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Characterization of transforming growth factor-beta growth regulatory effects and receptors on bovine mammary cells
T L Woodward1, N Dumont, M O'Connor-McCourt
1Department of Animal Science, McGill University, Montreal, Canada.
Abstract:
Transforming growth factor-beta (TGF-beta) has been shown to inhibit mammary morphogenesis, growth, and differentiation in murine studies. We have characterized TGF-beta receptors and their autoregulation, and the growth response to TGF-beta 1 and TGF-beta 2 in cultured bovine mammary epithelium (MAC-T) and fibroblasts. Affinity labelling studies revealed that fibroblast and epithelial cells contained type I, II, and III (betaglycan) receptors, with the type III receptor being the predominant binding component. On both fibroblasts and epithelial cells, TGF-beta 1 and TGF-beta 2 had equal binding affinities for the type I and II receptors, but TGF-beta 2 had a higher affinity for the type III receptor. Also, preincubation of MAC-T cells with 50 pM TGF-beta 1 or TGF-beta 2 markedly downregulated TGF-beta receptors. Proliferative response was measured using both total DNA and 3H-thymidine incorporation. Both TGF-beta isoforms were effective in inhibiting proliferation of MAC-T cells and fibroblasts. Inhibition of proliferation was not altered following immortalization of fibroblasts with SV-40 Large-T-antigen (LT), even when the cells acquired a transformed phenotype. Inhibition of proliferation was not a result of cytotoxicity, as TGF-beta at concentrations 1,000-fold higher than ED50 levels did not increase cell death. Moreover, the inhibition was reversible as shown by return of cellular proliferation to control levels following TGF-beta removal. Although growth inhibition was not transient as culture of MAC-T cells in TGF-beta resulted in sustained inhibition of proliferation for at least 144 h.
Insights
Transforming growth factor-beta (TGF-beta) inhibits bovine mammary cell and fibroblast proliferation. This growth inhibition is reversible and sustained, independent of cell transformation, and mediated by TGF-beta receptors.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Transforming growth factor-beta (TGF-beta) is known to inhibit mammary gland development and function in mice.
- Understanding TGF-beta receptor interactions and cellular responses is crucial for mammary biology.
- Bovine mammary epithelial cells (MAC-T) and fibroblasts provide a model to study these mechanisms.
Purpose of the Study:
- To characterize TGF-beta receptors and their autoregulation in bovine mammary epithelial cells and fibroblasts.
- To investigate the growth response of these cells to TGF-beta 1 and TGF-beta 2.
- To determine the impact of cell immortalization and transformation on TGF-beta's inhibitory effects.
Main Methods:
- Affinity labeling was used to identify TGF-beta receptor types (I, II, III/betaglycan) on MAC-T cells and fibroblasts.
- Binding affinities of TGF-beta 1 and TGF-beta 2 for different receptor types were assessed.
- Cell proliferation was measured by total DNA content and 3H-thymidine incorporation following TGF-beta treatment.
Main Results:
- Both cell types expressed TGF-beta receptors I, II, and III, with type III being the predominant binder.
- TGF-beta 1 and TGF-beta 2 exhibited similar affinities for type I/II receptors but differential affinity for type III.
- TGF-beta 1 and TGF-beta 2 effectively inhibited proliferation in both cell types, an effect that was reversible, non-cytotoxic, and sustained for over 144 hours, even in SV-40 LT-immortalized/transformed fibroblasts.
Conclusions:
- TGF-beta isoforms potently inhibit proliferation in bovine mammary epithelial cells and fibroblasts via receptor-mediated mechanisms.
- The inhibitory effect is sustained and not dependent on cell transformation status.
- These findings highlight TGF-beta's significant role in regulating mammary cell growth.