Evidence that IGF-binding protein-5 functions as a growth factor
Naohisa Miyakoshi, … , David J. Baylink, Subburaman Mohan
Naohisa Miyakoshi, … , David J. Baylink, Subburaman Mohan
Published January 1, 2001
Citation Information: J Clin Invest. 2001;107(1):73-81. https://doi.org/10.1172/JCI10459.
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Article

Evidence that IGF-binding protein-5 functions as a growth factor

Abstract

Recent studies support the concept that IGF-binding protein-5 (IGFBP-5) stimulates bone formation, at least in part, via IGF-independent mechanisms. To evaluate this hypothesis further, we evaluated in vitro and in vivo effects of IGFBP-5 on bone formation parameters using the IGF-I knockout (KO) mouse. Treatment of serum-free cultures of osteoblast clones derived from IGF-I KO mice with recombinant human IGFBP-5 increased both proliferation and alkaline phosphatase (ALP) activity in a dose-dependent manner, an effect comparable to that seen with IGF-I. IGF-II levels from media conditioned by osteoblasts derived from IGF-I KO mouse were below those detectable by RIA. To eliminate possible actions of IGF-II, if any was produced by osteoblasts derived from IGF-I knockout mice, the IGFBP-5 effect was studied in the presence of exogenously added IGFBP-4, a potent inhibitor of IGF-II actions in bone cells. Addition of IGFBP-4 blocked IGF-I– but not IGFBP-5–induced cell proliferation in osteoblasts derived from IGF-I knockout mice. Consistent with in vitro results, a single local injection of IGFBP-5 to the outer periosteum of the parietal bone of IGF-I KO mice increased ALP activity and osteocalcin levels of calvarial bone extracts. The magnitudes of IGFBP-5–induced increases in ALP and osteocalcin in parietal bone extracts of IGF-I KO mice were comparable to those seen in C3H mice. In contrast to IGFBP-5, local administration of IGFBP-4 had no significant effect on bone formation in C3H and IGF-I KO mice. These results provide the first direct evidence to our knowledge that IGFBP-5 functions as a growth factor that stimulates its actions in part via an IGF-independent mechanism.

Authors

Naohisa Miyakoshi, Charmaine Richman, Yuji Kasukawa, Thomas A. Linkhart, David J. Baylink, Subburaman Mohan

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Figure 1

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Displacement of IGF-II tracer from IGF-II antiserum. Bound to free ratio...
Displacement of IGF-II tracer from IGF-II antiserum. Bound to free ratios (B/Bo) on the y axis represent the relative levels of radiolabeled tracer bound to antiserum with and without competitor. Competition between the [125I]IGF-II tracer and purified recombinant human IGF-II (open circles), IGF pool from 7-day-old wild-type mice (filled squares), IGF pool from 56-day-old wild-type (filled triangles) or IGF-I KO mice (open squares), and IGF pool from conditioned medium of osteoblasts derived from wild-type (filled circle) or IGF-I knockout mice (×). A total of 50 μl of 1:5 diluted serum or 60× concentrated conditioned medium was applied to Bio-Gel P-10, and the IGFs were separated from IGFBPs by Bio-Spin in 1M acetic acid. The IGF pool was neutralized with an equal volume of 1.2M Tris base before RIA. For serum samples, 10, 20, or 40 μl of IGF pool was subjected to RIA at a final dilution of 1:1,500; 1:750; and 1:375, respectively. For conditioned media samples, 50 μl of IGF pool was subjected to RIA at a final concentration of 1× conditioned medium.