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

Abstract

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 n M staurosporine promotes an accelerated accumulation o f 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 n M 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.