THU0068 N-acylation and sulfation of glucosamine (glcn) differentially modulate chondrocyte (ch) proliferation and proteoglycan (pg) synthesis

Background GlcN and GlcN-sulfate (GlcN-S) are being used extensively for the treatment of Osteoarthritis (OA). These compounds were thought to serve as nutritional substrates for the repair of damaged articular cartilage. However, controversy exists because free GlcN appears in the serum in very small amounts after ingestion of recommended doses for OA. Further, we had observed that GlcN added to Ch cultures, in concentrations similar to medium glucose, decreased Ch proliferation and PG synthesis. We now expand on these observations and report on the effects of N-acylation and sulfation of the GlcN molecule on these biological effects. Objectives To examine the effects of GlcN and related N-acylated and sulfated analogues on Ch proliferation and PG synthesis. Methods The compounds tested on isolated bovine, articular Ch, were: glucose (Glc), GlcN. HCl, N-acetyl GlcN (GlcNAc), N-acetyl galactosamine (GalNAc), N-butyryl GlcN, (GlcNBu) GlcN-3,6-disulfate (GlcN-3,6-diS), GlcN-3.4.6- trisulfate (GlcN-3,4,6-triS), adenosine triphosphate (ATP) or inosine. Following the various additions the Ch were harvested, at different time intervals, and enumerated or assessed for DNA synthesis by measuring [3H]-thymidine incorporation. For PG synthesis, confluent Ch cultures were treated with transforming growth factor (TFG)- b, [35S]-sulfate for 4 days. The media were analysed by Alcian blue and Toluidine blue precipitation, followed by gradient SDS-PAGE and autoradiography. Results The addition of GlcN. HCl (0.5–5.0 mM) to Ch cultures strongly inhibited cell proliferation, both in the absence and presence of TGF-b. This major antiproliferative effect of GlcN. HCl was reversed by GlcNAc, while GlcNBu resulted in a marked increase in Ch proliferation. Equivalent concentrations of HCl had no effect while sodium butyrate itself also showed antiproliferative effects. The addition of low GlcN. HCl concentrations (0.05–0.25 mM) resulted in minor stimulations of Ch proliferation and DNA synthesis. The antiproliferative effect of GlcN. HCl could not be reversed by the addition of ATP and inosine. Sulfation of GlcN in the 6-, 2-, and 2,6-positions, but not in the 3-, 2,3- and 3,6- positions reversed the GlcN. HCl effect. Sulfation at the 2- and 2,6-positions also reversed the GlcN. HCl inhibition on DNA synthesis. Inosine (2.5 mM) reversed the GlcN-3-S mediated antiproliferative effect on Ch at 8 d of treatment. The addition of GlcN also resulted in a dose dependent inhibition of the net PG synthesis over the range of 0.1–5.0 mM. N-acylation reversed, while sulfation partially reversed the GlcN. HCl mediated PG inhibition. Inosine partially reversed the GlcN. HCl-dependent inhibition on PG synthesis. Conclusion Free GlcN, in the form of GlcN. HCl, inhibited Ch proliferation and PG synthesis. These effects were apparently reversed by N-acylation and, in a more complex pattern, by certain sulfation substitutions. N-butyryl GlcN, in particular, seems promising for further studies on the up-regulation of Ch growth and PG synthesis and in articular cartilage repair. Available at Poster.