IGFBP-2 and IGFBP-6, two IGF-II carriers, in the foveolus/neck region, including the proliferative compartment. This study provides new evidence for the involvement of an intragastric IGF/IGFBP system in the fine regulation of epithelial cell division and also in the control of zymogen synthesis. Moreover, the specific influence of IGF-II as a mitogen appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells.