Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP-PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a ≈ 470 kDa PAPP-A form (PAPP-A₄₇₀) to a ≈ 400 kDa PAPP-A form (PAPP-A ₄₀₀). Since unreduced PAPP-A₄₀₀ co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A₄₇₀ and PAPP-A₄₀₀ co-migrated on SDS/PAGE, conversion of PAPP-A₄₇₀ to PAPP-A₄₀₀ is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A₄₀₀/PAPP-A₄₇₀ is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A₄₀₀ exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A₄₇₀. These data suggest that a novel mechanism, namely conversion of PAPP-A₄₇₀ to the less-active PAPP-A₄₀₀, could account for the TPA-induced suppression of PAPP-A activity.