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Abstract
Proliferation of mesangial cells requires platelet-derived growth factor receptor beta (PDGFR)-mediated signal transduction. We have previously shown that activation of phosphatidylinositol (PI) 3-kinase is necessary for PDGFR-induced DNA synthesis in these cells. The mechanism by which PI 3-kinase stimulates DNA synthesis is not known. One target of PI 3-kinase, Akt serine threonine kinase, regulates survival of many cells by inhibiting the actions of certain proapoptotic proteins. In this study, we investigated the role of Akt in PDGF-induced DNA synthesis in mesangial cells. PDGF increased Akt serine threonine kinase activity in a time- and PI 3-kinase-dependent manner. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGF-induced activation of endogenous Akt in mesangial cells, resulting in complete inhibition of DNA synthesis. On the other hand, inhibition of MAPK attenuated PDGF-induced DNA synthesis only partially. Inhibition of Akt also attenuated PDGF-induced c-fos gene transcription, with concomitant inhibition of Elk-1-dependent transcription, indicating positive regulation of this early response gene by Akt. To further determine the role of Akt in PDGF-induced DNA synthesis, we investigated its effect on cyclin-dependent kinase 2 (CDK2). PDGF stimulated CDK2 activity in mesangial cells and decreased the level of p27(kip1) cyclin kinase inhibitor protein. Expression of dominant negative Akt increased p27(kip1) protein and resulted in inhibition of CDK2 activity. The increase in p27(kip1) expression in response to Akt kinase inhibition was due to increased transcription of the p27(kip1) gene. p27(kip1) transcription similarly was decreased by expression of constitutively active Akt kinase in mesangial cells. These data provide the first evidence that Akt kinase regulates PDGF-induced DNA synthesis by regulating CDK2 activity and define Akt-mediated inhibition of transcription of p27(kip1) as one of the mechanisms for PDGF-induced DNA synthesis in mesangial cells.
Highlights
Proliferation of mesangial cells requires platelet-derived growth factor receptor  (PDGFR)-mediated signal transduction
These data indicate that in mesangial cells, Akt serine threonine kinase regulates p27kip1 gene transcription. This may be one of the mechanisms for high protein level observed when the Akt kinase activity was inhibited by dominant negative Akt expression (Fig. 5, B and C). These studies represent the first direct demonstration that Akt serine threonine kinase activity is required for PDGFinduced DNA synthesis and c-fos gene transcription in glomerular mesangial cells
Our data provide the first evidence that Akt regulates Platelet-derived growth factor (PDGF)-induced cyclin-dependent kinase 2 (CDK2) activity by modulating the expression of the p27kip1 cyclin kinase inhibitor
Summary
Vol 276, No 38, Issue of September 21, pp. 35636 –35643, 2001 Printed in U.S.A. From the Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas and Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas 78229-3900. Expression of dominant negative Akt by adenovirus-mediated gene transfer blocked PDGFinduced activation of endogenous Akt in mesangial cells, resulting in complete inhibition of DNA synthesis. The lipid-modifying enzyme PI 3-kinase, which contains Src homology 2 domains, binds directly to the phosphorylated tyrosines 740 and 751 of the PDGFR [2, 5] Mutational analysis of these two tyrosines demonstrated that binding and subsequent activation of PI 3-kinase is necessary for PDGF-induced DNA synthesis [9]. Inhibition of Akt kinase activity, by adenovirus-mediated gene transfer of a dominant negative Akt, blocks PDGF-induced DNA synthesis completely. We provide the first evidence that Akt regulates PDGF-induced cyclin-dependent kinase 2 (CDK2) activity by modulating p27kip cyclin kinase inhibitor (CKI) gene transcription and protein expression
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