Pregnancy‐associated plasma protein‐A proteolytic activity in rat vertebral cell cultures: Stimulation by dexamethasone–a potential mechanism for glucocorticoid regulation of osteoprogenitor proliferation and differentiation

Abstract

Glucocorticoids (GCs) at physiological concentrations stimulate osteoprogenitor proliferation and differentiation in rat bone cell populations, and this is mediated in part by an increased response to insulin-like growth factors (IGFs). Since IGF binding proteins (IGFBPs) modulate IGF actions, we evaluated whether the increased IGF responsiveness might be associated with decreased inhibitory IGFBP-4 peptide levels. Rat vertebral cells were cultured for up to 20 days with or without dexamethasone (Dex). Cell layer proteins were extracted at day 6, 8, 14, and 20, conditioned media (CM) collected at day 8, 14, and 20, and total RNA isolated at day 14 and 20 of culture. Western blotting showed that cell layer IGFBP-4 levels were lower, while IGFBP-4 protease activity in CM was higher, in Dex-treated cultures. Addition of pregnancy-associated plasma protein-A (PAPP-A) antibody to CM abrogated IGFBP-4 proteolysis. PAPP-A mRNA levels were the same in control and Dex-treated cultures as evaluated by RT-PCR. Our data demonstrate that activity of the IGFBP-4 protease, PAPP-A, in rat bone cell cultures is increased by Dex via post-transcriptional mechanisms. Since IGFBP-4 mRNA levels in Dex-treated cultures were the same as in controls at day 8, slightly lower than in controls at day 14, and higher than in controls at day 20 as shown previously, the decreased IGFBP-4 peptide levels in Dex-treated cultures likely result from increased IGFBP-4 proteolysis by the elevated PAPP-A enzymatic activity. Our findings underscore a novel mechanism whereby GCs increase IGF responses in rat bone cells via PAPP-A-induced IGFBP-4 proteolysis.

Impact of Pregnancy-Associated Plasma Protein-A Deletion on the Adult Murine Skeleton

“…Our in vivo data, as well as previous in vitro data, suggest that PAPP‐A regulates bone formation by modulating IGF‐I bioavailability in the local bone environment. In agreement, bone‐specific IGF‐I receptor KO mice exhibited decreased trabecular bone volume, trabecular number, trabecular thickness, and connectivity, along with increased mineralization lag time. Deletion of IRS‐1, a major mediator of IGF‐I signaling, resulted in low turnover osteopenia, similar to what we see in the PAPP‐A KO mice.…”

“…(18,19) The IGFBP-4 protease expressed by human osteoblasts was identified as pregnancy-associated plasma protein-A (PAPP-A). (20)(21)(22) To address the physiological role of PAPP-A, we developed a mouse model with targeted disruption of the PAPP-A gene achieved through homologous recombination in embryonic stem cells. These mice showed a diminished growth response during early embryogenesis when heightened IGF-II activity is important, resulting in a delay in ossification and reduced overall body size.…”

Pregnancy-associated plasma protein-A (PAPP-A): A local regulator of IGF bioavailability through cleavage of IGFBPs

“…IGFBP-4 is an inhibitory IGFBP and binds IGFs with high affinity, thus preventing their interaction with the IGF-I receptor, which mediates cell growth and survival signals. PAPP-A cleaves IGFBP-4 in the middle of the protein, markedly reducing its affinity for IGFs and, thus, ''freeing'' them for receptor binding and activation [4,6,7,[34][35][36]. A signature characteristic of PAPP-A's interaction with IGFBP-4 is IGF-dependence [6].…”

“…Likewise, IGF-I is an important skeletal growth factor and PAPP-A is expressed by osteoblasts and chondrocytes [4,34,36,64]. Fracture healing is markedly delayed in PAPP-A KO mice [65].…”

Insulin-like growth factor-binding proteins and bone metabolism

“…The IGFBP-4 protease expressed by human fibroblasts and osteoblasts was identified as pregnancy-associated plasma protein-A (PAPP-A) (28). PAPP-A is the major, if not the only, IGFBP-4 protease produced by bone and enhances IGF bioactivity in bone cells in vitro (8,22). Moreover, PAPP-A has been shown to be important for bone growth in vivo.…”