Youngman Oh

Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics.

The IGFBPs are a family of homologous proteins that have co-evolved with the IGFs and that confer upon the IGF regulatory system both functional and tissue specificity. IGFBPs are not merely carrier proteins for IGFs, but hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, IGFBPs appear to have intrinsic biological activity independent of IGFs. The current status of research on IGFBPs is reviewed herein. Following a brief introduction to the entire IGF/IGFBP system, separate sections for each of the six cloned mammalian IGFBPs, the most extensive for IGFBP3, cover selected topics that emphasize the dynamics of IGFBPs--that is, their regulation in cells, their functionally important post-translational modifications, and their interactions in the cellular microenvironment--and how these dynamics influence physiological function.

Synthesis and Characterization of Insulin-like Growth Factor-binding Protein (IGFBP)-7 RECOMBINANT HUMAN mac25 PROTEIN SPECIFICALLY BINDS IGF-I AND -II

The mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells. The deduced amino acid sequence of the human mac25 propeptide shares a 20-25% identity to human insulin-like growth factor-binding proteins (IGFBPs), suggesting that mac25 could be another member of the IGFBP family. In the present study, we have generated recombinant human mac25 (rh-mac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene in human tissues. Binding of 125I-IGF-I and 125I-IGF-II to rh-mac25 was demonstrated by Western ligand blotting after nondenaturing polyacrylamide gel electrophoresis and by affinity cross-linking with as little as 2 nM rh-mac25. Specificity of rh-mac25 binding to 125I-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity for IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fold lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. mac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines. In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in normal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IGF-binding protein.

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor β subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3(1–87)), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.

IGF-independent regulation of breast cancer growth by IGF binding proteins.

The human IGFBP family consists of at least seven proteins, designated as IGFBP-1, -2, -3, -4, -5, -6, and-7. IGFBPs 1-6 bind IGF-I and IGF-II with high affinity whereas IGFBP-7, a newly identified IGFBP, binds IGFs with lower affinity and constitutes a low-affinity member of the IGFBP family. IGFBPs serve to transport the IGFs, prolong their half-lives, and modulate their biological action. At the cellular level, IGFBPs can either potentiate or inhibit the mitogenic effects of IGFs, depending upon cell types and IGFBP species (IGF-dependent action of IGFBPs). However, recent studies have indicated that IGFBPs, especially IGFBP-3, potently inhibit breast cancer cell growth in an IGF-independent manner. The IGF-independent action of IGFBP-3 requires interaction with cell-surface association proteins, presumably putative IGFBP-3 specific receptors, and is responsible for growth inhibitory action of the known growth suppressing factors such as TGF-β, retinoic acid, and antiestrogens in breast cancer cells. Thus, IGFBP-3 appears to be a major factor in a negative control system involved in regulating human breast cancer cell growth in vitro. IGFBP-7, representing a low affinity IGFBP, appears to function as an IGF-independent cell growth regulator in breast cancer cells. Overall structural similarity between IGFBP-7 and classical high affinity IGFBPs 1-6 suggests that the mechanisms of action and signaling pathways used by IGFBP-7 may provide insight into the IGF-independent actions of the high affinity IGFBPs.A fuller understanding of the IGF-independent action of IGFBPs will allow us to understand how the growth of neoplastic cells can be modulated by the IGF/IGFBP system, and how other growth factors or pharmacological agents can interface with this system.

ANTIPROLIFERATIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP)-3 IN HUMAN BREAST CANCER CELLS

A number of lines of evidence suggest that IGFs are important mitogens in human breast cancer: (1) IGFs are the most potent growth factor in human breast cancer cells; (2) estrogen stimulates expression of IGF-II and the type 1 IGF receptor; and (3) stromal cells express IGFs, which may act in a paracrine manner. Numerous studies have demonstrated that IGFBPs modulate the mitogenic effects of IGFs in the local environment. In particular, we have recently demonstrated that IGFBP-3 inhibits the growth of Hs578T and MDA-MB-231 human breast cancer cells in an IGF-independent manner. Further studies revealed the existence of cell surface-associated IGFBP-3 receptors. Receptor binding and the subsequent antiproliferative action of IGFBP-3 was inhibited by IGFs, owing to the formation of an IGF-IGFBP-3 complex that prevents the binding of IGFBP-3 to its receptors. In addition, exogeneously added soluble heparin or heparan sulfate inhibited the binding of IGFBP-3 to the cell surface in a dose-dependent manner. However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically remove, IGFBP-3 binding was only minimally affected. These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting receptor binding of IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of IGFBP-3. Additionally, the role of IGFBP-3 in the antiproliferative effects of transforming growth factor (TGF)-beta and retinoic acid (RA) is supported by our observations that: (1) inhibition of IGFBP-3 gene expression using an IGFNBP-3 antisense oligodeoxynucleotide not only blocks TGF-beta and RA simulation of IGFBP-3 production by up to 90%m but also inhibits their antiproliferative effects by 40-60%; and (2) treatment with IGF-II and IGF-II analogs diminish TGF-beta effects by blocking TGF-beta induced binding of IGFBP-3 to the cell surface. Taken together, our results support the hypothesis that IGFBP-3 is an important antiproliferative factor in human breast cancer, acting in an IGF-independent manner in addition to its ability to modulate the binding of IGF peptides to IGF receptors.

INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS : A PROPOSED SUPERFAMILY

Hwa V, Oh Y, Rosenfeld RG. Insulin‐like growth factor binding proteins: a proposed superfamily. Acta Pzdiatr 1999; Suppl 428:37‐45. Stockholm. ISSN 0803‐5326

Characterization of Insulin-Like Growth Factor-Binding Protein-Related Proteins (IGFBP-rPs) 1, 2, and 3 in Human Prostate Epithelial Cells: Potential Roles for IGFBP-rP1 and 2 in Senescence of the Prostatic Epithelium

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPs) are newly described cysteine-rich proteins that share significant aminoterminal structural similarity with the conventional IGFBPs and are involved in a diversity of biological functions, including growth regulation. IGFBP-rP1 (MAC25/Angiomodulin/prostacyclin-stimulating factor) is a potential tumor-suppressor gene that is differentially expressed in meningiomas, mammary and prostatic cancers, compared with their malignant counterparts. We have previously shown that IGFBP-rP1 is preferentially produced by primary cultures of human prostate epithelial cells (HPECs) and by poorly tumorigenic P69SV40T cells, compared with the cancerous prostatic LNCaP, DU145, PC-3, and M12 cells. We now show that IGFBP-rP1 increases during senescence of HPEC. IGFBP-rP2 (also known as connective tissue growth factor), a downstream effector of transforming growth factor (TGF)-beta and modulator of growth for both fibroblasts and endothelial cells, was detected in most of the normal and malignant prostatic epithelial cells tested, with a marked up-regulation of IGFBP-rP2 during senescence of HPEC. Moreover, IGFBP-rP2 noticeably increased in response to TGF-beta1 and all-trans retinoic acid (atRA) in HPEC and PC-3 cells, and it decreased in response to IGF-I in HPEC. IGFBP-rP3 [nephroblastoma overexpressed (NOV)], the protein product of the NOV protooncogene, was not detected in HPEC but was expressed in the tumorigenic DU145 and PC-3 cells. It was also synthesized by the SV40-T antigen-transformed P69 and malignant M12 cells, where it was down-regulated by atRA. These observations suggest biological roles of IGFBP-rPs in the human prostate. IGFBP-rP1 and IGFBP-rP2 are likely to negatively regulate growth, because they seem to increase during senescence of the prostate epithelium and in response to growth inhibitors (TGF-beta1 and atRA). Although the data collected on IGFBP-rP3 in prostate are modest, its role as a growth stimulator and/or protooncogene is supported by its preferential expression in cancerous cells and its down-regulation by atRA.

Insulin-like growth factor binding protein-3 and-5 are regulated by transforming growth factor-β and retinoic acid in the human prostate adenocarcinoma cell line PC-3

The family of insulin-like growth factor binding proteins (IGFBPs) can affect cell proliferation by modulating the availability and bioactivity of insulin-like growth factors (IGFs), or by mechanisms independent of IGFs. To understand better the role(s) of IGFBPs in prostate growth and malignancy, we examined the regulation of IGFBPs in PC-3 cells, a human prostatic adenocarcinoma epithelial cell line that is androgeninsensitive. Both transforming growth factor-β (TGF-β) and retinoic acid (RA), known inhibitors of cellular proliferation, significantly changed the IGFBP profile in PC-3 cells. In cells that were treated with transforming growth factor β-2 (TGF-β2) (0.5–10 ng/mL), IGFBP-3, and IGFBP-5 protein and mRNA increased in a time-and dose-dependent manner. At 10 ng/mL TGF-β, IGFBP-3, and IGFBP-5 protein concentrations were 14- and 9-fold, respectively, over that of controls. Cells treated with Ra (0–1 μM) also showed a time- and dose-dependent increase in IGFBP-3 protein and mRNA levels. However, in contrast to TGF-β2, high concentrations of RA (1 μM) negatively regulated IGFBP-5 expression, with IGFBP-5 mRNA levels downregulated to 20% of that of the control, and protein levels were decreased by 50%. Since both TGF-β and RA increased IGFBP-3 expression and both are known to inhibit prostate cell growth, we speculate that the inhibition of growth is mediated, at least in part, by IGFBP-3.

IGFBPs and neoplastic models: New concepts for roles of IGFBPs in regulation of cancer cell growth

The insulin-like growth factor binding proteins (IGFBPs) are a family of seven structurally homologous proteins that bind insulin-like growth factor 1 (IGF-1) and IGF-II with high affinity, thereby modulating the actions of IGFs. Several lines of recent evidence from various cell systems have suggested that IGFBPs, especially IGFBP-3, may play more active, IGF-independent, roles in growth regulation of cancer cells. In support of this hypothesis, the author has recently shown that IGFBP-3 binds specifically and with high affinity to the surface of various cell types and directly inhibits monolayer growth of these cells in an IGF-independent manner, presumably by specific interaction with cell membrane proteins that function as an IGFBP-3 receptor. The author’s current studies demonstrate that a new class of IGFBP, IGFBP-7, constitutes a low affinity member of the IGFBP family, but primarily functions as a modulator of cell growth in an IGF-independent manner, similar to the action observed with IGFBP-3 in breast cancer cells. The author’s studies on the mechanisms of action of the low affinity IGFBPs will provide insight into the IGF-independent actions of the classical high affinity IGFBPs and their impact on cancer cell growth.

CTGF (IGFBP-rP2) is specifically expressed in malignant lymphoblasts of patients with acute lymphoblastic leukaemia (ALL)

Connective tissue growth factor (CTGF) is a major chemotactic and mitogenic factor for connective tissue cells. The amino acid sequence shares an overall 28–38% identity to IGFBPs and contains critical conserved sequences in the amino terminus. It has been demonstrated that human CTGF specifically binds IGFs with low affinity and is considered to be a member of the IGFBP superfamily (IGFBP-rP2). In the present study, the expression of CTGF (IGFBP-rP2) in human leukaemic lymphoblasts from children with acute lymphoblastic leukaemia (ALL) was investigated. RNA samples from tumour clones enriched by ficoll separation of bone marrow or peripheral blood mononuclear cells (MNC) from 107 patients with childhood ALL at diagnosis and 57 adult patients with chronic myeloid leukaemia (CML) were studied by RT-PCR. In addition MNC samples from children with IDDM and cord blood samples from healthy newborns were investigated as control groups. Sixty-one percent of the patients with ALL (65 of 107) were positive for CTGF (IGFBP-rP2) expression. In the control groups, no expression of CTGF (IGFBP-rP2) in peripheral MNC was detected, and in the group of adult CML patients only 3.5% (2 of 57) were positive for this gene. The role of CTGF (IGFBP-rP2) in lymphoblastic leukaemogenesis requires further evaluation, as does its potential utility as a tumour marker.