Proteolysis of insulin-like growth factor binding protein-3 in serum from pregnant, non-pregnant and fetal rats by matrix metalloproteinases and serine proteases

The insulin-like growth factors (IGFs) in adult mammalian plasma circulate predominantly in 150-kDa complexes that also contain IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit. Proteolysis of IGFBP-3 within the 150 -kDa complex decreases its affinity for IGFs, facilitating their release to the tissues. By contrast,150-kDa complexes are not detected in serum from fetal or pregnant adult rats. Decreased complex formation results from insu fficient availability of IGFBP-3 due to increased IGFBP-3 proteolysis. The present study characterizes IGFBP-3 protease activity in serum from fetal, pregnant and non-pregnant adult rats by comparing the effect of different p rotease inhibitors. Proteolysis of exogenous recombinant human IGFBP-3 (for fetal and pregnancy serum) or endogenous ICFBP-3 (for non-pregnant adult r at serum) following incubation at 37 degrees C was measured by ligand blott ing. In all three sera, ICFBP-3 proteolysis was inhibited completely by: (i ) EDTA, a chelator of divalent cations. Inhibition was reversed by zinc, bu t not by calcium ions; (ii) 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBS F), an inhibitor of serine proteases; and (iii) a specific tissue inhibitor of matrix metalloproteinases (TIMP-1). Recombinant human matrix metallopro teinase-3 (MMP-3) proteolyzed recombinant human IGFBP-3 or endogenous rat I GFBP-3 in nonpregnancy serum pretreated with AEBSF to inactivate endogenous serine proteases. These results suggest that serine proteases initiate the activation of latent MMP precursor, and that the activated MMP directly de grades IGFBP-3.