PKCdelta is a positive regulator of chondrogenesis in chicken high density micromass cell cultures

We aimed to elucidate the role of the Ca-independent PKC isoenzyme PKCdelta in the regulation of spontaneous in vitro chondrogenesis occurring in a 6-day-long culturing period in chicken limb bud-derived high density cell cultures (HDC). PKCdelta expression and activity were detectable throughout the entire culturing period with a peak on days 2 and 3, when most of the chondroblasts differentiate. To inhibit the activity of PKCdelta, either the natural compound rottlerin was transiently applied to the culture medium of HDC in 2.5, 5 or 10 μM concentrations, or gene silencing was performed by using PKCdelta shRNA. Rottlerin significantly reduced the overall PKC activity in enzyme activity assays of cell-free samples of untreated control HDC, probably via the inhibition of PKCdelta. On the contrary, we were unable to detect any consistent change of PKC enzyme activity assayed in samples of HDC treated with rottlerin during culturing. PKCdelta gene silencing resulted in a significantly lower PKC activity. Both rottlerin and PKCdelta shRNA caused a severe reduction in cartilage formation, furthermore protein and phospho-protein levels of Sox9, the key transcription factor of chondrogenesis, were also significantly decreased. Rottlerin lowered, while PKCdelta gene silencing elevated the phosphorylation status of ERK1/2. Our data suggest that PKCdelta stimulates chondrogenesis via influencing Sox9 and ERK1/2 phosphorylation, but the inhibition of cartilage formation in the rottlerin-treated HDC is probably PKCdelta independent and rottlerin might have different effects when applied to cells or to an in vitro enzyme activity assay.