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Control of Insulin-Like Growth Factor Binding Protein-5 Protease Synthesis and Secretion by Human Fibroblasts and Porcine Aortic Smooth Muscle Cells

Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., Department of Medicine, Division of Endocrinology, CB# 7170, 6111 Thurston-Bowles, University of North Carolina, Chapel Hill, North Carolina 27599-7170. E-mail: endo{at}med.unc.edu.

IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types.

Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1ß or TNF resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNF (50 ng/ml) and IL-1ß (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10^-7 M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells.

To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNF, IL-ß, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C₁ inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNF inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNF and IL-1ß. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.

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