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The 25-KiloDalton Insulin-Like Growth Factor (IGF)-Binding Protein Inhibits both Basal and IGF-I-Mediated Growth of Chick Embryo Pelvic Cartilage in Vitro

WARNER M. BURCH, JAVIER CORREA, JOHN E. SHIVELY^* and DAVID R. POWELL

Departments of Medicine and Pharmacology, Duke University Medical Center Durham, North Carolina 27710
Division of Immunology, Beckman Research Institute of the City of Hope (J.E.S.) Duarte, California 91010
Department of Pediatrics, Baylor College of Medicine (D.R.P.) Houston, Texas 77054

Address all correspondence and requests for reprints to: David R. Powell, M.D., Baylor College of Medicine, Diabetes Research Center, 8080 North Stadium Drive, Houston, Texas 77054.

Insulin-like growth factor (IGF)-I stimulates the growth of many tissues, including growth plate cartilage. However, the role of IGF-binding proteins in the growth process is controversial. We purified a 25-kDa IGF-binding protein (BP-25) from amniotic fluid. We tested the effect of this BP-25 preparation on both basal and IGF-I-stimulated growth of chick embryo pelvic cartilages maintained in serum-free organ culture. Cartilage wet weight was 4.1 ± 0.3 mg/cartilage initially; after 3 days, BP-25 inhibited both basal and IGF-I-stimulated growth. Control cartilages weighed 7.4 ± 0.7 mg/cartilage, while those incubated with 100 nM BP-25 weighed 5.8 ± 0.5 mg/cartilage (P < 0.001 vs. control); BP-25 concentrations as low as 0.2 nM significantly inhibited basal cartilage growth. Cartilages incubated with 1.25 nM IGF-I weighed 10.4 ± 0.8 mg/cartilage (P < 0.001 vs. control), while those incubated with both 100 nM BP-25 and 1.25 nM IGF-I weighed 8.1 ± 0.5 mg/cartilage (P < 0.001 vs. cartilage incubated with IGF-I alone); BP-25 concentrations as low as 0.4 nM significantly inhibited IGF-I-stimulated cartilage growth. BP-25 also inhibited basal and IGF-I-stimulated increases in cartilage dry weight, [3H]thymidine incorporation into DNA, and ³⁵SO₄ incorporation into proteoglycan. A second BP-25 preparation, which in the presence of 1% platelet-poor plasma acts synergistically with IGF-I to stimulate DNA synthesis and cell replication of fibroblasts and smooth muscle cells in tissue culture, inhibited IGF-I-stimulated cartilage growth to the same degree as did our BP-25 preparation.

In separate experiments, proteins present in serum-free medium conditioned for 3 days by chick cartilages were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with [¹²⁶I]IGF-I. This medium was found to contain two IGF-binding proteins; one appeared to be the chick equivalent of BP-25, while the other had a molecular mass similar to that of a poorly characterized human 34-kDa IGF-binding protein.

We conclude that purified BP-25 inhibits the growth of chick embryo pelvic cartilage in our serum-free organ culture system. Since conditioned medium from these cartilages contains both IGF-I-like peptides and IGF-binding proteins such as BP-25, we suggest that the IGF-binding proteins present may act to downregulate the growth-promoting effects of the local IGF peptides.

^* Supported by NIH Grant DK-33155.

Supported by FIRST Award DK-38773 from the NIH.

Received February 21, 1989.

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