Promotion of embryonic limb cartilage differentiation in vitro by staurosporine, a protein kinase C inhibitor.

Phorbol 12-myristate 13-acetate (PMA), a protein kinase C-activating phorbol ester, is known to inhibit chondrogenic differentiation by embryonic limb mesenchyme cells in vitro. The present study demonstrates that staurosporine, a potent inhibitor of protein kinase C, conversely stimulates cartilage differentiation in cultures of limb mesenchyme cells isolated from whole wing buds of stage 23/24 chick embryos or from the distal subridge region of stage 25 wing buds. In high density micromass cultures, in which limb mesenchyme cells undergo extensive spontaneous cartilage differentiation, exposure to 5-20 nM staurosporine promotes an accelerated accumulation of type II collagen and cartilage proteoglycan mRNA transcripts and a 2- to 3-fold increase in matrix glycosaminoglycan deposition. Even in low density, monolayer cultures in which the mesenchymal cells do not normally form cartilage, treatment with 5 nM staurosporine induces extensive Alcian blue-positive matrix production, a striking 4- to 18-fold rise in sulfated glycosaminoglycan accumulation, and a dramatic elevation of cartilage-characteristic gene transcript expression. Moreover, concurrent treatment with staurosporine overcomes the inhibitory effects of PMA on in vitro limb cartilage differentiation. The results suggest the hypothesis that protein kinase C might function as a negative modulator of chondrogenic differentiation during embryonic limb development.