Differential effects of transforming growth factor‐β and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

Abstract

We examined the effect of transforming growth factor (TGF‐β) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [³H]‐thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF‐β at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S‐phase (SQ or G2Q) in either 2% or 10% FCS‐containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF‐β addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose‐dependence inhibition of cell growth was detected, in contrast, for 2% FCS‐treated cells. Presence of TGF‐β during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet‐poor plasma at 10% produced the same effects as FCS, suggesting that platelet‐derived factors, such as platelet‐derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF‐β to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S‐phase but an augmentation of the percentage of cells in S‐ and G2M‐phases were observed. When combined, TGF‐β and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [³H]‐thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF‐β than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell‐number increases at 48 h in 10% FCS‐containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF‐β induces first a recruitment of chondrocytes in noncycling SQ‐ or G2Q‐blocked cells. Then, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS). This mechanism is quite different from that of EGF, which make more cells enter S‐phase, whatever the serum concentration in medium, and might be related to the transforming capacity of TGF‐β.