Transforming growth factor-β, osteogenin,and bone morphogenetic protein-2 inhibit intercellular communication and alter cell proliferation in MC3T3-E1 cells

Intercellular communication by gap junctions has been implicated to function in the control of cell growth and differentiation in osseous tissues—processes which are regulated, in part, by peptide growth factors, including transforming growth factor-beta (TGF-β) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-β and BMPs on cell proliferation may be correlated to changes in intercellular communication. In a series of proliferation assays, MC3T3-E1 cells were cultured in the presence of bone morphogenetic protein-2 (BMP-2) or TGF-β for up to 48 hr. Proliferation of cells during the linear log phase (days 2 to 4) was assessed by ³H-thymidine (³H-TdR) incorporation. After times ranging from 6 to 48 hr, BMP-2 significantly inhibited uptake of ³H-TdR at doses of 50–800 ng/ml. Similarly, TGF-β inhibited uptake of ³H-TdR at doses of 2–32 ng/ml. In a separate group of experiments, intercellular communication through gap junctions was demonstrated by cell-cell transfer of the fluorescent tracer, lucifer yellow, after microinjection. One series of experiments showed that the gap junctional intercellular communication (GJIC) of cells, incubated for 48 hr in the presence of the higher dose of osteogenin (OG) (5.0 vs. 0.5 μg/ml) or higher dose of TGF-β (2.0 vs. 0.2 ng/ml), was significantly inhibited compared to control. In another series of experiments, time and dose dependent effects of BMP-2 and TGF-β on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-β (2.0 ng/ml) demonstrated a statistically significant effect in inhibiting GJIC as early as 6 hr, while BMP-2 (50 ng/ml) inhibited GJIC after 24 and 48 hr of treatment. The dose-dependent effects of BMP-2 and TGF-β on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-β at 2 and 4 ng/ml. The cell count results and injection study performed at 12 hr, at a fixed cell density, confirmed that the inhibitory effect was not due to differences in cell density. The 50% effective inhibitory concentrations (EC₅₀) calculated for BMP-2 and TGF-β at 48 hr, showed no dose correlation between proliferation and GJIC, suggesting that these two events are independent occurrences. Additionally, marked morphological change was observed in the cells treated with TGF-β. The observation may suggest that TGF-β may have effects upon cytoskeletal elements in osseous tissues. © 1996 Wiley-Liss, Inc.