Activation of protein kinase C during newt limb regeneration: effect of denervation.

Abstract

Blastema cell proliferation during newt limb regeneration is a nerve-dependent process. The present study was undertaken to determine whether or not that process is mediated by protein kinase C (PKC) activation during limb regeneration in Pleurodeles walt. Analysis included evaluation of PKC activity and its subcellular localization at various stages of regeneration, both in vivo and in vitro. The data reveal an increase in PKC activity in both the cytosol and particulate fractions of whole blastemas reaching a maximum at the mid-bud stage, which correlates with blastema cell proliferation rate. Denervation significantly reduces blastema cell proliferation and also causes a reduction in membrane-associated PKC activity. The effect of PKC activity appears to be restricted to the blastemal mesenchyme, which exhibits a dramatic reduction in activity 96 h after denervation. In contrast, PKC activity in the epidermal cap did not change. Cultured whole blastemas likewise express a decrease in particulate PKC activity and therefore mimic denervated blastemas in this parameter. Co-culture of blastemas with spinal ganglia partially reduces the decline in PKC activity, and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a direct activator of PKC, also prevents the fall in membrane-bound PKC activity while stimulating blastema cell proliferation, in vitro. These data indicate that blastema cell (mesenchyme) proliferation is related to increased PKC activity and that PKC may therefore be involved in the nerve-dependent signalling pathway regulating the early phase of urodele limb regeneration.