RhoA/ROCK signaling regulates chondrogenesis in a context-dependent manner

The development of the cartilage template that precedes endochondral bone formation requires the condensation of mesenchymal cells and their subsequent differentiation to the chondrocytic lineage. We have previously shown that inhibition of the RhoA/ROCK signaling pathway or actin dynamics enhances Sox9 mRNA expression, increases glycosaminoglycan production, and transforms cell shape to a spherical, chondrocyte-like morphology. However, we demonstrate here that in three-dimensional micromass cultures of mesenchymal cells, increased expression of Sox9 in response to these manipulations is not sufficient to induce the expression of established Sox9 target genes. This is illustrated by a decrease in the transcript levels of collagen II and aggrecan as well as reduced activity of a Sox9-responsive reporter gene in response to ROCK inhibition and cytochalasin D. We also demonstrate a decrease in mRNA levels of the transcriptional co-activators L-Sox5 and Sox6 upon ROCK inhibition and cytochalasin D. The decrease in Sox9 activity is likely partially due to reduced L-Sox5 and Sox6 levels but also to a delay in Sox9 phosphorylation following ROCK inhibition. In contrast, inhibition of the RhoA/ROCK pathway and cytochalasin D treatment in monolayer culture results in the enhancement of a number of markers of chondrogenesis such as Sox9 activity and collagen II and aggrecan transcripts levels. These data demonstrate that the effects of RhoA/ROCK signaling and actin polymerization inhibitors on chondrogenic gene expression are dependent on the cellular context.