Targeted Expression of a Protease-resistant IGFBP-4 Mutant in Smooth Muscle of Transgenic Mice Results in IGFBP-4 Stabilization and Smooth Muscle Hypotrophy

Mingyu Zhang,Eric P Smith,Hiroaki Kuroda,Walter Banach,Steven D Chernausek,James A Fagin

doi:10.1074/jbc.m112082200

Abstract

The insulin-like growth factor-binding protein 4 (IGFBP-4), the most abundant IGF-binding protein produced by rodent smooth muscle cells (SMC), is degraded by specific protease(s) potentially releasing IGF-I for local bioactivity. IGFBP-4 protease(s) recognizes basic residues within the midregion of the molecule. We constructed a mutant IGFBP-4 with the cleavage domain substitution 119-KHMAKVRDRSKMK-133 to 119-AAMAAVADASAMA-133. Myc-tagged native and IGFBP-4.7A retained equivalent IGF-I binding affinity. Whereas native IGFBP-4 was cleaved by SMC-conditioned medium, IGFBP-4.7A was completely resistant to proteolysis. To explore the function of the protease-resistant IGFBP-4 in vivo, expression of the mutant and native proteins was targeted to SMC of transgenic mice by means of a smooth muscle alpha-actin promoter. Transgene expression was confined to SMC-rich tissues in all lines. Bladder and aortic immunoreactive IGFBP-4/transgene mRNA ratios in SMP8-BP4.7A mice were increased by 2- to 4-fold relative to SMP8-BP4 mice, indicating that the IGFBP-4.7A protein was stabilized in vivo. SMP8-BP4.7A mice had lower aortic, bladder, and stomach weight and intestinal length relative to SMP8-BP4 counterparts matched for protein expression by Western blotting. Thus, IGFBP-4.7A results in greater growth inhibition than equivalent levels of native IGFBP-4 in vivo, demonstrating a role for IGFBP-4 proteolysis in the regulation of IGF-I action.

Highlights

Insulin-like growth factors (IGFs)1 I and II regulate cellular proliferation, survival, and differentiation
Bladder and aortic immunoreactive insulin-like growth factor-binding protein 4 (IGFBP-4)/transgene mRNA ratios in SMP8-BP4.7A mice were increased by 2- to 4-fold relative to SMP8-BP4 mice, indicating that the IGFBP-4.7A protein was stabilized in vivo
Binding of IGF-I by IGFBP-4.7A—The goal of this study was to examine the contribution of IGFBP-4 degradation to IGF action in vivo

Summary

Introduction

Insulin-like growth factors (IGFs) I and II regulate cellular proliferation, survival, and differentiation. These experiments clearly identify a 13-amino acid domain within the middle of the molecule as the principal site of cleavage This region is largely conserved among mammalian species of this binding protein but is unique to IGFBP-4 among all IGFBP family members, offering a biochemical explanation for the apparent specificity of the IGFBP-4 proteases. This region is rich in basic amino acid residues that could be subject to tryptic-like cleavage. These differences could be caused by secondary site use by the protease, intraspecies differences, or more than one IGFBP-4 protease that attacks this highly charged domain

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Conclusion