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.     

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.     

Expression of a lactogen-dependent insulin-like growth factor-binding protein in cultured mouse mammary epithelial cells.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.     

Transforming growth factor-beta induces growth inhibition and IGF-binding protein-3 production in prostatic stromal cells: abnormalities in cells cultured from benign prostatic hyperplasia tissues

The IGF axis has been implicated in the pathogenesis of benign prostatic hyperplasia (BPH) via the paracrine action of IGFs and IGF-binding proteins (IGFBPs). In this study, we examined the regulation of cell growth and IGFBP-3 secretion by transforming growth factor-beta (TGF-beta) in prostatic stromal cell (PC-S) cultures from histologically normal tissues and tissues from BPH. PC-S cultures were treated with varying doses of TGF-beta1. Forty-eight hour conditioned media (CM) from these cultures were subjected to Western immunoblotting and ligand blotting for detection and quantification of IGFBPs. IGFBPs-2, -3 and -4 were detected in the CM from normal PC-S cultures. In CM from BPH PC-S, IGFBP-3 levels were 2-fold lower at baseline than in the normal PC-S CM, in addition to the differences in IGFBPs-2 and -5 which we have previously reported. In response to TGF-beta1, a 15-fold increase in the levels of IGFBP-3 was observed in normal PC-S CM, while a mere 2-fold increase was observed in BPH PC-S CM (P<0.001). These findings were confirmed by specific immunoblotting and immunocytochemistry. IGFBP-3 mRNA levels detected by Northern blotting of total RNA extracted from similar cultures showed the induction of IGFBP-3 expression by TGF-beta1 in normal PC-S and its lack of induction in BPH PC-S. Cell growth inhibition in response to TGF-beta1 correlated with the IGFBP-3 concentrations found in CM. Normal PC-S showed a 60% decrease in cell number after 10 days in media with 1 ng/ml TGF-beta1, compared with the untreated control. The decrease in proliferation observed in comparably treated BPH cells was only 20% (P<0.001). In conclusion, BPH PC-S had a reduced IGFBP-3 response to TGF-beta1 and demonstrated decreased TGF-beta1-induced growth inhibition relative to normal PC-S. We hypothesize that in normal PC-S, TGF-beta exerts its anti-proliferative effects by stimulating the production of IGFBP-3, which acts as an inhibitory factor, either by inhibiting IGFs or directly by interacting with cells, and that this process is altered in BPH PC-S.     

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.     

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)     

The relationship between the circulating IGF system and the presence of retinopathy in Type 1 diabetic patients.

BACKGROUND: Patients with proliferative diabetic retinopathy (PDR) have increased vitreous levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding proteins (IGFBPs). This accumulation is probably caused by increased leakiness of the blood-retina barrier and influx of circulating IGFs and IGFBPs. To date, interest has focused on the role of circulating total IGF-I in the development of PDR, and there are only sparse data on circulating levels of free IGF-I and IGFBPs. METHODS: We compared fasting serum samples from matched groups of Type 1 diabetic patients with no retinopathy (n = 29), non-PDR (n = 13) and PDR (n = 16). We also included matched controls (n = 26). Serum was analysed for free and total IGF-I and -II, free plus dissociable IGF-I, IGFBP-1, -2 and -3, IGFBP-1-bound IGF-I as well as IGFBP-3 proteolysis. RESULTS: When compared with controls, diabetic patients (n = 58) showed reduced (P < 0.0005) levels of free and total IGFs, free plus dissociable IGF-I and IGFBP-3, whereas levels of IGFBP-1, IGFBP-1-bound IGF-I and IGFBP-2 were elevated. IGFBP-3 proteolysis remained unaltered. When comparing diabetic patients with different degrees of retinopathy, IGFBP-2 and IGFBP-1-bound IGF-I were the only parameters that differed significantly. Patients with retinopathy (non-PDR as well as PDR) had elevated IGFBP-2 (P < 0.03) and reduced IGFBP-1-bound IGF-I (P < 0.03), when compared with patients without retinopathy. Noteworthy, both parameters correlated (0.11 < r2 < 0.14, P < 0.02) with the severity of retinopathy. CONCLUSION: Our study gives no evidence of a direct role of circulating free IGF-I in the development of PDR, whereas IGFBP-2 and IGFBP-1-bound IGF-I showed a relationship with the degree of retinopathy. However, further investigations are needed in order to clarify the basis and clinical relevance of this finding.     

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.     

The insulin-like growth factor system in the GT1-7 GnRH neuronal cell line

Evidence suggests that insulin-like growth factors (IGFs; IGF-I and IGF-II) are involved in the regulation of reproductive function including the development of the gonadotropin-releasing hormone (GnRH) neuronal system and the modulation of GnRH secretory activities. To further characterize the regulatory role of the IGF system on GnRH neuronal function, we have examined the gene expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-binding proteins (IGFBPs) in a GnRH neuronal cell line (GT1-7 cells). The relative effects of IGFs and insulin on GnRH secretion by these cells was also investigated. RT-PCR analysis demonstrated IGF-I, IGF-II and IGF-IR mRNAs in GT1-7 cells. The mRNAs for IGFBP-2, -3, -4, -5 and -6 but not IGFBP-1 were also detected. Immunoreactive protein bands for IGFBP-2, -4 and -5 but not for other IGFBPs were demonstrated by Western blot with IGFBP-5 appearing to be the most abundant IGFBP secreted by GT1-7 cells. IGFBP-5 production by GT1-7 cells was stimulated by both IGF-I and IGF-II in a dose-dependent manner with approximately equal potency, whereas insulin caused no significant effect. GnRH secretion by GT1-7 cells treated with IGF-I or IGF-II but not insulin showed an increase (80-100%) at 2 h of treatment followed by a decrease (46%) at 6 h that continued up to 24 h. We conclude that the expression of IGFs, IGF-IR and IGFBPs and their interactions in the regulation of GnRH secretion by GT1-7 cells as demonstrated by our study provide a basis for an autocrine regulatory role for the IGF system in GnRH neuronal secretory activities.     

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.     

Inflammation-related neutrophil proteases, cathepsin G and elastase, function as insulin-like growth factor binding protein proteases.

Over the past few years, several proteolytic enzymes have been identified as insulin-like growth factor binding protein (IGFBP) proteases. It has been suggested that proteolytic cleavage of IGFBPs is associated with regulation of the proliferative effects of IGFs on their target cells. In this study, we have demonstrated that two neutrophil proteases, cathepsin G and elastase, effectively cleave IGFBPs in vitro and in vivo at concentrations lower than previous described IGFBP proteases. Purified leukocyte cathepsin G and elastase cleaved all six well-characterized IGFBPs into distinct fragments in a concentration-dependent manner. Under similar experimental conditions, cathepsin G preferentially cleaved IGFBP-5, followed by BP-2, BP-3, BP-4, BP-1, and BP-6. In comparison, elastase equally preferred IGFBP-3 and IGFBP-4, followed by BP-1, BP-5, BP-6, and BP-2. Proteolysis of rh(125)I-IGFBP-3 by cathepsin G was blocked by alpha(1)-antichymotrypsin, while elastase proteolytic activity was blocked by alpha(1)-proteinase inhibitor as expected. Elastase, but not cathepsin G, cleaved free IGF-I into a smaller molecular weight fragment in vitro, possibly designating unique functions for each protease within the IGF axis. Sequence analysis of IGFBP-3 fragments produced by cathepsin G and elastase demonstrated that each protease cleaved IGFBP-3 at unique sites within its midregion. More importantly, extracts from purified neutrophils have demonstrated significant proteolytic cleavage of IGFBP-3 that resembles elastase proteolysis of IGFBP-3. Recent studies using a monocyte-like cell model have also shown significant cleavage of IGFBP-3. These in vitro and in vivo data suggest that the neutrophil proteases, cathepsin G and elastase, in addition to their previously described functions as extracellular matrix-degrading enzymes, may potentially act as IGFBP proteases involved in regulation of IGFs and IGFBPs during inflammation and wound healing.     

Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.

The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.     

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Transgenic overexpression of pregnancy-associated plasma protein-A increases the somatic growth and skeletal muscle mass in mice

Although IGFs are indispensable to skeletal muscle development, little information is available regarding the mechanisms regulating the local action of IGFs in skeletal muscle tissues. Here we tested the hypothesis that pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, promotes skeletal muscle formation in vivo through degrading IGF binding proteins (IGFBPs), which increases the bioavailability of IGFs. Expression of PAPP-A is significantly increased in muscle five days after muscle injury in mice. Targeted overexpression of PAPP-A using a muscle-specific promoter significantly increased the prenatal/postnatal growth, skeletal muscle weight, and muscle fiber area in mice. These anabolic effects were reproduced using F2/F3 progeny. Free IGF-I concentration was severalfold higher in the conditioned medium (CM) of ex vivo cultured muscle from the transgenic mice, compared with the wild-type littermate muscle. Accordingly, the proliferation of C2C12 myoblasts was significantly increased in the presence of CM from cultured skeletal muscle of the transgenic mice, compared with the controls. This observed increase in myoblast proliferation was abolished on addition of noncleavable IGFBP-4 peptide, which reduced free IGF-I concentration back to the basal level of the wild-type CM. Furthermore, proliferation and differentiation of C2C12 myoblasts was increased by transient overexpression of proteolytically active PAPP-A but not by inactive mutant PAPP-A (E483/A). Collectively, we identified PAPP-A as a novel regulator of prenatal/postnatal growth and skeletal muscle formation in vivo. Moreover, our studies provide the first experimental evidence that IGFBP degradation is a key determinant in modulating the local action of IGFs in muscle.     

Complex mediation of uterine endometrial epithelial cell growth by insulin-like growth factor-II (IGF-II) and IGF-binding protein-2

The coexpression of IGF (-I and -II) peptides, corresponding receptors, and IGF binding proteins (IGFBPs) in uterine endometrium suggests that a significant component of IGF action in this tissue is via autocrine or paracrine pathways, or both. The present study examined whether IGF-II and a major uterine-expressed IGF-II binding protein, IGFBP-2, modulate endometrial epithelial cell mitogenesis. Serum-deprived porcine endometrial glandular epithelial (GE) cells of early pregnancy were treated with various concentrations of IGFs, recombinant porcine (rp) IGFBP-2, or both, and examined for changes in cellular mitogenesis by incorporation of [(3)H]thymidine into DNA. Recombinant human (rh) IGF-II stimulated DNA synthesis in a dose-dependent manner. Human [Leu(27)]-IGF-II, an analog with selective affinity for the IGF-II (type II) receptor, increased thymidine uptake by twofold compared with untreated GE cells. When added in combination with an equimolar concentration of rhIGF-I, [Leu(27)]-IGF-II or rhIGF-II stimulated thymidine incorporation to a greater extent than did rhIGF-I alone. Ligand blot analysis of GE cell conditioned medium revealed the presence of four IGFBPs with molecular masses of 48, 31, 23, and 15 kDa. Physiological concentrations of rpIGFBP-2 (nM range) increased both basal and IGF-induced DNA synthesis in GE cells. At equimolar concentrations, Des(1-6)IGF-II (an IGF-II analog with much reduced affinity for IGFBPs) and rpIGFBP-2 had additive effects on GE cell mitogenesis, suggesting that the IGFBP-2 modulation of uterine cell growth may involve both IGF-dependent and IGF-independent pathways. Our results demonstrate the complex interplay of IGF system components in uterine endometrial epithelial growth regulation in vitro, identify IGF-II and IGFBP-2 as locally coexpressed uterine epithelial cell mitogens, and suggest the presence of a functional signaling pathway by which IGF-II stimulates epithelial cell proliferation via the type II receptor.     

Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity.

The cDNAs encoding three major insulin-like growth factor-binding proteins (IGFBPs) have been cloned and sequenced. We have examined, by Western ligand blotting, the profiles of these binding proteins in human female serum in the normal menstrual cycle, throughout pregnancy, and during the postpartum period. There was no change in the serum profile of any of the binding proteins in early pregnancy compared to that in the secretory phase of the menstrual cycle. However, there was a marked decrease in circulating levels of the main serum IGFBP, IGFBP-3, after 6 weeks of gestation, continuing progressively to term and returning to nonpregnant levels by 5 days postpartum. IGFBP-2 decreased steadily throughout gestation. In contrast, IGFBP-1 levels were found to rise by the second trimester. Endoglycosidase-F digestion did not enhance detection of IGFBP-3 by ligand blotting. Immunoprecipitations with two separate antibodies against IGFBP-3 and IGFBP-2, followed by Western ligand blotting, confirmed the marked decrease in IGFBP-3 levels after 6 weeks of gestation and the more gradual decrease in IGFBP-2. In contrast, immunoprecipitations with IGFBP-1 monoclonal antibodies confirmed the increase in IGFBP-1 during gestation. Endogenous serum IGFs were separated from serum IGFBPs by acid chromatography, and an 80% decrease in total IGF-binding activity in the IGFBP fraction of chromatographed pregnancy vs. nonpregnancy serum was detected by charcoal absorption assay. Furthermore, immunoprecipitations of IGF affinity cross-linked IGFBP fractions with IGFBP-3-specific antiserum confirmed a marked diminution of IGFBP-3 in pregnancy compared to nonpregnancy serum, and revealed, only in pregnancy serum, the concomitant appearance of a band with a mol wt of 34K and three less intense bands with mol wt between 20-26K on sodium dodecyl sulfate gels. Incubation of nonpregnancy serum with 6-week pregnancy serum at 37 C for 5 h, followed by Western ligand blotting, showed only a slight reduction in the amount of IGFBP-3 in the mixture compared to that in controls. However, incubation of term pregnancy with nonpregnancy serum at 37 C for 5 h revealed a marked reduction of IGFBP-3 in the mixture. When iodinated recombinant IGFBP-3 was incubated with term pregnancy serum under the same conditions, the appearance of a 29K protein was identified by gel electrophoresis and autoradiography, along with three less intense bands with mol wt between 17-22K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells.

The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.     

Synthesis of placental protein 12 by human endometrium

We have previously shown that placental protein 12 (PP12) is synthesized and secreted by human term pregnancy decidua in vitro. In the present study, fragments of proliferative and secretory phase endometrium were cultured in media in the presence and absence of progesterone (P) and 17 beta-estradiol (E2) for 96 h. The PP12 concentrations in the media and tissues were measured by RIA, and de novo synthesis was investigated by measuring the incorporation of [35S]methionine into PP12. Before culture, PP12 could not be detected in any proliferative endometria, whereas all secretory endometria contained PP12. All secretory endometria released PP12 into the medium in the presence and absence of added P and E2. Secretory endometria released significantly more PP12 than proliferative endometria. Three of seven proliferative endometria did not release PP12 in the absence of P, but all did so after P had been added. The addition of P to culture medium caused a 2.4-to over 71-fold increase in PP12 secretion over control values in proliferative endometria and up to a 3.5-fold increase in secretory endometrium. E2 had no significant effect. Cycloheximide totally inhibited the PP12 release induced by P from proliferative endometrium, and in secretory endometrium, it either totally blocked PP12 release or inhibited the stimulation due to P. [35S]Methionine was incorporated into immunoprecipitable PP12 in cultures of secretory and P-treated proliferative phase endometria. These results demonstrate de novo synthesis of PP12 by nonpregnant endometrium in tissue culture and suggest that the biosynthesis and secretion of PP12 by nonpregnant endometrium are regulated by P.