The role of nucleoside diphosphate kinase and phosphatidylinositol 3-kinase in the expression and cellular localization of the glial fibrillary acidic protein upon cAMP-induced differentiation of rat C6 glioma.

摘要

This thesis studies the role of PI 3-K and Nm23/NDPK on the expression and cellular localization of GFAP upon induction of differentiation in rat C6 glioma cells.; The first part outlines a hypothetical model describing how Nm23 proteins could be involved in the regulation of the expression and the incorporation of GFAP in IFs of normal and neoplastic glial cells. It also reviews the current status on the involvement of PI 3-K's in several aspects of tumorigenesis.; The second part of the thesis provides evidence for a function of PI 3-K and upstream protein tyrosine kinases in the induction of GFAP upon cAMP-induced differentiation of rat C6 glioma cells. Inhibition of PI 3-K activity with specific PI 3-K inhibitors or transfection of C6 cells with a dominant negative PI 3-K construct, resulted in an inhibition of the cAMP-dependent expression of GFAP. In addition, the cellular distribution of GFAP was changed, leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. Moreover, herbimycin A also reduced GFAP mRNA and protein expression upon induction of differentiation concomitant with abolished PI 3-K activity and attenuated cAMP synthesis.; The third part of the thesis describes the expression and localization of C6 glioma-expressed Nm23 isoforms and their differential association with vimentin, GFAP and gamma-tubulin. Nm23-R1 mRNA and protein was dose-dependently and transiently upregulated upon induction of differentiation. In contrast, the expression of Nm23-R2 remained unchanged. Cytosolic Nm23-isoforms revealed a punctate reticular staining pattern in the cytosol and processes of the cells which was particularly intense in the perinuclear region. Moreover, an intense Nm23-R1/-H1 staining was observed in the centrosome of different dividing and non-dividing cells. While Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in non-differentiated cells, both isoforms were associated with GFAP in differentiated cells. The centrosomal enzyme was enzymatically active and coimmunoprecipitated with gamma-tubulin.; In conclusion, the data demonstrate a function for PI 3-K and Nm23 in the transcriptional and the post-translational regulation of GFAP. As a consequence, the activity of both proteins regulates the composition and architecture of IFs, which determines astroglial cell shape. The results of this study may identify new therapeutic targets which may help in the treatment of disorders caused by aberrant cytoskeletal structure e.g. astroglial tumor formation or neurodegenerative diseases.