Effect of growth factors on hyaluronan and proteoglycan synthesis by retroocular tissue fibroblasts of Graves' ophthalmopathy in culture. Acta Endocrinol 1992;126:541-52. ISSN 0001-5598 serum albumin (BSA) from Daiichi Pure Chemicals Co., Ltd.; insulin-like growth factor-1 (IGF-1) from Kabigen AB; platelet-derived growth factor (PDGF) from R&D System Inc.; hondroitin sulfate AC and ABC lyase from Seikagaku-kogyo, Tokyo. Sephadex G-50,

No abstract

nor ophthalmopathy. Human skin fibroblasts were incubated with the IgGs and labeled with [35S]sulfate. The medium and cell layer were separated and the proteoglycan was extracted. The 35S radioactivity in the proteoglycan fraction was measured. Compared with normal IgGs or with those of Graves' disease without PTM or ophthalmopathy, PTM IgGs significantly increased the incorporation of the 35S into the proteoglycan. The effect of PTM IgG was dose-dependent. As PTM IgG did not alter degradation of the 35S labeled proteoglycan, an increase in 35S incorporation reflects increased synthesis. The effect was mediated through a mechanism other than adenylate cyclase activation. The present study demonstrates the presence of an autoantibody in PTM IgG that stimulates proteoglycan production through human skin fibroblasts. This is not correlated with the thyroid stimulating antibody activity. It is suggested that the activity of this antibody leads to the development of PTM.Circumscribed pretibial myxedema (PTM) is a cutaneous complication occasionally associated with Graves' dis¬ ease. Histochemical examination of the biopsied speci¬ men showed the accumulation of hyaluronic acid and proteoglycan (1, 2). In a previous paper, we examined the proteoglycans extracted from the affected skin and found that a large amount of dermatan sulfate proteog¬ lycan had accumulated (3). It is widely accepted that fibroblasts are the source of hyaluronic acid and proteo¬ glycan synthesis, because skin fibroblasts actively syn¬ thesize both of them in culture. It is tempting to speculate that an autoantibody IgG produced in Graves' disease patients may stimulate skin fibroblasts to synthesize excess amounts of hyaluronic acid and proteoglycan.However, attempts to show that the sera of PTM patients stimulate proteoglycan synthesis in human skin fibro¬ blasts in culture have yielded conflicting results (3-13). For example, Joliffe et al. (4) and Cheung et al. (5) reported that serum components of PTM patients could stimulate glycosaminoglycan synthesis in human skin fibroblasts in culture. On the other hand, Rotella and his associates reported that the IgG of PTM patients stimu-lated glycosaminoglycan synthesis in rat thyroid cells but not in human skin fibroblasts, although they showed that PTM IgG stimulates collagen synthesis in human skin fibroblasts in culture (6-12). A recent report by Tao et al. also showed that glycosaminoglycan synthesis in cultured human skin fibroblasts was not stimulated by the sera of PTM patients which contained autoantibodies capable of stimulating thyroid tissue (13). We wondered if there was an antibody IgG in patients with PTM capable of stimulating proteoglycan synthesis in human skin fibroblasts in culture, but that the effect was masked either by serum-derived growth factors or by limitations of the methods employed. In the present study, we examined this aspect-no serum component other than IgG was included, except for bovine serum albumin (BSA)-employing the recently developed methods for proteoglycan...

The serum concentration of hyaluronan

Retro-ocular tissue fibroblasts are supposed to be responsible for the deposition of glycosaminoglycan in Graves' ophthalmopathy. We have reported in a preliminary fashion that interleukin 1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta) increased the rate of [35S]sulfate incorporation into proteoglycans two to five times the control in culture of retro-ocular tissue fibroblasts. The increase in the rate of [35S]sulfate incorporation into proteoglycan will occur as a result of: (a) net increase of proteoglycan synthesis; (b) elongation of glycosaminoglycan chains; (c) increased number of glycosaminoglycan chains; (d) oversulfation of glycosaminoglycan chains; (e) increase in cell number; (f) decreased rate of degradation. We have analyzed which mechanism is important for the increase of [35S]sulfate into proteoglycans observed in human retro-ocular tissue fibroblasts under the influence of cytokines. The last two possibilities (e, f) were ruled out because during the observation period there was no consistent proliferation of the cells and no decrease in the rate of degradation of proteoglycan examined by pulse-chase experiment. Cytokines did not change the size of glycosaminoglycan chains released from proteoglycan as measured by alkaline borohydride treatment, ruling out (b). Disaccharide analysis by HPLC after chondroitin sulfate ABC digestion revealed that glycosaminoglycan mainly contains monosulfated chondroitin disaccharides and that oversulfation was not observed under the influence of IL-1 beta or TGF-beta, ruling out (d). The capacity to synthesize glycosaminoglycan chain in the presence of an artifical acceptor of chain elongation, beta-D-xylodide, was increased significantly by IL-1 beta but not obviously so by TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)