Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

Inhibition of Caco-2 cell proliferation by all-trans retinoic acid: role of insulin-like growth factor binding protein-6.

The present study examined the effects of all-trans retinoic acid (tRA) on proliferation and expression of the IGF system in Caco-2 human colon adenocarcinoma cells. tRA inhibited Caco-2 cell proliferation in a dose-dependent manner, with a 40 +/- 2% decrease in cell number observed 48 h after the addition of 1 microM tRA. Ligand blot analysis of IGFBPs in conditioned media revealed that Caco-2 cells produced three IGFBPs of M(r): 34,000 (IGFBP-2), 24,000 (IGFBP-4), and 32,000 (IGFBP-6). The concentrations of IGFBP-2 and IGFBP-4 decreased by 48 +/- 6 and 70 +/- 13%, respectively, whereas that of IGFBP-6 increased by 698 +/- 20% with 1 microM tRA. tRA decreased mRNA levels of IGFBP-2 and IGFBP-4 by 20 +/- 3 and 50 +/- 8%, respectively, whereas tRA increased IGFBP-6 mRNA by 660 +/- 20%. tRA did not alter levels of IGF-II mRNA or peptide. To examine if endogenous IGFBP-6 inhibits cell proliferation, Caco-2 cells were transfected with an IGFBP-6 cDNA expression construct or pcDNA3 vector only and stable clones were selected. Clones overexpressing IGFBP-6 grew more slowly than vector controls and achieved final densities 30-55% lower than those of vector controls. Accumulation of IGFBP-6 mRNA and concentrations of IGFBP-6 peptide in conditioned media were increased by 200-250 and 220-250%, respectively, in the IGFBP-6 clones compared with controls. Increased expression of IGFBP-6, which has a high binding affinity for IGF-II, following tRA treatment suggests that the decreased proliferation caused by tRA may result, at least in part, from IGFBP-6-mediated disruption of the IGF-II autocrine loop in these colon cancer cells.     

Production of insulin-like growth factor II (IGF-II) and different forms of IGF-binding proteins by HT-29 human colon carcinoma cell line.

The serum-free medium conditioned by the human colon cancer cell line HT-29 contains insulin-like growth factors (IGF) that are entirely complexed to binding proteins (IGF-BP). Gel filtration in acid conditions of the cell-conditioned medium permits separation of IGF-BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF-BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF-I and IGF-II radioreceptor assays, IGF-I radioimmunoassay (RIA), and competitive protein-binding assay specific for IGF-II, it is shown that the IGF-type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF-II. Its concentration is approximately 6 ng/10(6) HT-29 cells with 60% present as a high-molecular-weight form of IGF-II. This large 15 K IGF molecule is devoided of any IGF-binding activity and might represent incomplete processing of pro-IGF-II peptide. By contrast, the level of IGF-I detected by RIA is barely measurable and considered negligible (0.57 pg/10(6) HT-29 cells). Although these IGF-II-like peptides exhibit a growth-promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF-I or IGF-II, 3H-thymidine incorporation into DNA of HT-29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT-29-D4 clonal cells, derived from the parental HT-29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.     

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

We have previously shown that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP-4, -5, and -6. Since we found that the temporal expression pattern of IGFBP-4 and IGFBP-5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP-4 and IGFBP-5 on IGF- stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP-4 had only inhibitory actions, inhibiting IGF-I and IGF-II- stimulated proliferation and differentiation. In contrast, IGFBP-5 exhibited both inhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF-I, IGFBP-5 (250 ng/ml) inhibited all markers of the early proliferative response: the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS-1 and Shc, the activation of the MAP kinases, ERK1 and 2, the elevation of c-fos mRNA, the early inhibition of the elevation in myogenin mRNA, and the increase in cell number. In contrast, IGFBP-5 stimulated all aspects of the myogenic response to IGF-I: the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes. This dual response to IGFBP-5 was greatest when it was added at a molar ratio of IGFBP-5 to IGF-I of 2:1. In contrast, when IGFBP-5 was added in the presence of IGF-II, it inhibited both proliferation and differentiation. Neither IGFBP had any effect when added in the presence of R3 IGF-I, an analog with substantially reduced affinity for IGFBPs. Our results suggest that the role of IGFBP-4 is mainly to sequester excess IGFs, and thus inhibit all actions. IGFBP-5, however, is capable of eliciting a dual response, possibly due to its unique ability to associate with the cell membrane. J. Cell. Physiol. 177:47–57, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of insulin-like growth factor binding proteins (IGFBPs) secreted by bovine adrenocortical cells in primary culture: regulation by insulin-like growth factors (IGFs) and adrenocorticotropin (ACTH).

In previous studies, we have shown that insulin-like growth factor II (IGF-II) stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I [1]. The steroidogenic effect of both IGFs is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we therefore characterized the IGFBPs secreted by bovine adrenocortical cells in primary culture, and investigated the effect of corticotropin (ACTH) and recombinant human IGF-I and IGF-II on the regulation of IGFBP synthesis. By Western ligand blotting, four different molecular forms of IGF-binding proteins were identified in conditioned medium of bovine adrenocortical cells with apparent molecular weights of 39-44 kDa, 34 kDA, 29-31 kDa, and 24 kDa. In accordance to their electrophoretic mobility, glycosylation status and binding affinity, these bands were identified by immunoprecipitation and immunoblotting as IGFBP-3, IGFBP-2, IGFBP-1, and deglycosilated IGFBP-4, respectively. Quantification of the specific bands by gamma counting revealed that, in unstimulated cells, IGFBP-3 accounts for approximately half of the detected IGFBP activity, followed by IGFBP-1, IGFBP-2 and IGFBP-4. ACTH treatment predominantly increased the abundance of IGFBP-1 and to a lesser extent IGFBP-3 in a time and dose-dependent fashion. In contrast, IGF-I or IGF-II (6.5 nM) preferentially induced the accumulation of IGFBP-3 (1.9-fold) and to a lesser extent of IGFBP-4, but did not show any effect on IGFBP-1. When ACTH and IGFs were combined, an additive stimulatory effect on the accumulation of IGFBP-3 and IGFBP-4 was observed. In contrast to their different steroidogenic potency, no significant difference in the stimulatory effect of IGF-I and IGF-II on IGFBP secretion was found. In conclusion, bovine adrenocortical cells synthesize IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4, and their secretion is regulated differentially by ACTH and IGFs. These results, together with earlier findings, suggest that IGF-binding proteins play a modulatory role in the regulation of differentiated adrenocortical functions. Therefore, bovine adult adrenocortical cells provide a useful tissue culture model in which the complex interactions between two IGF-ligands, at least four IGF binding proteins and two IGF-receptors can be evaluated.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.