Sue M. Firth

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-xL protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G1 arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G1 arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

The IGF axis and programmed cell death.

Insulin‐like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of high‐affinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP‐3, may have its own pro‐apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

Growth Inhibition by Insulin-like Growth Factor-binding Protein-3 in T47D Breast Cancer Cells Requires Transforming Growth Factor-β (TGF-β) and the Type II TGF-β Receptor

This study explores the relationship between anti-proliferative signaling by transforming growth factor-β (TGF-β) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-β1. To investigate the mechanism, we used T47D cells that lack type II TGF-β receptor (TGF-βRII) and are insensitive to TGF-β1. After introducing the TGF-βRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-β1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-β1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-βRII-expressing cells when exogenous TGF-β1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-β1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-βRII and exogenous TGF-β1. To investigate this synergism, the phosphorylation of TGF-β signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-β1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-β signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Nuclear Insulin-Like Growth Factor Binding Protein-3 Induces Apoptosis and Is Targeted to Ubiquitin/Proteasome–Dependent Proteolysis

Insulin-like growth factor binding protein-3 (IGFBP-3), the product of a tumor suppressor target gene, can modulate cell proliferation and apoptosis by IGF-I-dependent and IGF-I-independent mechanisms. IGFBP-3 controls the bioavailability of IGFs in the extracellular environment and is known to be subject to degradation by various extracellular proteases. Although nuclear localization and functions of IGFBP-3 have been described in the past, we show as the novel features of this study that the abundance of nuclear IGFBP-3 is directly regulated by ubiquitin/proteasome-dependent proteolysis. We show that IGFBP-3 degradation depends on an active ubiquitin-E1 ligase, specific 26S proteasome inhibitors can efficiently stabilize nuclear IGFBP-3, and the metabolic half-life of nuclear IGFBP-3 is strongly reduced relative to cytoplasmic IGFBP-3. Nuclear IGFBP-3 is highly polyubiquitinated at multiple lysine residues in its conserved COOH-terminal domain and stabilized through mutation of two COOH-terminal lysine residues. Moreover, we show that IGFBP-3, if ectopically expressed in the nucleus, can induce apoptotic cell death. These results suggest that ubiquitin/proteasome-mediated proteolysis of IGFBP-3 may contribute to down-regulation of apoptosis.

THE ROLE OF GLYCOSYLATION IN THE ACTION OF IGFBP-3

There are three potential N-glycosylation sites (Asn-X-Ser/Thr) located in the nonconserved central region of the human IGFBP-3 sequence (Asn89, Asn109, Asn172-sites 1, 2 and 3, respectively). Upon ligand blotting with IGFs, IGFBP-3 appears as two bands (40-45 kDa) representing different glycosylated forms. We have mutated the N-glycosylation sites in permutations of three single, three double and one triple mutations and expressed these variant cDNAs. Each mutant protein was detected by radioimmunoassay, indicating that glycosylation is not required for the secretion of the protein from CHO cells. Ligand blotting using [125I]IGF-I indicated that all seven mutants retained IGF-I binding. Based on the molecular weights of the variant proteins, there are approximately 4, 5 and 6 kDa of carbohydrate on sites 1, 2 and 3, respectively. Furthermore, the two forms of IGFBP-3 represent the protein glycosylated either at all three sites or at Asn89 and Asn109 only. There appears to be no difference between the mutants and the fully-glycosylated rhIGFBP-3 in their acid-labile subunit (ALS) binding. Analysis of variance confirmed that the association constant for ALS was not significantly changed by any mutation [Ka (fully-glycosylated) = 12.5 +/- 4.1 nM-1; mean Ka (all mutants) = 22.1 +/- 3.0 nM-1]. While glycosylation does not appear to play a role in IGFBP-3 ligand binding, it may affect the turnover rate of the protein or be involved in rendering the protein resistant to proteolysis.

The Acid-labile Subunit of the Serum Insulin-like Growth Factor-binding Protein Complexes STRUCTURAL DETERMINATION BY MOLECULAR MODELING AND ELECTRON MICROSCOPY

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potentialN-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.

Role Of N- And C-Terminal Residues Of Insulin-Like Growth Factor (IGF)-Binding Protein-3 In Regulating IGF Complex Formation And Receptor Activation

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu77, Leu80, and Leu81 in the N terminus and Gly217 and Gln223 in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF·IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.

IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments.

Despite the established role of insulin-like growth factor binding protein-3 (IGFBP-3) as a growth inhibitor in vitro, a high level of IGFBP-3 in breast tumor tissue is associated with the stimulation of xenograft growth in mice and poor prognosis in patients. To understand the contribution of IGFBP-3 to breast cancer progression, tandem affinity purification was used to identify novel interacting proteins. The endoplasmic reticulum protein, glucose-regulated protein 78 (GRP78), was shown to bind to IGFBP-3, confirmed by colocalization, coimmunoprecipitations, glutathione S-transferase (GST) pulldowns and a nanomolar binding affinity. GST pulldowns also indicated that the GRP78 ATPase domain mediated the interaction with IGFBP-3. The critical roles of GRP78 in the unfolded protein response and macroautophagy led to an investigation of possible links between IGFBP-3, GRP78 and cellular stress responses. IGFBP-3 was found to stimulate the survival of breast cancer cells subjected to glucose starvation and hypoxia. Pharmacological inhibitors and small interfering RNA knockdown established that the increased survival of IGFBP-3-expressing cells was dependent on an intact autophagy response, as well as GRP78. The contribution of autophagy was confirmed by the demonstration that IGFBP-3 expression increases both the formation of autophagic puncta and flux through the system. In conclusion, we have shown that IGFBP-3 stimulates autophagy and thereby promotes the survival of breast cancer cells exposed to conditions that represent the adverse microenvironments encountered by solid tumor cells in vivo.

Involvement of Pregnancy-Associated Plasma Protein-A2 in Insulin-Like Growth Factor (IGF) Binding Protein-5 Proteolysis during Pregnancy: A Potential Mechanism for Increasing IGF Bioavailability

CONTEXT During pregnancy, circulating IGF binding protein-5 (IGFBP-5) undergoes substantial molecular redistribution from ternary complexes to either binary complexes or the uncomplexed protein. OBJECTIVE This study aimed to characterize the proteolysis of circulating IGFBP-5 during pregnancy and to determine whether it can increase IGF bioavailability. DESIGN Biochemical methods were used to purify and characterize IGFBP-5 fragments and IGFBP-5-specific proteolytic activity from pregnancy plasma. RESULTS Circulating IGFBP-5 was fully proteolyzed at all stages of pregnancy. Cleavage after either Ser143 or Lys144 resulted in two complementary fragments. Of two pools of proteolytic activity (>150 kDa and approximately 40 kDa) identified in pregnancy plasma, only the greater than 150-kDa proteolytic activity was specific to pregnancy. The approximately 40-kDa proteolytic activity, also present in nonpregnancy plasma, appeared largely inactive against IGF-I-complexed IGFBP-5. The greater than 150-kDa proteolytic activity was inhibited by alpha-PAPP-A2 but not alpha-PAPP-A1 antibody, cleaved recombinant IGFBP-5 at Ser143-Lys144 similar to PAPP-A2, and was inactive against IGFBP-5 (Ala128), a PAPP-A2-resistant analog. Compared to nonpregnancy plasma, incubation with pregnancy plasma resulted in release of more bioactive IGF-I from IGF-I-IGFBP-5 complexes as measured by stimulation of IGF-I receptor phosphorylation. CONCLUSIONS Circulating IGFBP-5 is proteolyzed by PAPP-A2 during pregnancy, resulting in increased IGF bioavailability, which may have important consequences for the development of the fetus and/or the well-being of the mother.