Regulation of plasminogen activator and plasminogen activator inhibitor production by growth factors and cytokines in rat calvarial cells

Summary Fetal rat osteoblast-enriched calvarial cells were used to study the effects of various growth factors and cytokines on plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities and the possible relationship of these effects to bone resorption. Confluent cultures were exposed to various factors under serum-free conditions, and levels of PA and PAI activities were examined in both conditioned medium (CM) and cell layer using the¹²⁵I-fibrin plate assay, fibrin zymogram, and reverse fibrin zymogram. According to the¹²⁵I-fibrin plate assay or zymogram, incubation of cells with acidic fibroblast growth factor (aFGF), basic FGF (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) elevated the PA activity in the CM as well as in the cell layer extract. Incubation with interleukin 1α (IL-1α), tumor necrosis factor α (TNFα), and insulin-like growth factor I (IGF-I) produced no change in PA activity in either CM or cell layer. Addition of transforming growth factor β (TGFβ) to calvarial cells resulted in nearly undetectable PA activity in CM with the fibrin plate assay but increased PA activity on the fibrin zymogram after PAI was separated from PA by SDS-PAGE. A reverse fibrin zymogram indicated that PAI activity was greatly enhanced in TGFβ-treated CM. TGFβ treatment also increased PA activity in the cell layer of calvarial cells. Treatment of calvarial cells with bFGF and PDGF slightly increased PAI secretion into medium. This increase, however, was not as dramatic as the increase of PA induced by these two agents. IL-1α and TNFα did not change PAI concentration in CM. No detectable PAI activity was found in the cell layer in control and treated groups. The PA found in the CM and cell layer of rat calvarial cells was the urokinase type; the PAI stimulated by TGFβ was the endothelial cell type, PAI-1. The regulation of PA activity by growth factors and cytokines did not correlate with their resorption-stimulating activities. Thus, PA secreted by osteoblasts may not be the only factor involved in the initiation of bone resorption. Delineation of the function of PA and PAI in the physiology of bone tissue awaits further studies.