To investigate the effects of increasing extracellular osmolality on aggrecan gene expression and cell size in cultured chondrocytes.
Aggrecan promoter activity and mRNA levels were measured in bovine monolayer chondrocytes subjected to hyperosmotic loading for different time periods, using transient transfection assays or RT-PCR. Cell size changes were also determined using an epifluorescence microscopy system.
Hyperosmotic loading for 24 h suppressed aggrecan promoter activity and mRNA levels approximately two-fold. However no suppression of promoter activity was observed when exon 1 was deleted from the human aggrecan promoter construct. Osmotic regulation of aggrecan gene expression was time-dependent and found to correlate with cell shrinking and swelling. No suppression in promoter activity was observed when the hyperosmotic stimulus was applied in a cyclic manner, or when serum was present in the culture medium.
Hyperosmotic loading regulates aggrecan gene expression and cell size in isolated chondrocytes. Osmotic regulation of gene expression is also affected by the time-varying nature of loading and the presence of serum.