Abstract
Pregnancy‐Associated Plasma Protein‐A (PAPP‐A) proteolyses insulin‐like growth factor binding protein‐4 (IGFBP‐4), thereby regulating local IGF availability. Reduced PAPP‐A mRNA expression has been reported in ovarian cancer specimens compared to normal ovarian surface epithelial cells (OSE). To characterize PAPP‐A expression and proteolytic activity in OSE, we developed a lifespan‐extended human cell model using a temperature‐sensitive mutant of the SV40 large T antigen (SV40LT). These OSE(tsT) cells proliferate at 34°C (i.e., when SV40LT‐positive), but not at 39°C, a temperature at which the SV40LT is unstable (SV40LT‐negative). Proteolysis of radiolabeled IGFBP‐4 in conditioned media from OSE(tsT) lines was IGF‐dependent and blocked by anti‐PAPP‐A antisera. Temperature shifts that eliminated stable SV40LT induced a 7‐fold increase in PAPP‐A mRNA and a 4‐fold increase in protein. The converse experiment (shifting to SV40LT‐positive conditions) resulted in decreased levels of PAPP‐A mRNA but little change in PAPP‐A protein. Nevertheless, there was a marked reduction in IGF‐BP‐4 proteolytic activity in medium of SV40LT‐positive OSE‐(tsT) cells. This decreased PAPP‐A activity coincided with a nearly 20‐fold increase in mRNA encoding a physiological inhibitor of PAPP‐A, the precursor form of eosinophil Major Basic Protein (proMBP), and 4‐ to 5‐fold increases in proMBP protein. Primary cultures of unmodified OSE expressed high levels of PAPP‐A and undetectable proMBP, and therefore produced abundant IGFBP‐4 protease activity. Short‐term ovarian tumor cell cultures expressed variable levels of PAPP‐A and high levels of proMBP, and consequently secreted little or no IGFBP‐4 protease activity. The concurrent regulation of PAPP‐A and its inhibitor, proMBP, suggests that IGFBP‐4 proteolysis and local regulation of IGF availability may be altered in malignant ovarian epithelial cells. © 2004 Wiley‐Liss, Inc.