CREATION OF AN AUTOCRINE MODEL OF INSULIN-LIKE GROWTH FACTOR-I ACTION IN TRANSFECTED FRTL-5 CELLS

Although there is much evidence that insulin-like growth factor-I (IGF-I) is delivered to its target tissues via the circulation from distal sites of synthesis, many other observations suggest that it is synthesized in or near its target tissues and acts by autocrine and/or paracrine modalities. Studies of the mechanisms of such local actions, however, have been problematic, because in vivo studies of a single tissue are technically difficult and confounded by many variables, whereas in vitro studies of autocrine/paracrine actions have been limited by low levels of IGF-I expression and/or lack of dramatic or clearly defined responses to IGF-I. We, therefore, set about to create IGF-I expression in FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells that have been extensively studied as a model of TSH action. The modest increase in thymidine incorporation stimulated by TSH in wild type FRTL-5 cells is markedly increased in the presence of exogenous IGF-I. By transfecting these cells with a chimeric IGF-IA gene, driven either by the mouse metallothionein-1 or IGF-II 5' genomic regulatory regions, we were able to generate stable cell lines that synthesize and secrete mature IGF-I. This was demonstrated by RIA, by Northern analysis, and by polyacrylamide gel electrophoresis characterization of the radiolabeled intracellular and extracellular products that reacted with an IGF-I antibody. The mitogenic responses to TSH in IGF-I-expressing transfected FRTL-5 cells were indistinguishable from those stimulated by TSH and IGF-I in wild type or control-transfected cells (FRTL-5 cells stably transfected with a similar transgene that does not encode IGF-I). Basal DNA synthesis was higher and the peak of thymidine incorporation was earlier in IGF-I-expressing transfected FRTL-5 cells than in wild type or control cells (18-24 h vs. 30-36 h). The concentrations of TSH that maximally stimulate the incorporation of thymidine were not altered by IGF-I expression, and transfected cells did not appear to be transformed, as judged by their inability to form colonies in soft agar. TSH-stimulated DNA synthesis was blocked in IGF-I-expressing FRTL-5 cell by a monoclonal antibody to IGF (Sm 1.2). Thus, secretion of IGF-I appears to be required for the autocrine effects observed. These IGF-I-expressing FRTL-5 cell lines provide a model in vitro system to study the intracellular processing of IGF-I and the mechanisms by which IGF-I acts in an autocrine manner.