TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: possible involvement of pertussis toxin-sensitive pathway.

This study was undertaken to gain more insight into the mechanism whereby TGF-beta influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum-containing medium, we have previously shown that TGF-beta induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time-course incorporation of [3H]-thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF-beta provoked a decrease of cAMP content (0.5-1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]-thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF-beta. All these results suggest that TGF-beta may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi-protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re-entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary–for example, in the repair of tissue damage.