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

We aimed to elucidate the role of the Ca-independent isoenzyme 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). duanyu1531delta 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 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 duanyu1531delta shRNA. Rottlerin significantly reduced the overall duanyu1531 activity in enzyme activity assays of cell-free samples of untreated control HDC, probably via the inhibition of On the contrary, we were unable to detect any consistent change of duanyu1531 enzyme activity assayed in samples of HDC treated with rottlerin during culturing. duanyu1531delta gene silencing resulted in a significantly lower duanyu1531 activity. Both rottlerin and duanyu1531delta 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 duanyu1531delta gene silencing elevated the phosphorylation status of ERK1/2. Our data suggest that duanyu1531delta stimulates chondrogenesis via influencing Sox9 and ERK1/2 phosphorylation, but the inhibition of cartilage formation in the rottlerin-treated HDC is probably duanyu1531delta independent and rottlerin might have different effects when applied to cells or to an in vitro enzyme activity assay.

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