Regulation of human fibroblast insulin-like growth factor (IGF)-binding proteins by IGF-1 and cytokines, mechanisms of action and effects upon IGF bioactivity
The insulin-like growth factors, IGF-I and IGF-II, are ubiquitous polypeptide molecules that have mitogenic and metabolic actions in a wide variety of cell types, and consequently play a major role in mammalian growth and development. Unlike many other peptide hormones, IGF levels and their bioactivity are highly dependent upon the secretion of a family of six specific binding proteins, named IGFBPs. In this thesis, we have developed a normal human fibroblast in vitro cell culture model to investigate both factors that affect IGFBP secretion, the mechanisms behind such regulation, and also the effect of IGFBP modulation upon subsequent IGF-I mitogenic activity. Firstly, we have examined the possible role that the recently discovered IGFBP proteases may have in determining the effect of IGF-I on IGFBP-3 abundance in fibroblast conditioned media. We show that IGF-I increased IGFBP-3 when assessed by ligand blotting, but did not increase immunoreactive IGFBP-3, a discrepancy that could be explained by further data showing an inhibitory effect of IGF-I on the activity of the fibroblast IGFBP-3 protease. Thus, IGF-I protection of IGFBP-3 from enzymatic degradation may help explain the post-transcriptional, non-receptor mediated 'stimulation' of IGFBP-3 by IGF-I in these cells. We also show for the first time the ability of a number of cytokines to regulate IGFBP secretion, perhaps indicating a novel pathway of communication between these immune cell molecules and the IGFs. The inflammatory cytokine interleukin 113(] L-16) inhibited Hs68 fibroblast IGFBP-3 secretion by down-regulating its gene expression, whilst tumour necrosis factor oc (TNF(x) had a similar inhibitory effect on IGFBP-3 and IGFBP-4 but acted via a post-transcriptional mechanism. The exact nature of the TNF(x effect remains to be determined as no evidence was found to suggest TNF(x increased IGFBP protease activity, or that TNF(x removed IGFBPs from the conditioned media by increasing the proportion immobilised on the cell surface. The inhibition of IGFBP secretion by TNF(x was observed to have marked effects upon the mitogenic activity of IGF-I in these cells, with a five-fold increase in sensitivity to the growth factor seen in a novel cytochemical bioassay. 1 Inhibition of fibroblast IGFBP secretion appeared to be restricted to certain cytokines as IL-6 had no effect, whilst high doses of interferon gamma abolished the TNF(X effect. Conversely, transforming growth factor 6 (TGFB) directly stimulated fibroblast IGFBP-3 secretion, via an increase in gene expression, and subsequently resulted in the reduction in the mitogenic activity of IGF-I. These data indicate that a variety of mechanisms can be employed by a number of factors to elicit changes in fibroblast IGFBP secretion, and that these changes may have direct consequencesin determining IGF-I bioactivity. Such is the importance given to the IGFs in maintaining normal somatic growth, changes in IGFBP secretion may contribute to the altered cellular growth and metabolism seen associated with cytokines in conditions as diverse as chronic infection, rheumatoid arthritis, cancer and the wound healing process.