Influence of Novel Nano-Mesoporous Bioactive Glass on the Regulation of IGF-II Gene Expression in Osteoblasts

To investigate whether the mesoporous bioactive glass (MBG) exerts any in vitro bioactivity on rat osteoblasts and the potential regulatory mechanism of this bioactivity. Rat osteoblasts were incubated in the presence and absence (control) of ionic dissolution product of MBG in minimal essential medium. The osteoblast proliferation and differentiation were measured using MTT and alkaline phosphatase methods. The IGF-II mRNA expression in osteoblasts was measured by RT-PCR. IGF-II protein and IGFBP were measured by ELISA. The level of alkaline phosphatase activity was increased to 125% of control. Expression of IGF-II mRNA was increased to 125% of control. There was a 175 and 237% increase in the concentration of unbound IGF-II protein and IGFBP, respectively, in the conditioned media of treated osteoblasts. The osteoblast proliferation was 92% of control. The ionic dissolution product of MBG was able to promote the differentiation of osteoblasts, probably by inducing IGF-II expression at both mRNA and protein level.     

Regulation of insulin-like growth factor-II expression and its role in autocrine growth of human neuroblastoma cells

Insulin-like growth factor-II (IGF-II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH-SY5Y human neuroblastoma cell line also expresses IGF-II and its receptors and responds to exogenous IGF-II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF-II mediates autocrine growth of SH-SY5Y cells in serum-free media. SH-SY5Y cells plated at high densities proliferated in serum-free media, whereas sparsely plated cells did not. IGF-II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF-II protein. Exogenous addition of IGF-II increased ³H-TdR incorporation and cell number in a dose- and time-dependent fashion. By nuclear labelling experiments using 5-Bromo-2′ deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24-hour incubation in IGF-II. Treatment of SH-SY5Y cells with anti-IGF-II antibodies in serum-free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF-II. Collectively, our results indicate that IGF-II mediates an autocrine growth mechanism in SH-SY5Y cells that is associated with increased IGF-II expression. © 1993 Wiley-Liss, Inc.     

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.     

Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of human bone marrow stromal cell growth on biodegradable polymer/bioglass composites

Bone tissue engineering using human bone marrow mesenchymal stem cells (HBMCs) and biocompatible materials provides an attractive approach to regenerate bone tissue to meet the major clinical need. The aim of this study was to examine the effects of novel porous biodegradable composite materials consisting of a bioactive phase (45S5 Bioglass, 0, 5, and 40 wt%) incorporated within a biodegradable poly(dl-lactic acid) matrix, on HBMCs growth. Cell adhesion, spreading, and viability was examined using Cell Tracker Green/Ethidium Homodimer-1. Bone formation was assessed using scaffolds seeded with stro-1 positive HBMCs in nude mice. In vitro biochemistry indicated that with minimal scaffold pre-treatment osteoblast activity falls with increasing Bioglass content. However, 24h scaffold pre-treatment with serum resulted in a significant increase in alkaline phosphatase specific activity in 5 wt% Bioglass composites relative to the 0 and 40 wt% Bioglass groups. In vivo studies indicate significant new bone formation throughout all the scaffolds, as evidenced by immunohistochemistry.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Insulinlike growth factor II expression and oval cell proliferation associated with hepatocarcinogenesis in woodchuck hepatitis virus carriers.

Insulinlike growth factor II (IGF-II) is a highly mitogenic fetal growth factor suspected of regulating the growth of a wide spectrum of tissues via an autocrine or paracrine mode of action or both. High steady-state levels of IGF-II RNA were detected in 45% of hepatocellular carcinomas (HCCs) arising from woodchuck livers with persistent woodchuck hepatitis virus (WHV) infection. Analysis of WHV RNA in the same HCCs revealed that HCCs with high levels of IGF-II RNA contained low or undetectable levels of WHV RNA and HCCs with low levels of IGF-II RNA contained high levels of WHV RNA. Integrated WHV DNA was present in HCCs from both groups, but viral DNA replicating forms were present, predominantly in HCCs with low levels of IGF-II. Several IGF-II RNAs, the most prominent of which were poly(A) species of approximately 3.75 and 1.1 to 1.3 kilobases, were detected only in precancerous nodules and HCCs. Levels of IGF-II were elevated two- to three-fold in the serum of woodchucks with chronic active hepatitis preceding the occurrence of HCC. Proliferation of a population of oval cells, which arise from portal tract regions in the liver, preceded the development of HCC and was a prominent feature of livers from which tumors with high levels of IGF-II occurred. The HCCs tended to have distinct histological features according to their growth factor status. Tumors with low levels of IGF-II were generally highly differentiated acinar-trabecular HCCs, whereas tumors with high levels of IGF-II were more anaplastic, with regions of fibrosis and fatty accumulation. A model to relate the pathology of WHV infection to oval cell proliferation and IGF-II expression in the development of these heterogeneous HCCs is presented.     

Potentiating role of IGFBP-2 on IGF-II-stimulated alkaline phosphatase activity in differentiating osteoblasts

The insulin-like growth factor (IGF) system plays an important role in the autocrine and paracrine regulation of bone formation and remodeling. The aim of this study was to evaluate the role of the autocrine IGF system during osteogenic differentiation in rat tibial osteoblasts (ROB) in culture. In this in vitro model, the stages of osteogenesis studied were S1, corresponding to the onset of alkaline phosphatase (AP) expression (days 0-3); S2, coincident with the peak of AP expression in differentiation culture conditions (days 4-6), and S3, corresponding to the onset of mineral deposition in the extracellular matrix (days 7-9). The results showed that conditioned medium of ROB contains greater amounts of IGF-II than IGF-I at all differentiation stages. Both peptides showed the highest concentrations on day 3 of differentiation (end of S1). All IGF-binding proteins (IGFBPs), except IGFBP-1 and -6, were detected, and IGFBP-2 was the most abundant IGFBP present in the conditioned media, and its degradation increased from S1 to S3. By semiquantitative RT-PCR, IGF-I and IGF-II were highly expressed on days 3 and 6, whereas IGFBP-2 was constantly expressed. We focused our study on the role of IGF-II and IGFBP-2 on the synthesis of AP, an early marker of osteoblast maturation. The results showed that a significant increase in AP expression was induced by IGF-II added to the differentiating osteoblasts continuously or in S1 but not in S2 or S3. IGFBP-2 was able to potentiate endogenous and exogenous IGF-II-dependent stimulation of AP activity, and its proteolytic degradation in late stages of osteogenesis (S2 and S3) was highly correlated with the increase of active matrix metalloproteinase-2 in the CM and with the decreased efficacy of IGF-II action. These data suggest that IGFBP-2, at nearly equimolar concentration with IGF-II, plays a potentiating role in IGF-II action on ROB differentiation in vitro.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.