Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit

Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin beta nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[(228)KGRKR --> MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215-232] and IGFBP-5 [201-218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin beta and the alpha/beta heterodimer but only poorly by importin alpha. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-beta-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components.     

Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism

The insulin-like growth factor (IGF) signaling pathway plays a crucial role in the regulation of cell growth, differentiation, apoptosis, and aging. IGF-binding proteins (IGFBPs) are important members of the IGF axis. IGFBP-5 is up-regulated during cellular senescence in human dermal fibroblasts and endothelial cells, but the function of IGFBP-5 in cellular senescence is unknown. Here we show that IGFBP-5 plays important roles in the regulation of cellular senescence. Knockdown of IGFBP-5 in old human umbilical endothelial cells (HUVECs) with IGFBP-5 micro-RNA lentivirus caused partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, increases in cell proliferation, and decreases in senescence-associated beta-galactosidase (SA-beta-gal) staining. In addition, treatment with IGFBP-5 protein or up-regulation of IGFBP-5 in young cells accelerates cellular senescence, as confirmed by cell proliferation and SA-beta-gal staining. Premature senescence induced by IGFBP-5 up-regulation in young cells was rescued by knockdown of p53, but not by knockdown of p16. Furthermore, atherosclerotic arteries exhibited strong IGFBP-5-positive staining along intimal plaques. These results suggest that IGFBP-5 plays a role in the regulation of cellular senescence via a p53-dependent pathway and in aging-associated vascular diseases.     

Paradoxical actions of endogenous and exogenous insulin-like growth factor-binding protein-5 revealed by RNA interference analysis

Insulin-like growth factor-binding protein-5 (IGFBP-5) is abundantly expressed in bone cells. To determine the physiological role(s) of endogenous IGFBP-5 in regulating bone cell growth, differentiation, and survival, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in human osteosarcoma cells. The IGFBP-5 siRNA, targeting against a sequence unique to the IGFBP-5 middle domain, efficiently reduced IGFBP-5 mRNA and protein levels. The IGFBP-5 siRNA did not change the levels of IGFBP-4, a structurally related protein, or glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene. Knock-down of IGFBP-5 resulted in a significant increase in the number of transferase-mediated dUTP nick end labeling-positive cells and a decrease in a bone differentiation parameter (alkaline phosphatase activity) but had little effect on basal or insulin-like growth factor I-induced proliferation. Overexpression of a siRNA-resistant IGFBP-5 mutant in the IGFBP-5 knock-down cells restored the levels of survival to the control level; overexpression of IGFBP-4 or wild type IGFBP-5 had no such effect. Paradoxically, the addition of exogenous IGFBP-5 not only failed to rescue IGFBP-5 knock-down-induced apoptosis, it caused a further increase in apoptosis. Furthermore, the addition of exogenous IGFBP-5 alone increased apoptosis. This pro-apoptotic action of exogenous IGFBP-5 was abolished when IGF-I was added in excess, suggesting that exogenous IGFBP-5 increases apoptosis by binding to and inhibiting the activities of insulin-like growth factors. These results indicate that endogenous and exogenous IGFBP-5 exhibits opposing biological actions on cell survival and underscore the necessity and utility of studying IGFBP functions through loss-of-function approaches.     

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005). An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8. These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2. Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.     

Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin-like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin-like growth factor binding protein 5 (IGFBP-5) and the type-1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP-5 mRNA and protein expression. We also show that IGF-I increases IGFBP-5 expression in CGCs and that the downregulation of IGFBP-5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF-related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP-5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF-triggered survival signalling.     

Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IGFBP-5 (changing one charged residue, Lys(68), to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an approximately 1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.     

Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5

The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known about the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased twofold during the process of differentiation. IGF-binding protein-3 (IGFBP-3) expression showed a biphasic pattern of with a twofold increase at the onset of differentiation and a downregulation in late differentiating chondrocytes to 25% of baseline levels; the expression patterns of IGFBP-2, -4 and -6 were not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2 and -6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. Although IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol (PI) 3-kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.     

Insulin-like growth factor-binding protein-5 inhibits growth and induces differentiation of mouse osteosarcoma cells

The precise role of insulin-like growth factor-binding protein-5 (IGFBP-5) in regulating the growth of tumor cells, especially of bone-derived malignant cells, is not well understood. We have investigated the biological activity of IGFBP-5 by transfecting OS/50-K8 mouse osteosarcoma cells with an expression vector containing the osteocalcin promoter and the complete mouse IGFBP-5 cDNA (OC-IGFBP-5). Overexpression of IGFBP-5 mRNA and secretion of increased amounts of bioactive protein in conditioned media were demonstrated in different clones. For the analysis of cell proliferation, three clones exhibiting high levels of IGFBP-5 expression were selected and compared to a mock clone and to nontransfected parental cells. IGFBP-5-secreting clones displayed reduced proliferation under both anchorage-dependent and -independent conditions (P < 0.05). The increase in proliferation observed in IGFBP-5-secreting clones after addition of exogenous IGF was significantly lower than that observed in mock-transfected or parental cells. A similar result was obtained with long[R3]IGF-I which has a low affinity for all IGFBPs, suggesting that the inhibitory effect of IGFBP-5 is only partially IGF-dependent. OC-IGFBP-5-transfected clones expressed significantly higher amounts of osteocalcin mRNA (P < 0.05) and secreted more osteocalcin protein than a mock clone or parental OS-50/K8 cells. Thus, part of the growth-inhibiting effect of IGFBP-5 may be due to an induction of differentiation in these cells.     

Quantitative analysis of insulin-like growth factor-modulated proteolysis of insulin-like growth factor binding protein-4 and -5 by pregnancy-associated plasma protein-A

The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) specifically cleaves insulin-like growth factor binding protein (IGFBP)-4 and -5. Regulation of insulin-like growth factor (IGF) bioavailability through cleavage of these inhibitory binding proteins is an important mechanism for the control of growth and development of vertebrate cells. Although proteolysis of IGFBP-4 and -5 by PAPP-A has been extensively studied in many systems, quantitative analyses have been lacking. We have characterized the cleavage of its natural substrates, IGFBP-4 and -5, in the absence and presence of IGF-I or -II and determined the kinetic parameters (Km and kcat) for the different combinations of IGFBP and IGF. The rate of IGFBP-4 proteolysis is dramatically increased upon addition of IGF-I or -II. Kinetic analysis revealed that IGF-II was a more potent activator of IGFBP-4 proteolysis than IGF-I. Proteolysis of IGFBP-5 is slightly inhibited by IGF, and we find that IGF-I and -II display a similar degree of inhibition of IGFBP-5 cleavage. We show that the mechanism of IGF-modulated proteolysis of IGFBP-4 and -5 involves changes in both the recognition of substrate (Km) and the turnover rate (kcat). In addition, we have devised a novel method of revealing potential consequences of substrate modification for kinetic analysis, and we have used this method to establish that there is no apparent difference in the behavior of radiolabeled IGFBP-4 and -5 compared to the behavior of the unmodified protein substrates. We also propose experimental conditions for the proper analysis of IGFBP proteolysis, and we demonstrate their usefulness by quantitatively evaluating the effect of inhibitory compounds on the rate of proteolysis. Finally, we have compared PAPP-A to other proteinases thought to have IGFBP-4 or -5 as a substrate. This emphasizes the potential of PAPP-A to specifically and efficiently function as a regulator in the IGF system.     

Fibronectin binds insulin-like growth factor-binding protein 5 and abolishes Its ligand-dependent action on cell migration

Insulin-like growth factor-binding protein 5 (IGFBP-5) is a secreted protein that binds to insulin-like growth factors (IGFs) and modulates IGF actions on cell proliferation, differentiation, survival, and motility. IGFBP-5 also regulates these cellular events through IGF-independent mechanisms. To elucidate the molecular mechanisms governing these diverse actions of IGFBP-5, we screened a human cDNA library by a yeast two-hybrid system using IGFBP-5 as bait and identified fibronectin (FN) as a potential IGFBP-5-interacting partner. The complex formation of IGFBP-5 and FN was established by glutathione S-transferase pull-down, solution, and solid phase binding assays using glutathione S-transferase-IGFBP-5 and native IGFBP-5 in vitro and by co-immunoprecipitation in vivo. Binding assay using deletion mutants indicated that the IGFBP-5 C domain binds to the 10th and 11th type I repeats of FN. IGFBP-5 potentiated IGF-I-induced cell migration in FN-null, but not in wild-type, mouse embryonic cells. When FN was reintroduced either as an adhesive substrate or in solution to the FN-null cells, the potentiating effect of IGFBP-5 on IGF-I-induced cell migration was abolished. Binding of IGFBP-5 to FN had no effect on the ability of IGFBP-5 to bind IGF-I, but it increased the proteolytic degradation of IGFBP-5. Inhibition of IGFBP-5 proteolysis restored the potentiating effect of IGFBP-5. These results suggest that FN and IGFBP-5 bind to each other, and this binding negatively regulates the ligand-dependent action of IGFBP-5 by triggering IGFBP-5 proteolysis.     

Insulin-like growth factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation

Schwann cells (SCs) are the myelin producing cells of the peripheral nervous system. During development, SCs cease proliferation and differentiate into either a myelin-forming or non-myelin forming mature phenotype. We are interested in the role of insulin-like growth factor-I (IGF-I) in SC development. We have shown previously SCs proliferate in response to IGF-I in vitro. In the current study, we investigated the role of IGF-I in SC differentiation. SC differentiation was determined by morphological criteria and expression of myelin proteins. Addition of 1 mM 8-bromo cyclic AMP (cAMP) or growth on Matrigel matrix decreased proliferation and induced differentiation of SCs. IGF-I enhanced both cAMP and Matrigel matrix-induced SC differentiation, as assessed by both morphological criteria and myelin gene expression. Cultured SCs also express IGF binding protein-5 (IGFBP-5), which can modulate the actions of IGF-I. We examined the expression of IGFBP-5 during SC differentiation. Both cAMP and Matrigel matrix treatment enhanced IGFBP-5 protein expression and cAMP increased IGFBP-5 gene expression five fold. These findings suggest IGF-I potentiates SC differentiation. The concomitant up-regulation of IGFBP-5 may play a role in targeting IGF-I to SCs and thus increase local IGF-I bioavailability. J. Cell. Physiol. 171:161–167, 1997. © 1997 Wiley-Liss, Inc.     

Insulin-like growth factor-binding protein-3 plays an important role in regulating pharyngeal skeleton and inner ear formation and differentiation

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypoglycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IGFBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IGFBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner ear development and growth in zebrafish.     

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 μM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or, alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley-Liss, Inc.