Insulin-like Growth Factor-binding Protein-5 Activates Plasminogen by Interaction with Tissue Plasminogen Activator, Independently of Its Ability to Bind to Plasminogen Activator Inhibitor-1, Insulin-like Growth Factor-I, or Heparin

Gordon J Allan,Matteo Gamberoni,Elizabeth Tonner,James Beattie,David J Flint,Alice M Sorrell,Pier A Accorsi,John H Shand

doi:10.1074/jbc.m508505200

Abstract

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.

Highlights

The remodeling of the mammary gland, which occurs at the end of lactation, involves an initial phase in which there are dramatic increases in the rates of apoptosis and a second phase involving extracellular proteases, including the plasminogen system and matrix metalloproteinases, which are involved in degrading the extracellular matrix in the later stages of tissue remodeling [6]
This could provide a mechanism by which IGFBP-5 could coordinate the induction of cell death with the activation of extracellular matrix degradation by generation of the extracellular protease cascades initiated by plasminogen activators
Bovine insulin (I-6634), epidermal growth factor (E-4127), and urokinase plasminogen activator (uPA) were from Sigma. tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and plasminogen were from Calbiochem

Summary

Introduction

The remodeling of the mammary gland, which occurs at the end of lactation, involves an initial phase in which there are dramatic increases in the rates of apoptosis and a second phase involving extracellular proteases, including the plasminogen system and matrix metalloproteinases, which are involved in degrading the extracellular matrix in the later stages of tissue remodeling [6]. Recent studies have suggested that the effects of IGFBP-3 and -5 can be both IGF-dependent and IGF-independent and that interactions with particular extracellular matrix components might influence these responses [8, 9] These observations led us to consider whether the extremely high concentrations of IGFBP-5 in milk from involuting mammary glands (ϳ50 –100 ␮g/ml) were present solely to inhibit IGF actions or whether they were involved in additional and perhaps IGF-independent actions [10]. The aims of this study were to determine whether IGFBP-5 and IGFBP-5 mutants that are unable to bind to IGF-I or heparin are able to interact with PAI-1 and influence plasminogen activation If so, this could provide a mechanism by which IGFBP-5 could coordinate the induction of cell death (by sequestering IGF-I) with the activation of extracellular matrix degradation by generation of the extracellular protease cascades initiated by plasminogen activators.

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