Addition Of IGFBP-3 Research Articles

Regulation of Fetal Hemoglobin through the Insulin Signaling Pathway

Background: HbF induction is a key therapeutic strategy for sickle cell disease (SCD). Analysis of whole exome sequencing (WES) data from patients with SCD identified variants in two components of the insulin signaling pathway, FOXO3 and its activator, AMPK, to be associated with HbF levels; the association was confirmed by functional studies in hematopoietic stem and progenitor cells (HSPC) (Zhang, Blood 2018). This work has led to a clinical trial of metformin, an activator of FOXO3, as a novel HbF inducing agent in patients with SCA (NCT02981329). We then performed whole genome sequencing (WGS) on 567 samples from patients with SCA, and identified an association between another component of the insulin signaling pathway, IGFBP3, and HbF levels (p<1x10-6). Of note, IGFBP-3 expression is upregulated by several drugs also reported to increase HbF, including decitabine, metformin, and vitamin D. Methods: Plasma levels of IGFBP3 relative to IGF1 in patients with and without IGFBP3 variants were measured by ELISA. Three unique SCD patient-derived HSPC cultures were treated with metformin (100 µM), piceatannol (12.5 µM) compound C (1 µM), and exogenous IGFBP3 (1µg/ml); their effect on HbF, gamma-globin, known modifiers of HbF, protein levels and phosphorylation status of members of the FOXO3-AMPK pathway were assessed by HPLC, RT-qPCR and western blot at day 14 and 21 of culture. Results In vitro: Plasma IGFBP3 levels were higher in patients heterozygous for an IGFBP3 variant (p=0.01). Treatment of HSPCs with recombinant IGFBP3 resulted in a significant increase in %HbF (p=0.008). Adding IGFBP3 to erythroid culture altered the insulin signaling pathway; both total protein and activated phosphorylated (Ser 413) levels of FOXO3 increased (p=0.01 and p=0.03, respectively). Piceatannol induces HbF (Zhang, Blood 2018), however, this effect was abolished when AMPK specific inhibitor compound C was added (p=0.01). Neither IGFBP3 nor metformin altered erythroid maturation or expression of known gamma-globin regulators BCL11A, KLF1, and MYB; however, addition of IGFBP3 increased total NRF2 protein levels and Ser40 NRF2 phosphorylation. In vivo: In Table 1, we show the HbF response to metformin from our prospective clinical trial. Patients who demonstrated compliance with metformin showed an average 4 percentage point rise in HbF. Furthermore, retrospective chart review of HbF and vitamin D levels in patients with SCD indicate that HbF levels correlate strongly with vitamin D levels (R2=0.404), and that vitamin D supplementation increases HbF in patients with SCD (Figure 1). Conclusions: In vitro: We have shown that elevation or activation of IGFBP3, FOXO3, and AMPK induces HbF in HSPCs in vitro, without altering erythroid maturation or levels of BCL11A, KLF1, or MYB. These results show that manipulation of the insulin signaling pathway at several levels can induce HbF in vitro in HSPCs. We hypothesize that circulating IGFBP3 induces HbF via the insulin signaling pathway, by binding IGF1, preventing activation of the IGF1 receptor (IGF1R), a negative regulator of FOXO3. Thus, IGFBP3 may promote HbF production by inhibiting aFOXO3 inhibitor (Figure 2), and by activating a known positive regulator of HbF, NRF2. In vivo: Preliminary results from our clinical trial of metformin in patients with SCA shows a rise in HbF in adherent patients, providing in vivo support for the role of the insulin signaling pathway in HbF regulation. Correlations between HbF and vitamin D levels in patients with SCD suggest that agents that increase IGFBP3 like vitamin D, may increase HbF in patients with SCD. Our in vitro and in vivo data in combination indicates a role for the insulin signaling pathway in HbF regulation. We propose that the insulin signaling pathway can be pharmacologically targeted with safe, well-studied agents like metformin and Vitamin D along with other HbF inducers to maximize clinical benefit. Disclosures Weiss: Cellarity INC: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy.

Ascites from patients with alcoholic liver cirrhosis contains higher IGF‐I bioactivity than serum

Patients with liver cirrhosis have diminished hepatic IGF-I generation, resulting in low circulating levels, whereas data on IGF-I in ascites are sparse. Therefore, we compared the IGF-system in serum and ascites from cirrhotic patients. The study comprised 43 patients (12 females) with ascites and liver function of 58 +/- 10% of normal. Serum and ascites were collected concomitantly in the fasting state. In 11 patients, second serum and ascitic samples were collected within the first week. Eleven matched controls were also included. All samples were assayed for IGF-related parameters by immunoassays and by cell-based IGF-I bioassay. As compared with controls, serum total IGF-I, total IGF-II, pro-IGF-II and bioactive IGF-I were reduced in liver patients, whereas IGF-binding protein 1 (IGFBP-1), IGFBP-2 and the soluble IGF-II receptor were elevated (P < 0.005 for all). In ascites, all IGF-related peptides but pro-IGF-II were further reduced as compared with serum (P < 0.001). By contrast, bioactive IGF-I was fourfold elevated in ascites as compared with serum (2.20 +/- 0.33 vs. 0.55 +/- 0.08 microg/l, P < 0.001). In ascites, the IGF-I bioactivity signal was completely blocked by addition of IGFBP-3. Repetitive measurements (n = 11) in ascites showed that all peptides but IGFBP-1 remained unchanged within 1 week. It is a novel observation that the in vitro bioactivity of IGF-I can be higher in fluids from an extravascular compartment than in serum, in contrast to immunoreactive levels. This supports different roles for endocrine and paracrine/autocrine IGF-I, but the pathophysiological significance of our observation remains to be clarified.

Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes.

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.

Down-regulation of insulin-like growth factor binding proteins and growth modulation in hepatoma cells by retinoic acid.

We observed that all-trans-retinoic acid (RA) down-regulated insulin-like growth factor binding proteins (IGFBPs) in cultured human hepatoma cells (Hep 3B, PLC/PRF/5, and Hep G2); therefore, we characterized the role of this down-regulation in cell growth. Treatment with 10 micromol/L RA revealed a rapid decrease in IGFBP-3 within 2 days, and continued treatment with RA for 6 days resulted in a time-dependent stimulation of Hep 3B cell growth. However, RA treatment decreased IGFBP-1 in PLC/PRF/5 cells and in Hep G2 cells, and the growth-stimulatory activity of RA was transient and less prominent, and was finally obliterated in both cell lines. The addition of 5 ng/mL or 50 ng/mL insulin-like growth factors (IGFs) did not change the growth effects elicited by RA. The addition of IGFBP-3 (1,000 ng/mL) inhibited the growth of Hep 3B cells and counteracted the growth-stimulatory activity of RA, but not completely, suggesting that RA has direct growth-stimulatory activity and that this is enhanced by autocrine down-regulation of IGFBP-3. IGFBP-3 also inhibited the growth of PLC/PRF/5 cells and of Hep G2 cells. Treatment with phosphorylated IGFBP-1 (1,000 ng/mL) alone or with RA did not affect the growth of PLC/PRF/5 cells or Hep G2 cells. However, addition of dephosphorylated IGFBP-1, derived from in vivo dephosphorylation of the phosphorylated form, stimulated the growth of both cell lines, independent of interaction with IGF-I. From these observations, we propose that RA down-regulates IGFBPs, which in turn causes autocrine modulation of cell growth independent of IGF in hepatoma cells in vitro or in vivo. In addition, RA regulates IGFBPs at the posttranscriptional (Hep 3B cells and Hep G2 cells) or transcriptional level (PLC/PRF/5 cells) in a cell-specific manner.

Insulin-like growth factor binding protein-3 inhibits gonadotropin-induced ovulation, oocyte maturation, and steroidogenesis in rabbit ovary.

The effects of insulin-like growth factor binding proteins (IGFBPs) on human CG (hCG)-induced oocyte maturation, ovulation, steroidogenesis, and intrafollicular plasminogen activator (PA) activity were investigated in rabbit ovaries perfused in vitro. The addition of IGFBP-3, but not IGFBP-1, to the perfusate dose dependently inhibited hCG-induced ovulation, whereas ovulation failed to occur in any ovaries perfused with medium or IGFBP-3 alone. IGFBP-3 (100 ng/ml) significantly inhibited the resumption of meiosis in ovulated ova and follicular oocytes in hCG-treated ovaries, as well as the hCG-stimulated production of estradiol (E2), but not progesterone, by the perfused ovaries. Intrafollicular PA activity increased significantly within 1 h after exposure to hCG, reaching a maximum at 4 h; IGFBP-3 significantly inhibited hCG-stimulated intrafollicular PA activity. The blockade of hCG-induced ovulation by IGFBP-3 correlated with the reduction in intrafollicular PA activity. Treatment with hCG induced a 2.5-fold increase in intrafollicular IGF-I messenger RNA levels at 4 h. Although ovulation failed to occur in ovaries treated with IGF-I (100 ng/ml) in the absence of gonadotropin, IGF-I significantly increased the mean diameter of preovulatory follicles and stimulated the resumption of meiosis in follicular oocytes. These effects of IGF-I on follicular growth and oocyte maturation were significantly inhibited by IGFBP-3 (100 ng/ml). Furthermore, IGFBP-3 significantly inhibited the IGF-I-stimulated production of E2. In conclusion, IGFBP-3, but not IGFBP-1, blocked the stimulatory effects of hCG in the ovulatory process. These findings suggest that IGFBP-3 may contribute to the regulation of intrafollicular PA activity during follicular development and ovulation evoked by gonadotropin exposure, at least in part, via neutralizing endogenously produced IGF-I.

Insulin-Like Growth Factor (IGF)-Binding Protein-3 Blocks IGF-I-Induced Receptor Down-Regulation and Cell Desensitization in Cultured Bovine Fibroblasts*

Insulin-like growth factor-I (IGF-I) initiates its diverse biological effects by binding to type I IGF receptors on cells. In addition, IGF-I associates with distinct proteins that can modulate its actions. One of these IGF-binding proteins, IGFBP-3, is the major circulating form in adults and is produced by many cells in culture. We investigated the effect of purified bovine IGFBP-3 on IGF-I binding and IGF-I stimulation of amino acid uptake and DNA synthesis in cultured bovine fibroblasts, a cell culture system highly suitable for these types of studies. Incubation of cells with IGF-I resulted in time- and dose-dependent decreases in [125I]IGF-I binding and IGF-I stimulated [3H]aminoisobutyric acid uptake and [3H]thymidine incorporation. Preincubation with 4 nM IGF-I resulted in a 50-60% decrease in IGF-I receptor binding, accompanied by marked decreases in IGF-I-stimulated [3H]aminoisobutyric acid uptake (50-60%) and [3H]thymidine incorporation (80-90%). Preincubation with the IGF-I analog [QAYL]IGF-I (4 nM) or with 100 nM insulin, growth factors that bind and activate type I IGF receptor signalling but have little or no affinity for IGFBP-3, had effects comparable to IGF-I, decreasing both IGF-I binding and action 50-95%. The addition of IGFBP-3 during the preincubation period with IGF-I blocked the decrease in receptor availability and prevented the cells from becoming desensitized. IGFBP-3 did not prevent the [QAYL]IGF-I- or insulin-induced receptor loss and cellular resistance to IGF-I. These data indicate that IGFBP-3 can prevent IGF-I-induced receptor down-regulation, a process that renders cells refractory to further stimulation by IGF-I. Thus, cell-derived IGFBP-3 may function in a buffering capacity to restrict IGF-I and target cell interaction, thereby modulating the biological response to changes in local IGF-I levels.