Phorbol esters and PKC signaling regulate proliferation, vimentin cytoskeleton assembly and glutamine synthetase activity of chick embryo cerebrum astrocytes in culture

Abstract

We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC-α/β in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [ 35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.