Growth Factor Binding Protein Secretion Research Articles

Regulation of insulin-like growth factor binding protein secretion by human granulosa luteal cells in a polycystic ovary-like environment

Objective: To determine the effect of androstenedione (A), insulin, and LH on secretion of insulin-like growth factor binding proteins (IGFBPs) from human granulosa luteal cells. Design: Human granulosa cells were cultured for a total of 4 days in serum-free medium containing A (10 −6 mol/L), with or without insulin (100 μU/mL–800 μU/mL), LH (1 μU/mL–10 μg/L), and A (10 −5 mol/L). Setting: Granulosa cells were obtained from IVF procedures. Patient(s): Women undergoing IVF for tubal disease. Intervention(s): None. Main Outcome Measure(s): Immunoassay and autoradiographs of Western ligand blotting detected IGFBP accumulations in the medium. Result(s): Cultured granulosa cells secreted IGFBP-1 through IGFBP-4. Insulin (100 μU/mL–800 μU/mL), LH (1 μg/L–10 μg/L), and A (10 −5 mol/L) caused a significant decrease in IGFBP-1 accumulation in the medium both alone and when added in combination. The release of IGFBP-2 and IGFBP-4 was significantly stimulated by insulin, whereas LH had no effect. Elevated levels of androgen (10 −5 mol/L) significantly stimulated the secretion of IGFBP-2, whereas the release of IGFBP-4 was reduced. Conclusion(s): These results demonstrate that androgen and insulin are important regulators of IGFBP release and that elevated levels of the two hormones may contribute to the altered IGFBP profile found in PCOS follicles, compared with the case of estrogen-dominant follicles.

Growth Hormone has no Direct Effect on Human Adrenal Steroid and Insulin-Like Growth Factor-Binding Protein Secretion

Although it is known that growth hormone (GH) exerts its growth-promoting effects mainly via Insulin-like growth factor-I (IGF-I), an increasing number of direct effects of GH has been described in many tissues. In vivo, mice transgenic for human growth hormone (hGH) show significantly elevated levels of corticosterone, enlarged adrenal glands, and altered levels of insulin-like growth factor binding proteins (IGF-BPs). Recently, we have shown that IGF's induce the secretion of cortisol and IGF-BP's in adult human adrenocortical cells. However, since human adrenal glands express the intact GH-receptor, the objective of this study was to investigate whether GH exerts a direct effect on the steroidogenesis and IGF-BP synthesis in adult human adrenocortical cells. Primary cell cultures in monolayer were incubated under serum-free conditions with human growth hormone and/or ACTH for up to 72 hours. Cortisol was measured by specific RIA and the secretion of insulin-like growth factor binding proteins was analyzed by Western ligand blotting. hGH alone was unable to stimulate basal or ACTH-induced cortisol secretion. Additionally, neither hGH alone or in combination with ACTH did significantly alter the secretion of IGF-BP's. Therefore we conclude that hGH is unable to directly stimulate cortisol secretion and IGF-BP secretion in cultured human adrenocortical cells.

Growth factor regulation of insulin-like growth factor binding protein secretion by cultured human granulosa-luteal cells

Objective: To examine the effects of epidermal growth factor (EGF), transforming growth factor-β (TGF-β), and fibroblast growth factor (FGF) on insulin-like growth factor binding protein (IGFBP) secretion by cultured human granulosa-luteal cells. Design: Granulosa-luteal cells obtained at the time of oocyte harvest for IVF were cultured in serum-free medium in the presence or absence of EGF, TGF-β, or FGF. Conditioned medium then was analyzed by Western ligand blot and immunoradiometric assays. Setting: An academic medical center. Patient(s): Women undergoing IVF. Intervention(s): None. Main Outcome Measure(s): IGFBP-1 secretion. Result(s): By Western ligand blot analysis, IGFBP-1 levels were 1.4-fold to 7.4-fold higher in conditioned medium from cells cultured in the presence of EGF than in control medium. By immunoradiometric assay, IGFBP-1 levels increased from 1.6 to 9.9 times over control. The TGF-β had no apparent effect, and FGF did not consistently stimulate IGFBP-1 secretion. Conclusion(s): The EGF may decrease intrafollicular bioavailable IGF levels by increasing inhibitory IGFBPs, thereby leading to arrest of follicular development. Interactions between the EGF and IGF systems may be involved in the processes governing human ovarian follicle maturation and atresia.

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells.

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC3H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE1]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC3H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC3H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC3H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC3H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation.

Role for cyclic adenosine monophosphate in modulating insulin‐like growth factor binding protein secretion by muscle cells

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC3H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE1]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC3H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC3H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC3H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC3H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley-Liss, Inc.

Hormonal regulation of insulin-like growth factor binding protein secretion by a bovine mammary epithelial cell line

Hormonal regulation of insulin-like growth factor binding protein secretion by a bovine mammary epithelial cell line

Cytokines and growth factors modulate cell growth and insulin‐like growth factor binding protein secretion by the human salivary cell line (HSG)

The human salivary cell line (HSG) was investigated for the effect of various growth factors and cytokines on cellular proliferation and on the production of insulin-like growth factor binding proteins (IGFBPs). IGF-I increased cell growth by approximately 25%, and induced the appearances of three distinct protein bands on ligand blot of the cell culture. Two bands with molecular weights of 43 and 45 Kda, respectively, proved to be IGFBP-3 using a specific antibody, and the third was a 24 Kda species (probably, IGFBP-4). Similar IGFBPs were released by the cells following stimulation by EGF and insulin as well as following incubation with the IGF-I receptor antibody alpha IR3. Retinoic acid had an inhibitory effect (50%) on IGF-I-induced cellular proliferation and an attenuative effect on the 24 Kda band when it was combined with IGF-I, and to a lesser effect EGF; however, it enhanced IGFBP-3 production when incubated with IGF-I. The IGF-I receptor antibody had an agonistic effect on IGFBPs production when applied alone or together with IGF-I. TNF-alpha and INF-gamma had minimal effects on cell growth when added alone but when applied in combination, a marked inhibition of cellular proliferation was noted. These cytokines caused increased accumulation of IGFBP-3, -4, and -5. Addition of IGF-I to these cytokines enhanced the expression of these bands. These data demonstrate that growth factors and cytokines which modulate HSG cell growth, induce specific IGFBPs which may play a role in their effects on cell growth.

Insulin-like growth factor-binding proteins in the human fetus: Tissue-specific protein secretion, immunologic characterization, and gene expression

The objectives of this study were to investigate the profile of insulin-like growth factor-binding proteins secreted by human fetal tissues and their immunologic identification and tissue-specific gene expression. Explants of midgestational fetal tissues from seven fetuses were cultured with and without cycloheximide. Conditioned media were examined for insulin-like growth factor-binding proteins by Western ligand blot analysis, and insulin-like growth factor-binding proteins were identified by immunoprecipitation. Gene expression was analyzed by Northern analysis. Fetal liver and kidney explants secreted insulin-like growth factor-binding protein-1 to insulin-like growth factor-binding protein-4, with insulin-like growth factor-binding protein-1 being the most prominent in liver. Fetal lung secreted insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4 and lesser amounts of insulin-like growth factor-binding protein-3, whereas white matter explants secreted exclusively insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4. Cycloheximide inhibited secretion of binding proteins, suggesting de novo synthesis. Northern blot analyses were consistent with the protein studies. These data demonstrate that insulin-like growth factor-binding protein secretion by fetal tissues is tissue specific.

Phorbol esters showing selective activation of PKS isozymes in vitro regulate thyroid function and insulin-like growth factor binding protein secretion

We have examined the effects of phorbol derivatives which show selective activation of protein kinase C (PKC) isozymes in vitro, on several parameters of thyroid function. Functions examined were iodide uptake and organification, iodocompound secretion and insulin-like growth factor binding protein (IGFBP) secretion, all of which have been shown previously to be modulated by 12- O-gtetradecanoylphorbol 13-acetate (TPA), a pan activator of PKC isozymes. All of the agnes examined, including DOPPA (12-deoxyphorbol-13- O-phenylacetete-20 acetate), whis is speciffic for the β1 isozyme in vitro, were able to mimic the effects of TPA. These effects were evident by 2 h int he iodide uptake and organification assays, by 4 h in teh secretion assays and 8 h in the IGFBP secreton assay. the phorbol deivatives differed from TPA in their ability to down-regulated total PKC activity, DOPPA being weakly effective at 8 h (14% inhibition) when TPA had affected > 70% down-regulation of PKC. As the effects of DOPPA were detected by 8 h at the latest, these data indicated that the effects observed were due to PKC activation rather than donw-regulaton. Furthermore, the differences, in down-regulation profiles between DOPPA and TPA suggest that in vitro, DOPPa may maintain its in vitro specificity. We conclude that inhibition of thyroid iodide uptake and its organification, stimulation of iodocompound secretion and stimulation of IGFBP-2 and IGFBP- 3 secretion may be effected through the modulation of a limited of PKC isozymes and possibly initially, only through PKC β1.

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro.

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

Effects of cytokines on insulin-like growth factor-binding protein secretion by muscle cells in vitro

The insulin-like growth factors (IGF-I and IGF-II) stimulate muscle cell proliferation and/or differentiation (depending upon the culture conditions). They also increase IGF-binding protein (IGFBP) levels in muscle cell conditioned media and in some instances there is a direct correlation between the apparent rate of IGFBP secretion and muscle cell proliferation. We have investigated the effect of other cytokines on muscle cell IGFBP conditioned media levels using rat skeletal (L6), mouse myocytes (BC3H-I) and porcine vascular smooth (pVSM) muscle cells in vitro to determine if this relationship is maintained. IGFBP levels in conditioned media (CM) were measured by an [125I]IGF-I binding capacity assay and by western blot analysis. Immunoblots indicated that BC3H-1 and L6 cells secrete IGFBP-5 (31-32,000 M(r)), L6 cells secrete IGFBP-4, and pVSM cells secrete IGFBP-2 (34,000 M(r)). Both L6 and BC3H-1 cells responded to transforming growth factor beta 1 (TGF-beta 1), in a dose-dependent manner, with suppressed conditioned media levels of IGFBP-4 and IGFBP-5 but TGF-beta 1 did not affect IGFBP-2 levels in pVSM cell media. TGF-beta 1 (5 ng/ml) suppressed IGFBP levels (CM [125I]IGF-I binding capacity) in L6 and BC3H-1 cell media by 48% and 61%, respectively. IGFBP-5 levels, in BC3H-1 cell media, were decreased by treatment with either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Neither treatment affected IGFBP-2 levels. In contrast in L6 myoblasts, bFGF increased media levels of IGFBP-4 and IGFBP-5; L6 cells were not responsive to EGF. Insulin increased IGFBP-4 and IGFBP-5 levels in L6 and BC3H-1 cell media. This stimulatory effect was markedly suppressed by either TGF-beta 1 (L6 and BC3H-1 cells) or bFGF (BC3H-1 cells). L6 and BC3H-1 cell CM IGFBP levels were also suppressed 34% and 84% by 5 U/ml thrombin, respectively. The inhibitory activity of thrombin was specific, i.e. reversible by hirudin and was not due to direct IGFBP proteolysis. Since suramin and staurosporine increased media levels of the IGFBP, this suggests that constitutive secretion of TGF-beta 1, bFGF, or EGF might provide a tonic suppressive mechanism for controlling IGFBP secretion. Thus, our results support the conclusion that the secretion of IGFBP-4 and IGFBP-5, but not IGFBP-2, by muscle cells was suppressed by several cytokines. Depressed IGFBP secretion may play a key role in determining muscle cell responsiveness to either the mitogenic or differentiation stimulating activity of the IGFs.

Role of 3', 5' cyclic adenosine monophosphate and protein kinase C in the regulation of insulin-like growth factor-binding protein secretion by thyroid-stimulating hormone in isolated ovine thyroid cells.

Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10(-8)-10(-3) mol/l), dibutyryl cAMP (0.25-1 mmol/l), forskolin (5-20 mumol/l) or 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-11)-10(-6) mol/l), with or without exposure to TSH (200 microU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [gamma-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10(-6)-10(-3) mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10(-11)-10(-6) mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Insulin-like Growth Factor Binding Protein Secretion by a Murine Mammary Epithelial Cell Line

The regulation of insulin-like growth factor binding protein (IGFBP) secretion by mammogenic and lactogenic hormones was investigated in a mouse mammary epithelial cell line (COMMA-D/MME). Cells were grown to confluency on plastic culture plates in serum-containing medium. The confluent cells were exposed to the hormonal treatments in serum-free medium for 6 days. Conditioned medium was collected on Days 5 and 6 and analyzed for IGFBPs by 125 I-labeled insulin-like growth factor (IGF)-II ligand blotting and quantified by densitometry. IGFBP data were expressed as absorbance units × millimeters (AUxmm) that were corrected for DNA per well. In basal serum-free media, the COMMA-D/MME cells secreted predominately IGFBP-3, but also some IGFBP-2. The mammogenic growth factors, IGF-I (13.3 n M) and epidermal growth factor (EGF; 1.7 n M), both stimulated DNA synthesis ( P ≤ 0.05); however, their effects on IGFBP-3 secretion differed. IGF-I stimulated IGFBP-3 secretion whereas EGF was inhibitory. In addition, EGF inhibited IGFBP-2 secretion and IGF-I tended to increase it. No interaction was observed between IGF-I and EGF for any of the parameters measured. Three lactogenic hormones (insulin, 154 n M; prolactin, 4.3 n M; and cortisol, 1.4 μ M) in all combinations were tested to determine their effects on IGFBP secretion. Insulin stimulated IGFBP-3 secretion and DNA synthesis, but had no effect on IGFBP-2 secretion. Cortisol inhibited IGFBP-3 secretion and DNA synthesis, but increased IGFBP-2 secretion. Prolactin had no effect on any of the parameters examined. In summary, the COMMA-D/MME cells secrete IGFBP-2 and IGFBP-3 in serum-free media. Although the secretion of IGFBP-2 and IGFBP-3 was regulated by several of the hormones and growth factors tested, no clear distinction was observed between the mammogenic and lactogenic treatments.

Insulin-Like Growth Factor Binding Protein Secretion by Breast Carcinoma Cell Lines: Correlation with Estrogen Receptor Status*

Breast tumor cell lines have been shown to secrete several distinct polypeptide growth factors, although conflicting results exist for the insulin-like growth factors (IGFs). In contrast a limited number of breast tumor cell lines have definitely been shown to secrete the high affinity IGF binding proteins (IGFBPs) that modify IGF actions. To characterize the types of IGFBPs that are secreted by breast tumor cell lines, conditioned medium was collected from seven separate tumor cell lines, three of which were estrogen receptor (ER) negative, and four of which were ER positive. All three of the ER negative cell lines, MDA-231, MDA-330, and HS578T, secreted binding proteins of 49,000 and 43,000 Mr (IGFBP-3) as well as 29,000 (IGFBP-1) and 24,000 Mr. In contrast, all four ER positive cell lines secreted 34,000 (IGFBP-2) or 24,000 Mr forms, and none secreted the 49,000 and 43,000 or 29,000 Mr forms. BT-20, a cell line that is positive for ER messenger RNA (mRNA) but negative for ER protein, secreted predominantly a 34,000 Mr protein. The amount of total IGFBP activity released in 24 h ranged between 0.4 and 5.6 nM equivalents of IGFBP-1, and there was no significant difference between the ER positive and negative cell lines. The MCF-7 cells that produced predominantly 34,000 and 24,000 Mr forms showed a 1.8-fold increase in IGFBP secretion after estrogen stimulation. Immunoblotting and a specific RIA for IGFBP-1 showed that only the ER negative lines MDA-330, MDA-231, and HS578T secreted this form. Northern blotting analysis for the mRNA encoding this protein showed that both MDA-330 and MDA-231 contained a single 1.6 kilobase mRNA species that hybridized with an IGFBP-1 complementary DNA (cDNA) probe. Immunoblotting analysis of the other cell lines showed that only the 34,000 Mr form secreted by the ER positive cell lines reacted with IGFBP-2 antisera. Exposure of the conditioned media from the three ER negative cell lines to N-glycanase revealed that the 49,000 and 43,000 Mr forms of IGFBP were glycosylated and therefore probably represent IGFBP-3. We conclude that ER negative cell lines secrete three forms of IGFBPs, IGFBP-1, IGFBP-3, and a 24,000 Mr form. In contrast, the ER positive cell lines secrete predominantly IGFBP-2 and the 24,000 Mr form but do not secrete IGFBP-3 or 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.

Several growth hormone-independent 25-31,000 kD insulin-like growth factor binding proteins (IGF-BPs) have been identified in plasma, extravascular fluids, and various cell-conditioned media. Cultured human decidual cells release three IGF-BPs with 24,000, 30,000, and 34,000 Mr. Using ligand blot analysis and an RIA for the 30,000-Mr form (IGF-BP-1), we examined the effects of IGF-I (10-1,000 ng/ml), insulin (10-10,000 ng/ml), and relaxin (10-250 ng/ml) on decidual cell IGF-BP release after 120 h of hormone exposure. IGF-I inhibited release of both IGF-BP-1 and the 24,000 Mr form. Inhibition of IGF-BP-1 release was noted after 48 h of treatment and was progressive throughout the subsequent 120 h. Insulin stimulated a fourfold increase in release of the 24,000-Mr protein while inhibiting IGF-BP-1 release comparable to IGF-I, alpha-IR3, a monoclonal antibody to the IGF-I receptor, blocked approximately 33% of the IGF-I response but had no effect on insulin-mediated IGF-BP-1 inhibition. Relaxin stimulated a 2.4-fold increase in release of the 24,000-Mr form and a 16-fold increase in the 30,000-Mr protein after 120 h. Stimulation of the 30,000-Mr protein was inhibited by the addition of cycloheximide (50 micrograms/ml). Both IGF-I and insulin also blocked the relaxin-mediated increase in IGF-BP-1. These studies suggest that three structurally related proteins differentially regulate IGF-BP secretion possibly via activation of distinct receptor subtypes.

Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.

Differential regulation of insulin-like growth factor binding protein secretion from human decidual cells by IGF-I, insulin, and relaxin.

Insulin‐like growth factor binding protein secretion by muscle cells: Effect of cellular differentiation and proliferation

Several cell types have been shown to secrete insulin-like growth factor binding proteins (IGF-BP) in vitro. Since IGF-BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF-BP and to identify factors that regulate IGF-BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H-1 myocytes were used. IGF-BP activity in serum-free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF-BP activity into the medium. Insulin was a potent stimulant of IGF-BP secretion for each cell type. Specifically, 1 microgram/ml insulin increased the IGF-BP concentration in conditioned media from 10.5 +/- 1.3 to 15.0 +/- 1.5 ng/ml in confluent L6 myotubes, from 42.5 +/- 11.1 to 90.5 +/- 9.8 ng/ml in confluent BC3H-1 cells, and from 2.1 +/- 0.1 to 3.8 +/- 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 micrograms/ml) to achieve a half-maximal stimulation of IGF-BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H-1 cells, where half-maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40-fold more sensitive to insulin stimulation of IGF-BP secretion than L6 myotubes. IGF-I, although it interferes with the assay and thereby lowers the amount of detectable IGF-BP, stimulated the secretion of IGF-BP from all three cell types. Dexamethasone, (10(-7) M) decreased IGF-BP secretion into the media by approximately 50% for all three cell types. Affinity cross-linking and ligand blotting of 125I-IGF-I to conditioned media from each cell type showed (IGF-BP)-(IGF-I) complexes with molecular weights ranging 32-40 kDa (24-32 kDa for IGF-BP and 7.5 kDa for IGF-I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H-1 myocytes. This cell proliferative response was associated with an increase in IGF-BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF-BP secretion/cell. We conclude that IGF-BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin-stimulated secretion. Insulin and IGF-I are stimulators of IGF-BP secretion, whereas dexamethasone inhibits IGF-BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF-BP may play an important regulatory role in these processes.