Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells.

Abstract

In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.