Targeted expression of a protease-resistant IGFBP-4 mutant in smooth muscle of transgenic mice results in IGFBP-4 stabilization and smooth muscle hypotrophy.
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.
Zhang
Mingyu
M
Division of Endocrinology, University of Cincinnati College of Medicine and the Children's Hospital Medical Center, Cincinnati, Ohio 45267-0547, USA.
Smith
Eric P
EP
Kuroda
Hiroaki
H
Banach
Walter
W
Chernausek
Steven D
SD
Fagin
James A
JA
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D006984:Hypertrophy; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000235:genetics* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000473:pathology*; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D019076:Transgenes
D018076:DNA, Complementary; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D000409:Alanine