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.
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Acknowledgments
The authors thank Ms. Eri Ishihara for secretarial assistance. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid 20591751 for Scientific Research (C)], and by the Suzuken Memorial Foundation. Funding sources had no role in the study design, collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.
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Fig. S1
Effect of 4-MU on mRNA expression of UDP-glucose dehydrogenase (UGDH) in cultured ATDC5 cells at day 0–day 21. The data depict relative mRNA expression standardized by that of GAPDH. Error bars express ± standard error of mean (S.E.M.) (TIFF 112 kb)
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Yoshioka, Y., Kozawa, E., Urakawa, H. et al. Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells. Histochem Cell Biol 144, 167–177 (2015). https://doi.org/10.1007/s00418-015-1325-3
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DOI: https://doi.org/10.1007/s00418-015-1325-3