Figure 6.

Effect of 4-methylumbelliferone (4-MU), a hyaluronan synthase inhibitor, on bone morphogenetic protein 7 (BMP-7) signaling. A, Freshly isolated chondrocytes from wild-type (WT) mice and CD44^−/− mice, as well as bovine articular chondrocytes (BACs) and rat chondrosarcoma (RCS) cells were plated, cultured for 48 hours in the absence or presence of 1.0 mM 4-MU (an intracellular inhibitor of hyaluronan synthesis), stimulated with BMP-7, and analyzed by Western blotting for Smad1 phosphorylation (p-Smad1). GAPDH was used as a loading control for protein content. Results are representative of 2 independent experiments. The results demonstrate that hyaluronan is required for optimal BMP-7 responsiveness in chondrocytes derived from 3 different species. B, Mouse chondrocytes were treated for 48 hours with 0–5 mM 4-MU and then stained for live (red) and dead (green) cells. Images are representative of 2 independent experiments. C, Pericellular matrix retention was examined by a particle-exclusion assay to view the cell coats. Mouse chondrocytes were treated for 48 hours in the absence or presence of 1.0 mM 4-MU and then stained with the viability dye calcein AM. Representative images show parallel sets of chondrocytes exposed to 4-MU at the same time as those depicted in B. Original magnification × ••• in B and ••• in C.