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Identification of Rat Cell Lines that Preferentially Express Insulin-Like Growth Factor Binding Proteins rlGFBP-1, 2, or 3

Growth and Development Section, Molecular, Cellular and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland 20892

Address requests for reprints to: Dr. Yvonne W.-H. Yang, National Institutes of Health, Building 10, Room 8D14, Bethesda, Maryland 20892.

Abstract

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hlGFBP-1), human IGFBP-3 (hlGFBP-3), and rat IGFBP-2 (rlGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rlGFBP-2 and hlGFBP-1; RNAs were hybridized to cDNA probes for rlGFBP-2 and hlGFBP-1.1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rlGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rlGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rlGFBP-2 or hlGFBP-1. 2) BRL-3A, Clone 9, and TRL12-15 cell lines derived from normal rat liver express rlGFBP- 2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rlGFBP-2 but not by antibodies to hlGFBP-1. RNAs from these cells hybridize to a rlGFBP-2 cDNA probe, but not to a hlGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rlGFBP-1. It does not react with antibodies to rlGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hlGFBP-1. RNA from H35 cells hybridizes to a hlGFBP-1 cDNA probe, but not to a rlGFBP-2 probe. Expression of rlGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.

FOOTNOTES

A preliminary account of this work was presented at the 71st Annual Meeting of The Endocrine Society, 1989, Seattle, Washington.

Received for publication August 8, 1989. Revision received October 2, 1989. Accepted for publication October 12, 1989.

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