Modulation of articular chondrocyte proliferation and anionic glycoconjugate synthesis by glucosamine (GlcN), N-acetyl GlcN (GlcNAc) GlcN sulfate salt (GlcN.S) and covalent glucosamine sulfates (GlcN-SO4)

SummaryObjectivesTo investigate, in chondrocyte cultures under conditions for maximizing responses in proliferation and proteoglycan (PG) synthesis, the effects of glucosamine hydrochloride (GlcN.HCl) and glucosamine sulfate (GlcN.S) salts, N-acetyl glucosamine (GlcNAc), and covalently substituted GlcN-X,Y,Z(SO4)n (general formula).MethodsBovine articular chondrocytes (BAC) were studied under anchorage-independent (AI, alginate beads) and anchorage-dependent (AD, plastic surface) conditions. Differentiation markers were evaluated (e.g., cartilage-specific (V + C)− fibronectin). Varying concentrations of GlcN.HCl, GlcN.S, GlcNAc and GlcN sulfated at positions −2, −3, −6, (−2,3), (−3,6) and (−3,4,6), were tested. Cell proliferation, DNA synthesis and [35S]-sulfate incorporation into newly synthesized PG were determined.ResultsIncreasing GlcN.HCl or GlcN.S concentrations gave decreasing net PG synthesis. Compounds showed more pronounced effects in AD cultures (expressing the V−C+ fibronectin isoform) compared to AI cultures ((V + C)− isoform). Addition of GlcN.HCl or GlcN.S gave a concentration-dependent decrease in BAC proliferation, partially prevented by glucose (Glc). GlcNAc was not inhibitory. Addition of GlcN-2-SO4 or GlcN-2,6-diSO4 did not affect proliferation or DNA synthesis. The other GlcN-sulfates gave varying inhibitory effects, which for GlcN-3-SO4 were reversed by inosine.ConclusionsThe free amino group of GlcN seems responsible for inhibition of chondrocyte proliferation and PG synthesis. These effects were greater under higher concentrations of GlcN in AD vs AI conditions. GlcN.HCl behaves similarly to GlcN.S, but differential effects with GlcN-X,Y,Z(SO4)n isomers were observed. Acetylation or sulfation of the GlcN amino group reverses or partially reverses, respectively, anti-proliferative effects of GlcN. Sulfation of GlcN, at positions 3 and 6 results in complex effects on AC proliferation and PG synthesis.