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The Role of the Acid-Labile Subunit in Regulating Insulin-Like Growth Factor Transport across Human Umbilical Vein Endothelial Cell Monolayers

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Robert C. Baxter, Ph.D., D.Sc., Kolling Institute of Medical Research, Department of Molecular Medicine, Royal North Shore Hospital, University of Sydney (E25), St. Leonards, Sydney, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au.

We have established a human umbilical vein endothelial cell (HUVEC) monolayer system to study the role of complex formation with IGF binding protein (IGFBP) and acid-labile subunit (ALS) on the transendothelial transport of IGF. Incubation with recombinant human IGFBP-3 alone did not retard ¹²⁵I-IGF-I or -II transport, but addition of ALS caused marked inhibition. Transport of ¹²⁵I-des(1–3)IGF-I was more rapid than ¹²⁵I-IGF-I, suggesting the presence of some endogenous IGFBPs, although these were undetectable by affinity labeling of cells or medium. In the presence of ALS, recombinant human IGFBP-5 also retarded IGF transport, although significantly less than IGFBP-3, despite their similar ternary complex formation. In contrast, IGFBP-3 mutated in its ALS-binding domain was not inhibitory. To study IGF transport by pregnancy-proteolyzed IGFBP-3, we prepared [Tyr³¹]monoiodoIGF-I, the only iodoIGF-I form that reacts normally with proteolyzed IGFBP-3. In the presence of ALS, IGFBP-3 isolated by immunoaffinity chromatography from second-trimester pregnancy serum significantly retarded IGF transport, but to a lower extent than IGFBP-3 isolated from normal serum, despite normal ALS binding. This study demonstrates the key role of ALS in regulating transendothelial IGF transport, but indicates that other factors are also involved. Our data suggest that pregnancy-proteolyzed IGFBP-3, despite forming normal ternary complexes, is less effective than intact IGFBP-3 in retarding IGF egress from the circulation.

This work was supported by the National Health and Medical Research Council, Australia (Grant 990005 to R.C.B. and S.M.F.).

Abbreviations: ALS, Acid-labile subunit; HUVEC, human umbilical vein endothelial cell; IGFBP, IGF binding protein; IGFBP-3mut, IGFBP-3 mutant; ns, nonpregnancy serum; PC, permeability coefficients; ps, pregnancy serum; rh, recombinant human; SF, serum-free; TCA, trichloroacetic acid.

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