John I. Jones

The Roles of Integrins and Extracellular Matrix Proteins in the Insulin-like Growth Factor I-stimulated Chemotaxis of Human Breast Cancer Cells

The effects of insulin-like growth factor I (IGF-I) on the migration of two human breast cancer cell lines, MCF-7 and MDA-231, were examined using a modified Boyden chamber. 10 ng/ml was the optimal IGF-I concentration for stimulation of migration. The majority of IGF-I-stimulated migration in both cell types was due to chemotaxis. MCF-7 cells failed to migrate on membranes coated with gelatin or fibronectin and migrated only in small numbers on laminin. In contrast, when vitronectin- or type IV collagen-coated membranes were used, the MCF-7 cells migrated in large numbers specifically in response to IGF-I but not to 10% fetal calf serum, epidermal growth factor, fibroblast growth factor, or platelet derived growth factor-BB. An IGF-I receptor-blocking antibody inhibited IGF-I-stimulated migration in both cell types. In addition, a blocking antibody to the αvβ5 integrin (a vitronectin receptor) inhibited migration of MCF-7 cells in response to IGF-I through vitronectin but not through type IV collagen. Similarly, blocking antibodies specific for α2 and β1 integrins significantly inhibited migration of both cell types through type IV collagen-coated membranes but not through vitronectin-coated membranes. We conclude that: 1) IGF-I stimulates migration of these two cell types through the IGF-I receptor; 2) interaction of vitronectin with the αvβ5 integrin or collagen with the α2β1 integrin is necessary for the complete IGF-I response in MCF-7 cells, and 3) because migration represents an in vitro model for metastatic spread, integrins, extracellular matrix proteins, and IGF-I may play coordinated roles in the metastasis of breast cancer in vivo.

Cell migration: Interactions among integrins, IGFs and IGFBPs

The migratory behaviour of cells is fundamental to diverse biologic processes such as tumor metastasis, development of atherosclerotic plaques, embryonic development and wound healing. We have examined the effects of IGF-I and IGFBPs on the migration of Chinese Hamster ovary (CHO) cells, smooth muscle cells (SMC) and human breast cancer cells (HBC) and have studied the involvement of integrin receptors in migration induced by IGF-I and by IGFBPs. Using a monolayer wounding assay, we determined the effect of IGFBP-1 on SMC to be qualitatively similar to its effect we reported earlier on CHO cells, in that there is a direct stimulation of migration mediated by the alpha 5 beta 1 integrin. IGFBP-2 has no direct effect on SMC migration, and although it also contains the Arg-Gly-Asp sequence, we can detect no integrin binding. Unlike CHO cells, SMC are stimulated to migrate by IGF-I. IGFBP-2 and IGFBP-1 both inhibit this IGF-I receptor-mediated stimulation. We have also studied the migration of HBC using a Boyden chamber apparatus and have shown a potent chemotactic effect of IGF-I. We have investigated the mechanisms for IGF-I stimulation of SMC and HBC migration. IGF-I stimulation of SMC migration requires the presence of either 0.2% serum or vitronectin, because of a requirement for ligand binding by the alpha V beta 3 integrin (vitronectin receptor). MCF-7 HBC migrate toward a concentration gradient of IGF-I, the only growth factor that was able to stimulate these cells to migrate. Integrin ligand binding was also necessary for MCF-7 cells to migrate in response to IGF-I; alpha V beta 5 integrin was required for migration on vitronectin and alpha 2 beta 1 was required on collagen. These studies demonstrate that the stimulation of cell migration by IGFBP-1 and IGF-I involves signaling by members of the integrin family of receptors. The mechanisms by which the IGF-I receptor and integrin receptors interact are not yet known.

Role of insulin-like growth factor binding proteins in the control of IGF actions.

The insulin-like growth factor binding proteins have been shown to modify IGF actions. IGFBP-5 binds to extracellular matrix (ECM) and its ability to potentiate IGF activity is dependent upon the amount that is ECM associated. To determine the specific regions of IGFBP-5 that are required for ECM association, site directed mutagenesis has been used to prepare several forms of IGFBP-5. Mutants that have had the amino acids between positions 201 and 218 altered have been useful. Mutation of the lysine 211 resulted in no change in the affinity of IGFBP-5 for ECM or heparin Sepharose; however, it resulted in a major reduction in affinity for IGF-I following heparin binding. Other mutations which disrupted heparin binding also resulted in loss of this affinity shift. Most distruptive were mutations of amino acids 211, 214, 217 and 218 and 202, 206 and 207. Mutation of residues 201 plus 202 had some effect, but substitution for 207, 211, 217 and 218 had no effect. When binding to intact ECM was analyzed, similar results were obtained. This suggests that amino acids 202, 206 and 214 are definitely involved in heparin and ECM binding. When binding to proteoglycans such as tenascin and heparin sulfate proteoglycan was analyzed, similar results were obtained. IGFBP-5 also binds to other proteins in ECM, including type IV collagen and plasminogen activator inhibitor-I. Specific antisera for plasminogen activator inhibitor-1 can coprecipitate IGFBP-5. IGFBPs are degraded by specific proteases. Three proteases that degrade IGFBP-2, -4 and -5 have been characterized. They are serine proteases that cleave these proteins at basic residues. Although several well characterized serine proteases cleave IGFBP-4 or -5, the proteases in cell conditioned media appear to be distinct.

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin‐like growth factor binding protein‐I (IGFBP‐1) alters its binding affinity for insulin‐like growth factor I (IGF‐I) and thus regulates the bioavailability of IGF‐I for binding to the IGF‐I receptor. The kinase(s) responsible for the phosphorylation of IGFBP‐1 has not been identified. This study was designed to characterize the IGFBP‐1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP‐1 primarily as phosphorylated isoforms. IGFBP‐1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate Mr 150,000 (peak I kinase) and Mr 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP‐1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC50 = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI‐7 (IC50 = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma‐32P]ATP lowered the incorporation of 32P into IGFBP‐1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP‐1 was radiolabeled by the partially purified kinase activity when [gamma‐32P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP‐1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP‐1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases.