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 approximately 470 kDa PAPP-A form (PAPP-A470) to a approximately 400 kDa PAPP-A form (PAPP-A400). 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.
Sivanandam
Arun S
AS
Musculoskeletal Disease Center, J. L. Pettis Memorial Veterans' Medical Center, 11201 Benton Street, Loma Linda, CA 92357, USA.
Mohan
Subburaman
S
Kita
Hirohito
H
Kapur
Sanjay
S
Chen
Shin-Tai
ST
Linkhart
Thomas A
TA
Bagi
Gyorgy
G
Baylink
David J
DJ
Qin
Xuezhong
X
D000328:Adult; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001798:Blood Proteins / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D019281:Dimerization; D004789:Enzyme Activation; D047091:Eosinophil Granule Proteins; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011266:Pregnancy-Associated Plasma Protein-A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012260:Ribonucleases / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014161:Transduction, Genetic
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D017077:Culture Media, Conditioned; D047091:Eosinophil Granule Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D010703:Phorbol Esters; D020033:Protein Isoforms; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C008876:phorbol-12,13-didecanoate; D011493:Protein Kinase C; D012260:Ribonucleases; D011266:Pregnancy-Associated Plasma Protein-A; D013755:Tetradecanoylphorbol Acetate