Thyroid hormone inhibits fibronectin synthesis by cultured human skin fibroblasts

Fibronectin (Fn) is a glycoprotein composed of two different subunits, each with a mol wt of approximately 230K. Fn is important for cell adhesion and for maintaining normal cell morphology. Recent in vivo studies suggested that thyroid hormone affects the plasma Fn level in man. The aim of this study was to determine whether T3 regulates the synthesis of Fn in cultured human skin fibroblasts. Skin fibroblasts obtained from seven normal subjects were grown in Minimum Essential Medium supplemented with 10% fetal calf serum. After reaching confluence, the cells were exposed for 3 days to the same medium in which fetal calf serum was replaced by 10% thyroidectomized bovine serum without or with added T3. At the end of this period, the cultures were incubated with [35S]methionine (95 microCi/dish) for 4 h before harvesting. Cell lysates and corresponding media were combined, and 35S incorporation into total protein and Fn was determined by trichloroacetic acid precipitation and immunoprecipitation with antihuman Fn immunoglobulin, respectively. Analysis of the immunoprecipitated material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the 35S activity migrated as a 230K band which was displaced by excess unlabeled Fn. Addition of T3 did not affect trichloroacetic acid-precipitable 35S activity but decreased the 35S activity precipitated with anti-Fn in a dose-dependent manner. While addition of 10(-10) M T3 to the medium had no effect, 35S incorporation into Fn was inhibited by 22.5 +/- 7.4% (mean +/- SD) with 10(-9) M T3 (free T3, 6 X 10(-12) M) and by 31.3 +/- 5.8% with 10(-7) M T3. To assess whether the inhibition of Fn accumulation by T3 was due to suppression of Fn synthesis or enhanced Fn degradation, cells were labeled for 4 h with [35S]methionine and chased for another 4 h with excess unlabeled methionine. T3 had no effect on the rate of decline of [35S]Fn during the chase. We conclude that physiologic T3 concentrations inhibit the synthesis of Fn in normal human fibroblasts. This effect provides a new method to study the action of thyroid hormone which may prove useful in the tissue diagnosis of resistance to thyroid hormone.