The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A

Abstract

Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.

Figure 1

Purification of the IGF-dependent IGFBP-4 protease from HFCM. (a) Metal-chelating affinity chromatography. Bound proteins were eluted with a descending stepwise pH gradient, and 50 μl aliquots of each of these fractions were dialyzed and assayed for IGFBP-4 protease activity. IGF-dependent IGFBP-4 protease activity is defined as the loss of intact 24-kDa [¹²⁵I]IGFBP-4 and the appearance of 18- and 14-kDa radiolabeled fragments (denoted by arrows) in the presence (+), but not in the absence (-), of 5 nM IGF-II. HFCM, starting material; FT, flowthrough. (b) Wheat-germ agglutinin chromatography. Metal-chelating affinity chromatography fraction eluting at pH 5.0 was adjusted to pH 7.4 and immediately passed over a wheat-germ agglutinin column. Non- and weakly bound proteins came out in the flowthrough (FT) or were eluted with three washes of 0 mM and three washes of 25 mM N-acetylglucosamine. IGF-dependent IGFBP-4 protease activity was eluted with three washes of 500 mM N-acetylglucosamine.

Figure 2

Confirmation of IGF-dependent IGFBP-4 protease as PAPP-A. (a) Inhibition of IGFBP-4 protease activity in HFCM by PAPP-A/proMBP antibodies. HFCM was assayed for IGFBP-4 protease activity, as described in Fig. 1 and in Materials and Methods, in the presence of indicated titer of either PAPP-A/proMBP polyclonal antibody (anti-PAPP-A) or nonspecific rabbit IgG. (b) Immunodepletion of IGFBP-4 protease activity from HFCM by PAPP-A antibodies. HFCM was precleared with a 1:50 titer of nonspecific rabbit IgG. The precleared fractions were immunodepleted with indicated titer of PAPP-A/proMBP polyclonal antibody and assayed for IGFBP-4 protease activity. (c) IGF-dependent IGFBP-4 protease activity of purified PAPP-A. PAPP-A/proMBP (0.5 μg) purified from pregnancy sera was assayed for IGFBP-4 protease activity in the absence (-) and presence (+) of 5 nM IGF-II.

Figure 3

Expression and secretion of PAPP-A. (a) Northern analysis. Poly-A tailed mRNAs (10 μg) from cultured human cells were probed with ³²P-labeled PAPP-A cDNA. The lanes are: a, adult fibroblasts; b, adult osteoblasts; c, fetal osteoblasts; d, marrow stromal cells; e, MG63 osteosarcoma cells; f, U2 osteosarcoma cells; g, TE85 osteosarcoma cells. (b) Western immunoblot. a, HFCM (10 μl of 30× concentrate corresponding to 0.05 μg PAPP-A by ELISA) and b, PAPP-A/proMBP purified from pregnancy serum (0.05 μg) were separated by SDS/PAGE under both nonreducing and reducing conditions, transferred to membrane, and immunoblotted with PAPP-A-specific monoclonal antibodies.