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Transforming growth factor-beta complexes with thrombospondin
J E Murphy-Ullrich1, S Schultz-Cherry, M Höök
1Department of Biochemistry, University of Alabama, Birmingham 35294.
Abstract:
Thrombospondin (TSP) was demonstrated to inhibit the growth of bovine aortic endothelial cells, an activity that was not neutralized by antibodies to TSP or by other agents that block TSP-cell interactions but that partially was reversed by a neutralizing antibody to transforming growth factor-beta (TGF-beta). Similar to TGF-beta, TSP supported the growth of NRK-49F colonies in soft agar in a dose-dependent manner, which required epidermal growth factor and was neutralized by anti-TGF-beta antibody. Chromatography of a TSP preparation did not separate the TGF-beta-like NRK colony-forming activity from high molecular weight protein. However, when chromatography was performed at pH 11, this activity was dissociated from TSP. These results suggest that at least some growth modulating activities of TSP are due to TGF-beta associated with TSP by strong non-covalent forces. Most of the active TGF-beta released from platelets after degranulation was associated with TSP, as demonstrated by anti-TSP immunoaffinity and gel permeation chromatography. 125I-TGF-beta binds to purified TSP in an interaction that is specific in the sense that bound TGF-beta could be displaced by TGF-depleted TSP but not significantly by native TSP, heparin, decorin, alpha 2-macroglobulin, fibronectin, or albumin. Hence, TGF-beta can bind to TSP, and the complex forms under physiological conditions. Furthermore, TSP-associated TGF-beta is biologically active, and the binding of TGF-beta to TSP may protect TGF-beta from extracellular inactivators.
Insights
Thrombospondin (TSP) growth inhibition is partly due to associated transforming growth factor-beta (TGF-beta). TSP binding to TGF-beta may protect it from inactivation, influencing cell growth.
Area of Science:
- Cell Biology
- Protein Interactions
- Biochemistry
Background:
- Thrombospondin (TSP) exhibits growth-modulating activities.
- Transforming growth factor-beta (TGF-beta) is a key regulator of cell growth.
- The precise mechanisms of TSP's influence on cell growth require further elucidation.
Purpose of the Study:
- To investigate the role of TGF-beta in TSP-mediated inhibition of endothelial cell growth.
- To determine if TSP-associated factors contribute to its growth-modulating effects.
- To characterize the interaction between TSP and TGF-beta.
Main Methods:
- Cell culture assays using bovine aortic endothelial cells and NRK-49F cells.
- Soft agar colony formation assays.
- Chromatographic techniques (gel permeation, immunoaffinity) and pH-dependent dissociation.
- Binding assays using radiolabeled TGF-beta and purified TSP.
Main Results:
- TSP inhibited endothelial cell growth, an effect partially reversed by anti-TGF-beta antibodies.
- TSP supported NRK-49F colony growth, dependent on epidermal growth factor and neutralized by anti-TGF-beta.
- A TGF-beta-like activity co-eluted with TSP but dissociated at pH 11, suggesting non-covalent association.
- Specific binding of TGF-beta to TSP was demonstrated, with potential protection from inactivation.
Conclusions:
- TSP's growth-modulating activities are, in part, attributable to associated TGF-beta.
- TSP binds TGF-beta via strong non-covalent forces, forming a biologically active complex.
- This TSP-TGF-beta interaction may protect TGF-beta from degradation, influencing its bioavailability and function.