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.     

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.     

Purification of a 41-kDa insulin-like growth factor binding protein from serum of chinook salmon,Oncorhynchus tshawytscha

In salmon, at least three insulin-like growth factor binding proteins (IGFBPs) with molecular masses of 41, 28, and 22kDa exist in serum. The 41-kDa IGFBP is up-regulated by growth hormone treatment and down-regulated by fasting, suggesting that it is a homolog of IGFBP-3. We purified the 41-kDa IGFBP from chinook salmon serum by IGF-I affinity chromatography followed by reversed-phase high pressure liquid chromatography. Purified IGFBP appeared as doublet bands on electrophoresis and was N-glycosylated. Analysis of partial N-terminal amino acid sequence revealed that salmon 41-kDa IGFBP has the cysteine rich domain conserved among IGFBP family. In a binding assay using 125I-salmon IGF-I, purified 41-kDa IGFBP specifically bound salmon IGF-I, human IGF-I and human IGF-II, but neither Long R(3)IGF-I nor salmon insulin, showing that binding characteristics of the salmon IGFBP are similar to those of mammalian IGFBPs. Although the partial amino acid sequence of 41-kDa IGFBP showed highest homologies with zebrafish and seabream IGFBP-2, the highly conserved nature of the N-terminus makes it impossible to identify the type of IGFBP from partial sequence data. However, based on physiological responses, molecular weight and type of glycosylation, the 41-kDa IGFBP is most similar to mammalian IGFBP-3.     

Development of an RIA for salmon 41 kDa IGF-binding protein

Salmon plasma contains at least three IGF-binding proteins (IGFBPs) with molecular masses of 41, 28 and 22 kDa. The 41 kDa IGFBP is similar to mammalian IGFBP-3 in size, type of glycosylation and physiological responses. In this study, we developed an RIA for the 41 kDa IGFBP. The 41 kDa IGFBP purified from serum was used for antibody production and as an assay standard. Binding of three different preparations of tracer were examined: (125)I-41 kDa IGFBP, (125)I-41 kDa IGFBP cross-linked with IGF-I and 41 kDa IGFBP cross-linked with (125)I-IGF-I (41 kDa IGFBP/(125)I-IGF-I). Only binding of 41 kDa IGFBP/(125)I-IGF-I was not affected by added IGFs, and therefore it was chosen for the tracer in the RIA. Plasma 41 kDa IGFBP levels measured by RIA were increased by GH treatment (178.9+/-4.9 ng/ml) and decreased after fasting (95.0+/-7.0 ng/ml). The molarities of plasma 41 kDa IGFBP and total IGF-I were comparable, and they were positively correlated, suggesting that salmon 41 kDa IGFBP is a main carrier of circulating IGF-I in salmon, as is mammalian IGFBP-3 in mammals. During the parr-smolt transformation (smoltification) of coho salmon, plasma 41 kDa IGFBP levels showed a transient peak (182.5+/-10.3 ng/ml) in March and stayed relatively constant thereafter, whereas IGF-I showed peak levels in March and April. Differences in the molar ratio between 41 kDa IGFBP and IGF-I possibly influence availability of IGF-I in the circulation during smoltification.     

Measurement of circulating salmon IGF binding protein-1: assay development, response to feeding ration and temperature, and relation to growth parameters

Fish plasma/serum contains multiple IGF binding proteins (IGFBPs), although their identity and physiological regulation are poorly understood. In salmon plasma, at least three IGFBPs with molecular masses of 22, 28 and 41 kDa are detected by Western ligand blotting. The 22 kDa IGFBP has recently been identified as a homolog of mammalian IGFBP-1. In the present study, an RIA for salmon IGFBP-1 was established and regulation of IGFBP-1 by food intake and temperature, and changes in IGFBP-1 during smoltification, were examined. Purified IGFBP-1 from serum was used for as a standard, for tracer preparation and for antiserum production. Cross-linking (125)I-labelled IGFBP-1 with salmon IGF-I eliminated interference by IGFs. The RIA had little cross-reactivity with salmon 28 and 41 kDa IGFBPs (< 0.5%) and measured IGFBP-1 levels as low as 0.1 ng/ml. Fasted fish had significantly higher IGFBP-1 levels than fed fish (21.6 +/- 4.6 vs 3.0 +/- 2.2 ng/ml). Plasma IGFBP-1 was measured in individually tagged 1-year-old coho salmon reared for 10 weeks under four different feeding regimes as follows: high constant (2% body weight/day), medium constant (1% body weight/day), high variable (2% to 0.5% body weight/day) and medium variable (1% to 0.5% body weight/day). Fish fed with the high ration had lower IGFBP-1 levels than those fed with the medium ration. Circulating IGFBP-1 increased following a drop in feeding ration to 0.5% and returned to the basal levels when feeding ration was increased. Another group of coho salmon were reared for 9 weeks under different water temperatures (11 or 7 degrees C) and feeding rations (1.75, 1 or 0.5% body weight/day). Circulating IGFBP-1 levels were separated by temperature during the first 4 weeks; a combined effect of temperature and feeding ration was seen in week 7; only feeding ration influenced IGFBP-1 level thereafter. These results indicate that IGFBP-1 is responsive to moderate nutritional and temperature changes. There was a clear trend that circulating IGFBP-1 levels were negatively correlated with body weight, condition factor (body weight/body length(3) x 100), growth rates and circulating 41 kDa IGFBP levels but not IGF-I levels. During parr-smolt transformation of coho salmon, IGFBP-1 levels showed a transient peak in late April, which was opposite to the changes in condition factor. Together, these findings suggest that salmon IGFBP-1 is inhibitory to IGF action. In addition, IGFBP-1 responds to moderate changes in dietary ration and temperature, and shows a significant negative relationship to condition factor.     

Hormonal control of ovarian cell production of insulin-like growth factor binding proteins

To determine if the hormonal effects on insulin-like growth factor binding protein (IGFBP) production differed between granulosa and thecal cells, both cell types were collected and cultured in serum-free medium with various hormone treatments, arranged in three experiments. Following treatment, cells were enumerated and media were collected, concentrated 10-fold and subjected to ligand blotting. Experiment 1 revealed that > or =1.5 x 10(5) viable cells at plating were needed for maximal IGFBP production by granulosa and thecal cells. The major forms of IGFBPs produced were a 27-34-kDa IGFBP (IGFBP-2 and -5), and a 20-22-kDa IGFBP (IGFBP-4) by the granulosa cells and a 40-44-kDa IGFBP (IGFBP-3), 34-kDa IGFBP (IGFBP-2), 27-29-kDa IGFBP (IGFBP-5) and a 20-22-kDa IGFBP (IGFBP-4) by the thecal cells. In Experiment 2A, insulin stimulated production of IGFBP-5 by thecal cells, and basic fibroblast growth factor (bFGF) inhibited the insulin-induced increase in IGFBP-5 production; epidermal growth factor (EGF) and luteinizing hormone were without effect. The small amounts of IGFBP-2 and -3 produced by thecal cells of Experiment 2A were not affected by treatment. Production of IGFBP-2/-5 by granulosa cells in Experiment 2B was inhibited by insulin, with EGF and bFGF further enhancing insulin's inhibitory effect; follicle-stimulating hormone was without effect. In Experiment 3A, insulin enhanced production of IGFBP-5 by thecal cells whereas glucagon blocked insulin's stimulatory effect. In contrast, insulin or glucagon alone had no effect on production of the IGFBP-4 by thecal cells but when combined inhibited IGFBP-4 production. The small amounts of IGFBP-2 and -3 produced by thecal cells of Experiment 3A were not affected by treatment. In Experiment 3B, production of IGFBP-2/-5 by granulosa cells was attenuated in the presence of cortisol with or without insulin and insulin plus glucagon; glucagon and cortisol decreased production of IGFBP-4 by granulosa cells. These results suggest that production of IGFBP-2, -4, and -5 by granulosa and thecal cells are differentially affected by hormonal stimuli, and that IGFBP-3 is more consistently produced by thecal cells than granulosa cells of cattle although its production was not hormonally regulated.     

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.     

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.     

Follicular fluid insulin-like growth factor binding protein profiles in polycystic ovary syndrome.

Insulin-like growth factor binding proteins (IGFBPs) are believed to modulate the actions of IGF-I and IGF-II at the cellular level. We have examined, by Western ligand blot analysis, the IGFBP profiles in follicular fluid (FF) from patients with polycystic ovarian syndrome (a disorder of ovarian folliculogenesis), compared to FF from atretic and developing (estrogenic) follicles from normally cycling women. IGFBPs with apparent mol wts (Mr) of 41.5, 38.5, 31, 28, and 24kDa were detected in PCOS FF. The profile of IGFBPs in PCOS FF was indistinguishable from that seen in atretic follicles in cycling women. However, higher levels of the 31, 28, and 24kDa IGFBPs were observed in PCOS FF, compared to healthy, estrogenic follicles. Using specific antisera, the 41.5 and 38.5kDa IGFBPs were identified as IGFBP-3, and the 31kDa IGFBP as IGFBP-2. IGFBP-1, however, was not appreciably detectable in PCOS FF, by Western ligand blotting. Endoglycosidase F treatment of FF decreased the Mr of the 28kDa IGFBP to 24kDa, and neither the 28kDa nor the 24kDa IGFBP was immunoprecipitated by antibodies to IGFBP-1, -2, or -3. Elevated levels of 28kDa and 24kDa IGFBPs in PCOS FF may represent glycosylated and core forms of IGFBP-4. The data presented herein show that in PCOS FF, as well as in FF from atretic follicles from normally cycling women, IGFBP-2 and 28 and 24kDa IGFBPs are present in greater amounts, compared to levels in FF from healthy, developing, estrogenic follicles. One or more of the IGFBP species elevated in atretic and PCOS follicles may bind IGFs in FF, thereby inhibiting IGF action on the granulosa during normal folliculogenesis.     

Biochemical and functional analysis of a conserved IGF-binding protein isolated from rainbow trout (Oncorhynchus mykiss) hepatoma cells

Rainbow trout (Oncorhynchus mykiss) serum contains several IGF-binding proteins (IGFBPs) that specifically bind to IGFs. The structures of these fish IGFBPs have not been determined and their physiological functions are poorly defined. In this study, we identified a 30 kDa IGFBP present in rainbow trout serum and secreted by cultured trout hepatoma cells. This IGFBP binds to IGFs but not to insulin. This IGFBP was purified to homogeneity using a three-step procedure involving Phenyl-Sepharose chromatography, IGF-I affinity chromatography and reverse-phase HPLC. Affinity cross-linking studies indicated that this IGFBP binds to IGF-I with a higher affinity than to IGF-II. N-terminal sequence analysis of the trout IGFBP suggests that it shares high sequence identity with that of human IGFBP-1 in the N-terminal region. When added to cultured fish and human cells, the trout IGFBP inhibited IGF-I-stimulated DNA synthesis and cell proliferation in a concentration-dependent manner. The inhibitory effect of the fish IGFBP was comparable to those of human IGFBP-1 and -4. These results indicate that the IGFBP molecule is structurally and functionally conserved in evolutionarily ancient vertebrate species such as bony fish.     

Effects of homologous pituitary hormone treatment on serum insulin-like growth-factor-binding proteins (IGFBPs) in hypophysectomized tilapia, Oreochromis mossambicus, with special reference to a novel 20-kDa IGFBP

In the circulation, insulin-like growth factors (IGFs) bind to high-affinity-binding proteins. Insulin-like growth-factor-binding proteins (IGFBPs) appear to be present in all vertebrates. To examine the hormonal regulation of serum IGFBPs in a fish, tilapia (Oreochromis mossambicus) were hypophysectomized (Hx) and then treated with homologous tilapia growth hormone (tGH) or either form of tilapia prolactin (tPRL177, tPRL188). Hormones were administered at three doses: 15, 150, and 500 ng/g of body weight. Serum IGFBP profiles were analyzed by SDS-PAGE and Western ligand blotting using 125I-rhIGF-I as a probe. A prominent IGFBP (ca 20 kDa), termed IGFBP-20K, appeared after hypophysectomy. Administration of tGH at all dose levels suppressed this BP and restored levels back to those seen in sham-operated control fish. tPRL177 and tPRL188 were also effective in lowering IGFBP-20K levels. Levels of the 29-kDa IGFBP (termed IGFBP-29K) increased after hypophysectomy; tGH at all doses and tPRL177 at the two lower doses further increased these levels. All doses of tGH, tPRL177, and tPRL188 significantly increased levels of the 32-kDa IGFBP (termed IGFBP-32K). Hypophysectomy significantly lowered levels of the 40-kDa IGFBP (termed IGFBP-40K) below levels seen in the sham-operated controls. tGH treatment significantly raised IGFBP-40K levels at all doses examined, but not to the levels seen in intact tilapia. The 42-kDa IGFBP (termed IGFBP-42K) was not affected by hypophysectomy or hormone replacement. Our data suggest that the novel 20-kDa IGFBP and the 40-kDa IGFBP species may be similar in function to mammalian IGFBP-1 and IGFBP-3, respectively.     

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.     

Insulin-like growth factor binding proteins in the bovine anterior pituitary

Insulin-like growth factor binding proteins (IGFBPs) were characterized in bovine anterior pituitary tissue, pituitary conditioned media, and serum collected during the preovulatory and early luteal phases of the estrous cycle. Effects of in vitro treatments of pituitaries with luteinizing hormone-releasing hormone (LHRH), estradiol, and progesterone on IGFBP secretion were also evaluated. Predominant IGFBPs detected in anterior pituitary tissue by immunoprecipitation, ligand blotting, and Northern blotting were IGFBP-5 (29 kDa), IGFBP-2 (32 kDa), and IGFBP-3 (36 and 39 kDa doublet). Conditioned culture media contained IGFBP-5, a slightly larger form of IGFBP-3 (33 kDa), the 36- and 39-kDa forms of IGFBP-3, and a more extensively glycosylated form of IGFBP-3 (44 kDa). In serum, IGFBP-5 was not readily detected, and IGFBP-3 (40- and 44-kDa doublet) and IGFBP-2 (34 kDa) were larger than in pituitary tissue. Levels of IGFBP-2,-3, and-5 in pituitary tissue decreased during the preovulatory period and were lowest in the early luteal phase. Treatment with LHRH increased IGFBP-2 levels in media twofold. Estradiol or progesterone did not alter IGFBP secretion in vitro. Predominant IGFBPs produced and released by anterior pituitary tissue were IGFBP-2,-3 and-5. The activity of IGFBPs fluctuates in the pituitary in association with changes in stage of estrous cycle, implicating IGFBPs as potential regulators of gonadotrope function. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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.     

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.     

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.     

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.     

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.     

Local synthesis and developmental regulation of avian vitreal insulin-like growth factor-binding proteins: a model for independent regulation in extravascular and vascular compartments.

The expression and regulation of insulin-like growth factor-binding proteins (IGFBPs) in developing avian vitreous humor and serum were compared. Vitreal IGF-I-binding activity was highest on embryonic day 6 [E-6; bound/free ratio (B/F), 0.22 +/- 0.019/50 microliters), decreased 10-fold between E-6 and E-19, and then remained stable through the remainder of embryonic development. In contrast, serum IGF-I binding increased 2-fold over this period, from a B/F of 0.380 +/- 0.056 (E-6) to a B/F of 0.89 +/- 0.18 (E-19). After hatching, serum IGF-I-binding activity continued to increase through posthatching week 12, while vitreal IGF-I binding increased only slightly and then remained constant. Although IGF-II binding in the vitreous humor and serum is 2- to 3-fold higher than that of IGF-I, the same pattern of developmental regulation was observed as with IGF-I. Western ligand blots revealed a vitreal 24-kilodalton (kDa) IGFBP that was absent from both embryonic and adult sera. Likewise, posthatching serum was found to contain a 70-kDa IGFBP absent in vitreous humor. Deglycosylation of vitreal and serum IGFBPs followed by Western ligand blotting revealed unique glycosylation patterns for vitreal and serum IGFBPs. One of the IGFBPs that is differentially glycosylated in vitreous and serum is a 33-kDa IGFBP that is precipitated with human IGFBP-2 antiserum. Northern blot analysis revealed the presence of IGFBP-2 mRNA in several embryonic ocular tissues as well as liver. The observations that vitreal and serum IGFBP levels are independently regulated during development and that IGFBPs from these two compartments have different molecular weights and glycosylation patterns suggest that the vitreal IGFBPs are not derived from serum. The presence of IGFBP-2 mRNA in ocular tissue surrounding the vitreal chamber supports the view that certain vitreal IGFBPs may be synthesized locally.