Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1β and transforming growth factor-β

Yumi Imai; Kyomi Ibaraki; Ritsuko Odajima; Yoshimasa Shishiba

doi:10.1530/eje.0.1310630

Imai Y, Ibaraki K, Odajima R, Shishiba Y. Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1β and transforming growth factor-β Eur J Endocrinol 1994;131:630–8. ISSN 0804–4643 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 1β (IL-1β) and transforming growth factor-β (TGF-β) increased the rate of [ 35 S]sulfate incorporation into proteoglycans two to five times the control in culture of retro-ocular tissue fibroblasts. The increase in the rate of [ S]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 [ 35 S]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-1β or TGF-β, ruling out (d). The capacity to synthesize glycosaminoglycan chain in the presence of an artifical acceptor of chain elongation, β-d-xylodide, was increased significantly by IL-1β but not obviously so by TGF-β. Thus, an increased number of glycosaminoglycan chains (c) is possible for IL-1β. A preliminary northern blot analysis employing probes for mRNAs for various proteoglycan core proteins showed increased expression of versican and aggrecan in the presence of IL-1β or TGF-β. This result supports the possibility of (a). In conclusion, IL-1β and TGF-β increased [ 35 S]sulfate incorporation into proteoglycan by increasing the net increase of proteoglycan synthesis and possibly by increasing the number of glycosaminoglycan chains attached to core protein in the case of IL-1β. Y Shishiba, Division of Endocrinology, Department of Medicine, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, Japan

Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1β and transforming growth factor-β

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