Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies

Cheryl A Conover, M. C. Kiefer, J. Zapf

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Abstract

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4°C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

Original languageEnglish (US)
Pages (from-to)1129-1137
Number of pages9
JournalJournal of Clinical Investigation
Volume91
Issue number3
StatePublished - 1993

Fingerprint

Insulin-Like Growth Factor Binding Protein 4
Somatomedins
Fibroblasts
Insulin-Like Growth Factor II
Conditioned Culture Medium
Insulin-Like Growth Factor I
Pregnancy-Associated Plasma Protein-A
Proteolysis
Peptide Hydrolases
Aminoisobutyric Acids
Simian virus 40
Protease Inhibitors
Immunoblotting

Keywords

  • insulin-like growth factor binding protein-4
  • insulin-like growth factors I and II
  • protease
  • protease inhibitor

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies",
abstract = "Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85{\%} decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4°C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.",
keywords = "insulin-like growth factor binding protein-4, insulin-like growth factors I and II, protease, protease inhibitor",
author = "Conover, {Cheryl A} and Kiefer, {M. C.} and J. Zapf",
year = "1993",
language = "English (US)",
volume = "91",
pages = "1129--1137",
journal = "Journal of Clinical Investigation",
issn = "0021-9738",
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T1 - Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies

AU - Conover, Cheryl A

AU - Kiefer, M. C.

AU - Zapf, J.

PY - 1993

Y1 - 1993

N2 - Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4°C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

AB - Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4°C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

KW - insulin-like growth factor binding protein-4

KW - insulin-like growth factors I and II

KW - protease

KW - protease inhibitor

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