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 insufficient 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 protease inhibitors. Proteolysis of exogenous recombinant human IGFBP-3 (for fetal and pregnancy serum) or endogenous IGFBP-3 (for non-pregnant adult rat serum) following incubation at 37 degrees C was measured by ligand blotting. In all three sera, IGFBP-3 proteolysis was inhibited completely by: (i) EDTA, a chelator of divalent cations. Inhibition was reversed by zinc, but not by calcium ions; (ii) 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an inhibitor of serine proteases; and (iii) a specific tissue inhibitor of matrix metalloproteinases (TIMP-1). Recombinant human matrix metalloproteinase-3 (MMP-3) proteolyzed recombinant human IGFBP-3 or endogenous rat IGFBP-3 in non-pregnancy 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 degrades IGFBP-3.
Wu
H B
HB
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. hbwu@nih.gov
Lee
C Y
CY
Rechler
M M
MM
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000097:blood; D006224:Cricetinae; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D006023:Glycoproteins / Q000097:blood; D006031:Glycosylation; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000097:blood*; D019278:Matrix Metalloproteinase 3 / Q000097:blood*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000097:blood*; D013002:Somatomedins / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000097:blood*; D000511:alpha-Macroglobulins / Q000378:metabolism
D002352:Carrier Proteins; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D013002:Somatomedins; D019715:Tissue Inhibitor of Metalloproteinase-1; D000511:alpha-Macroglobulins; C075944:insulin-like growth factor binding protein, acid labile subunit; D012697:Serine Endopeptidases; D019278:Matrix Metalloproteinase 3