Transforming growth factor-beta, 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-beta) and the bone morphogenetic proteins (BMPs). Using the osteoblastic cell line MC3T3-E1, we tested the hypothesis that the effects of TGF-beta 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-beta 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-beta 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 microgram/ml) or higher dose of TGF-beta (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-beta on GJIC were investigated. In the time course experiments (3, 6, 12, 24, and 48 hr), TGF-beta (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-beta on cell couplings, determined at 48 hr, showed significant inhibitory effects with BMP-2 at 25 and 50 ng/ml and with TGF-beta 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-beta 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-beta. The observation may suggest that TGF-beta may have effects upon cytoskeletal elements in osseous tissues.

The isolation of a fibroblast growth inhibitor associated with perigraft seroma.

PURPOSE: Perigraft seroma is a rare complication of reconstructive vascular surgery characterized by a clear, sterile fluid collection confined within a fibrous pseudomembrane around a prosthetic graft. The exact cause of this disease is unknown but involves failure of surrounding connective tissue to incorporate the graft. To understand why this occurs, we studied sera from patients with perigraft seroma for their effect on human fibroblasts. Sera from control subjects, patients with uninfected prosthetic grafts, and patients with prosthetic grafts were tested for comparison. METHODS: Fibroblast growth was measured by radioactive thymidine uptake and hexosaminidase colorimetric cell proliferation assays. We fractionated sera with gel filtration columns and measured each fraction's effect on fibroblast growth. RESULTS: Serum samples from patients with perigraft seroma inhibited fibroblast growth in a dose-dependent manner. In contrast, a postseroma sample, normal human sera, and sera from patients with infected and uninfected grafts showed no significant fibroblast inhibition. An inhibitory factor with a molecular weight of 2000 d was isolated from serum of patients with perigraft seroma. CONCLUSIONS: Perigraft seroma is associated with a fibroblast inhibitor with a molecular weight of 2000 d. Further identification and characterization of this protein may lead to clinical applications in preventing and treating perigraft seroma.