Insulin-like growth factor binding protein profiles in human ovarian follicular fluid correlate with follicular functional status.

The insulin-like growth factors (IGF), IGF-I and IGF-II, influence ovarian follicular development. The IGF binding proteins (IGFBPs) comprise at least six distinct species which may modulate IGF action. We examined IGFBP profiles in follicular fluid (FF) from developing human ovarian follicles with aromatase activity as well as from androgen-dominant, atretic follicles. Western ligand blot analysis of FF from both atretic and active follicles revealed the presence of IGFBP-3 and IGFBP-2, confirmed by immunoprecipitation with specific antiserum, as well as 28 kilodalton (kDa) and 24 kDa IGFBPs. In FF specimens obtained during the follicular phase, IGFBP-2 and the 28 kDa and 24 kDa species were present in 3-, 6-, and 19-fold higher amounts in atretic compared to healthy developing follicles, respectively, and were present in greater amounts in atretic FF than in serum. In atretic FF obtained during the luteal phase, lower levels of IGFBP-2 and the 28 and 24 kDa IGFBPs were seen than in atretic follicles in the follicular phase. IGFBP-3 levels were comparable in all types of follicles and in serum. The 28 kDa IGFBP is glycosylated, and its level varied in parallel with the 24 kDa IGFBP, suggesting that these are variants of the recently identified IGFBP-4. IGFBP-1 was not detectable by immunoprecipitation and ligand blotting of FF obtained during the follicular phase. These data suggest that one or more IGFBPs present in higher levels in FF from atretic compared to healthy, developing follicles may play an important role in folliculogenesis and follicular atresia.     

Insulin-like growth factor binding proteins in ovarian follicles from women with polycystic ovarian disease: cellular source and levels in follicular fluid.

The follicular fluid (FF) of human ovaries contains insulin-like growth factor binding proteins (IGFBPs) which may regulate the bioavailability of the IGFs. Previously we showed discrete changes in IGFBP concentrations in FF which correlated with the physiological state of the follicle. The purpose of the present study was to test the hypothesis that IGFBPs in FF may be increased in polycystic ovarian disease (PCO). FF was aspirated from PCO follicles and size matched healthy and atretic follicles from normal ovaries of naturally cycling women. The IGFBPs in FF samples were studied by ligand blot analysis with 125I-IGF-II and by immunoblot analysis using specific antisera to five human IGFBPs. Of six IGFBP bands in PCO FF, three were identified as IGFBP-2, IGFBP-3, and IGFBP-4. Bands (29K and 31K) were not identified by any of the five IGFBP antisera. The total IGF binding capacity was increased in PCO FF relative to normal healthy or atretic FF. IGFBP-3 was the predominant BP present in FF from PCO follicles as well as normal healthy and atretic follicles with no significant difference in any of the groups of follicles studied. IGFBP-2, IGFBP-4, and the 29K BP were present in smaller amounts, but there were significantly higher levels of each of these BPs in PCO follicles than in normal healthy follicles. Only IGFBP-4 was elevated in PCO follicles relative to normal atretic follicles indicating that the pattern of IGFBP expression in PCO FF was very similar to the pattern observed in atretic follicles. To determine the source of the IGFBPs in FF, granulosa or theca cells were cultured (up to 6 days) in serum-free medium. Ligand blot analysis of the conditioned medium revealed basal secretion of IGFBP-3 by both theca and granulosa cells. FSH inhibited granulosa cell IGFBP-3 production but increased the 29K BP in the medium. Transforming growth factor-beta stimulated basal IGFBP-3 secretion and reversed the FSH effects. Human CG (100 ng/mL) inhibited theca cell IGFBP-3 production but did not stimulate any other IGFBP. The results of our studies indicate that IGFBP-2, IGFBP-4, and the 29K BP are significantly increased in PCO FF and that gonadotropins and TGF-beta regulate the production of IGFBPs by human theca and granulosa cells.     

INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS IN FOLLICULAR FLUID FROM NORMAL DOMINANT AND COHORT FOLLICLES, POLYCYSTIC AND MULTICYSTIC OVARIES

There is now considerable evidence that the insulin-like growth factors (IGFs) play an important role in the human ovary. It has also recently become apparent that the physiological activity of the IGFs is modulated by a number of specific binding proteins (IGFBPs). In order to understand the role of the IGFs in ovarian physiology, the presence and functions of these IGFBPs will need to be characterized. As an initial step towards this we have investigated the presence of the various binding proteins by Western ligand blotting and have measured the levels of one of them, IGFBP-1, in follicular fluid (FF) obtained from unstimulated dominant and cohort follicles in 19 normal women and in eight patients with polycystic and one with multicystic ovaries. In normal women, IGFBP-1 levels in dominant follicles were similar to matched serum levels but were significantly lower in cohort follicles. IGFBP-1 levels correlated with FF-volume (r = 0.58, P less than 0.001) and with paired serum levels (r = 0.63, P less than 0.001). In post-LH surge dominant follicles this relationship with serum levels no longer held and in three out of nine subjects FF levels were higher than in serum. Thus IGFBP-1 in normal human FF appears to be partly derived from the circulation but with additional local production in the larger developing dominant follicles. Western ligand blotting revealed five IGF-binding proteins in FF running parallel with those identified in serum, suggesting that the IGFBP species previously identified in serum may also be present in FF. The two bands in positions corresponding to the components of the large (150kDa) binding complex were, as in serum, the predominant forms and in most FF samples these were even more prominent than in the accompanying serum sample. This contrasts with previous studies in lymph which suggested that the 150kDa complex was largely retained in the circulation. All three small IGFBPs varied considerably between FF samples even within an individual; each IGFBP varied independently of the other IGFBPs. Our results demonstrate that at least four discrete IGFBPs are present in FF and suggest that each may be produced independently within the ovary.     

Characterization of insulin-like growth factor-binding proteins in rat serum, lymph, cerebrospinal and amniotic fluids, and in media conditioned by liver, bone and muscle cells.

Insulin-like growth factor-binding proteins (IGFBPs) in rat serum, lymph, amniotic fluid and cerebrospinal fluid (CSF), and in rat cell-conditioned media were characterized using a combination of gel-permeation chromatography, Western immunoblots and Western-ligand analysis. Adult serum and abdominal lymph contained a 200 kDa IGFBP (the putative type-II IGF receptor) and a 150 kDa IGFBP that contained subunits of 40-50 kDa aligning with porcine IGFBP-3 on Western-ligand blots. In addition, both fluids contained the smaller IGFBPs: a 30 kDa IGFBP which was immunoreactive with IGFBP-2 antiserum, a 28 kDa IGFBP which electrophoresed with human IGFBP-1, and a 24 kDa IGFBP. In contrast, fetal serum and amniotic fluid lacked the 150 kDa and the 28 kDa IGFBPs. CSF contained only a 30 kDa IGFBP, but this was not IGFBP-2. Several IGFBPs were detected in media conditioned by liver, bone and muscle cells. Liver-derived cells and some hepatoma cell lines produced similar patterns upon ligand blot analysis, i.e. IGFBPs of 30 kDa (which reacted with IGFBP-2 antiserum), 28 kDa and 24 kDa. A hepatoma cell line, HTC, and a smooth muscle cell line contained only an IGFBP of 26 kDa. Skeletal muscle-derived cells (L6 myoblasts) produced a 28 kDa, a 26 kDa and a 24 kDa IGFBP. Both calvarial osteoblasts and osteogenic sarcoma cells produced an IGFBP of 30 kDa that cross-reacted with IGFBP-2 antisera. In addition, osteogenic sarcoma cells produced a 28 kDa and a 24 kDa IGFBP. These results allow us partially to classify and to compare the IGFBPs in rat fluids and those produced by cultured cells.     

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)     

Production of insulin-like growth factor binding proteins (IGFBPs) by porcine granulosa cells: identification of IGFBP-2 and -3 and regulation by hormones and growth factors.

Using ligand blotting and immunoprecipitation we have characterized the insulin like growth factor binding proteins (IGFBPs) produced by cultured porcine granulosa cells. Ligand blot analysis of granulosa cell conditioned medium revealed 5 bands of IGF binding activity with apparent molecular sizes of 44, 40, 34, 29, and 22 kilodaltons (kDa). The 40-44 kDa bands of granulosa- conditioned medium were identified by immunoprecipitation with an antibody to porcine IGFBP-3, the acid-stable subunit of the 150 kDa GH-dependent serum IGFBP complex. The 34 kDa band was immunoprecipitated by an antibody to the rat IGFBP-2, the major IGFBP found in fetal rat serum and in BRL-3A cell cultures. To date we have been unable to immunoprecipitate the 29 and 22 kDa bands with any of the antibodies tested including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGFBP. The pattern of secretion varied with size of the follicles from which granulosa cells were obtained and the culture conditions. With cells from small (2-4 mm) follicles, short term cultures secreted mainly IGFBP-3 and IGFBP-2, while IGFBP-2 and the 29 and 22 kDa bands were pronounced in longer term cultures. In short term culture, granulosa cells from medium sized (4-6 mm) porcine follicles produced IGFBPs in substantially greater amounts than did those from small (1-3 mm) follicles, but exhibited comparable band patterns. The production of IGFBPs was inhibited by cycloheximide. IGFBP production by granulosa cells in culture was regulated by hormones and growth factors. The most striking effects were the inhibition of IGFBP-3 secretion by transforming growth factor beta and FSH. In contrast, IGFBP-3 levels were enhanced by epidermal growth factor. The IGFBPs produced by cultured porcine granulosa cells are identical in size and immunoreactivity to those previously found in porcine follicular fluid. Thus, follicular cells may be the source of follicular fluid IGFBPs. The IGFBPs may be important modulators of the IGF autocrine/paracrine system in the ovary.     

Inhibition of human fibroblast insulin-like growth factors binding protein (IGFBP) production by IGFBP-3.

Human neonatal fibroblasts in monolayer culture secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [125I]IGF-II ligand blotting, human neonatal fibroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs.     

Expression patterns of insulin-like growth factor-binding proteins 1, 2, 3, 5, and 6 in the mid-cycle monkey ovary

IGFs and IGF-binding proteins (IGFBPs) are thought to play important roles in ovarian follicular growth and selection. To elucidate the role of IGFBPs in primate ovarian function, we analyzed IGFBP mRNA expression patterns in ovaries from mid-cycle rhesus monkeys using in situ hybridization. IGFBP-1 mRNA was concentrated in theca-interstitial cells and was present at low levels in granulosa cells of atretic follicles. IGFBP-2 mRNA was expressed in the ovarian surface epithelium and granulosa cells of all antral follicles, including obviously atretic as well as dominant follicles. IGFBP-3 mRNA was localized in oocytes and in the ovarian vascular endothelium; this mRNA was also concentrated in the superficial cortical stroma in which it was distinctly more abundant in the nondominant ovary. Granulosa cells of mature dominant and ovulatory follicles selectively expressed IGFBP-5 mRNA. IGFBP-5 mRNA was also widely expressed in the ovarian stroma, in which, in contrast to IGFBP-3, it was distinctly more abundant in dominant, compared with nondominant, ovary. IGFBP-6 mRNA was present at low levels in the ovary interstitium and theca externa and was more abundant in the ovary surface epithelium. These novel data reveal distinctive cellular expression patterns for IGFBPs 1, 2, 3, 5, and 6 in the nonhuman primate ovary, suggesting distinct roles for each binding protein in ovarian function.     

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.     

Most of the circulating insulin-like growth factors-I and -II are present in the 150 kDa complex during human pregnancy.

The aim of this study was to assess the molecular size distribution of insulin-like growth factors (IGFs) complexed to IGF-binding proteins (IGFBPs) in serum of non-pregnant and pregnant women. Sera were fractionated on a size-exclusion column at pH 7.4 to resolve different IGF-IGFBP complexes from unbound IGFs. Each fraction was further chromatographed on a size-exclusion column under acid conditions to dissociate IGFs from binding proteins prior to measurement of the IGF content of the complexes. The IGF-containing fractions from the acid column were assayed specifically for IGF-I and IGF-II. Serum pooled from pregnant women contained more IGF-I and IGF-II than serum from non-pregnant women. In both groups most of the IGF-I and IGF-II was found in a large (150 kDa) complex in serum and the remainder was present in complexes eluting in the 40-50 kDa region at pH 7.4. Some free IGF-I was also detected. Serum fractions collected by size-exclusion chromatography at pH 7.4 were also analysed by Western-ligand blotting to characterize the IGFBPs in the two main IGFBP size classes. In serum pooled from non-pregnant women, the 150 kDa IGF-IGFBP complexes contained a 40-50 kDa IGFBP doublet following Western-ligand blot analysis. This IGFBP co-migrated with a pure IGFBP-3 standard. IGFBPs of 34, 28 and 24 kDa all contributed to the 40-50 kDa IGF-IGFBP complexes of the pH 7.4 chromatograph.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exogenous porcine growth hormone on serum insulin-like growth factor-binding proteins in growing pigs.

The insulin-like growth factor-binding proteins (IGFBPs) in sera of growing pigs were partially characterized with respect to their size, immunological relationships to other known IGFBPs and their regulation by porcine (p) GH. Castrated male pigs (14-16 weeks of age) were treated with either vehicle or pGH (up to 100 micrograms/kg body weight per day) by daily i.m. injection for 7 days. Blood samples were collected by jugular venepuncture at the time of injection. Five IGFBPs of 43, 40, 34, 30 and 26 kDa were identified on ligand blots of porcine sera. A 30 kDa IGFBP, in addition to the 43 and 40 kDa IGFBPs, was immunoprecipitated by antiserum to pIGFBP-3 and found to contain N-linked carbohydrate suggesting that it is a fragment of pIGFBP-3 as has been noted for a 29 kDa N-glycosylated IGFBP in rat sera. The 34 kDa IGFBP in pig sera was precipitated by antisera to rat IGFBP-2 and contained no N-linked carbohydrate. Administration of pGH to normal growing pigs not only increased pIGFBP-3 levels but elicited a dose-dependent suppression of levels of the 34 kDa IGFBP as well. In summary, the Mr pattern of IGFBPs in the sera of growing pigs is similar to that observed in fetal and maternal pig sera and in other species. Furthermore, we report that administration of pGH to normal pigs suppresses the expression of an IGFBP-2-like IGFBP in pig sera while increasing expression of pIGFBP-3.     

Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal aminoacid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5'- and 3'-rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23.8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3'-untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1.     

Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: effects of hypophysectomy and an IGFBP-specific serum protease activity.

Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I]IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42%, 17%, and 10% of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93% of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor binding proteins in human breast cancer cells: relationship to hIGFBP-2 and hIGFBP-3.

Human breast cancer cells (HBCC) secrete at least four different forms of IGFBPs. We have previously demonstrated that hIGFBP-1 is a minor component of IGFBPs secreted by Hs578T cells and is absent in CM from MCF-7 cells. In our present report, we describe the immunological and structural relationship of HBCC IGFBPs to hIGFBP-2 and hIGFBP-3. Analysis of conditioned media (CM) from Hs578T by Western ligand blotting revealed three IGFBPs of apparent Mr = 38K, 28K, and 24K; CM from MCF-7 revealed only two IGFBPs, of apparent Mr = 31K and 24K. Immunoprecipitation studies with polyclonal antibodies raised against hIGFBP-2 and hIGFBP-3 demonstrated that the 38K IGFBP in Hs578T CM is immunologically related to hIGFBP-3, while the 31K IGFBP in MCF-7 cells is related to the hIGFBP-2. Analysis by Northern blot demonstrated that MCF-7 cells contained mRNA for hIGFBP-2, while Hs578T cells contained the mRNA characteristic of the hIGFBP-3. The identity of the 24K IGFBP remains unknown, and may represent a distinct IGFBP. Of note, assay of CM following removal of BPs by acid chromatography demonstrated no detectable IGF-I or -II. The role of these IGFBPs in HBCC is of interest in view of the potential modulation of IGF actions by these proteins.     

The expression of insulin-like growth factor binding proteins is tissue specific during human fetal life and early infancy.

The insulin-like growth factors (IGFs) are bound to multiple IGF binding proteins (IGFBPs) that are present both in the circulation and in extracellular fluids. There are at least six different IGFBP species that have been fully characterized in terms of molecular structure and amino acid sequence. The tissue distribution and local production of these proteins as well as the regulation of IGFBP production in different tissues have not been elucidated. We have studied the distribution of multiple IGFBP species in protein extracts from human kidney, skeletal muscle, lung, liver and brain by ligand blotting employing [125I]IGF-2 as the radiolabeled hormone. Five distinct IGFBP species with a respective molecular weight of 43, 38, 34, 30 and 20 kDa were detected on the ligand blots in tissues from human fetuses and infants (23 weeks of gestation till 24 months of postnatal age). The 34 kDa species and a 30-32 kDa IGFBP species were predominant in brain, whereas a 30 kDa IGFBP species was mainly detected in skeletal muscle. Immunoblotting experiments using an anti IGFBP-2 antiserum showed that the 34 kDa IGFBP species from human brain was presumably related to IGFBP-2. We conclude that IGFBPs are differentially expressed in different tissues throughout human fetal life and early infancy. Local production or accumulation of the different IGFBPs could modulate IGF action at a local level or alternatively have differential functions during development.     

Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification.

Isolated sheep thyroid follicles release specific insulin-like growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.     

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.     

Circulating salmon 41-kDa insulin-like growth factor binding protein (IGFBP) is not IGFBP-3 but an IGFBP-2 subtype

In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150 kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50 kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates.     

Characterization of insulin-like growth factor binding proteins produced in the rat central nervous system.

Insulin-like growth factor binding proteins (IGFBP) are thought to modulate the biological actions of the insulin-like growth factors (IGF), including possible regulatory roles in the growth and differentiation of the central nervous system. Extracellular fluids usually contain a mixture of IGFBPs, three of which have been cloned, sequenced, and designated IGFBP-1, -2, and -3. We used Western ligand blotting, immunoprecipitation, and competitive binding analysis to characterize IGFBPs found in fetal and adult rat cerebrospinal fluid (CSF) and IGFBPs produced by cultures of neonatal rat choroid plexus, astrocytes, and C6 glial cells. Pooled rat CSF contains primarily IGFBP-2 (a narrow band at Mr = 29,000), lesser quantities of IGFBP-3 (a multicomponent broad band at Mr = 37,500-43,000), and trace amounts of low mol wt IGFBPs. Conditioned medium from cultures of choroid plexus cells contained a single binding protein corresponding to IGFBP-2, whereas C6 cells made predominately an IGFBP corresponding to IGFBP-3. Astrocytes secreted two IGFBPs corresponding to IGFBP-2 and -3, primarily IGFBP-3. Neonatal CSF contained substantially more binding activity corresponding to IGFBP-2 than did adult CSF. In all samples showing Western ligand binding profiles corresponding to IGFBP-2, identification was established by immunoprecipitation. Competitive binding analysis performed on choroid plexus IGFBP showed preferential high affinity binding for IGF-II compared with that for IGF-I. In conclusion, CSF contains a mixture of distinct IGFBPs, primarily IGFBP-2. The other IGFBPs found in CSF are capable of being synthesized locally within the central nervous system by glial cells and neurons, suggesting that they are not derived from plasma by transport across the blood-brain barrier.