EGF STIMULATES MESANGIAL CELL MITOGENESIS VIA PI 3 KINASE- MEDIATED MAPK-DEPENDENT AND AKT KINASE-INDEPENDENT MANNER: Involvement of c-fos and p27

Epidermal growth factor (EGF) is a potent mitogen for mesangial cells. The mechanism by which EGF induces DNA synthesis is not precisely understood. We investigated the role of phosphatidylinositol (PI) 3 kinase in regulating mitogenesis. EGF increased PI 3 kinase activity resulting in stimulation of PDK-1 and Akt kinase activities. Blocking of PI 3 kinase activity using Ly 294002 or adenoviral expression of PTEN, which dephosphorylates PI 3,4,5-tris-phosphate (PIP3) and thus inactivates PI 3 kinase signaling, significantly inhibits EGF-induced DNA synthesis. Expression of dominant negative Akt kinase, however, had no effect on DNA synthesis. But it inhibited EGFinduced phosphorylation of FoxO3a transcription factor, thus demonstrating its functional consequences. These data indicate that EGF increases the DNA synthesis in a PI 3 kinase-dependent but Akt-independent manner. In addition to activating PI 3 kinase signaling, EGF increased Erk1/2 MAPK activity, leading to transcriptional activation of its nuclear target Elk-1, and resulting in c-fos expression. Inhibition of MAPK activity by MEK inhibitor U0126 abolished EGF-induced DNA synthesis. Since EGF activates PI 3 kinase, which also regulates DNA synthesis, the effect of PI 3 kinase on MAPK activity was also examined. Inhibition of PI 3 kinase signaling blocked EGF-induced MAPK activity as well as Elk-1 dependent reporter transcription and c-fos gene transcription. To further determine the mechanism of EGF-induced DNA synthesis, we investigated the effect of EGF on the cyclin-dependent kinase inhibitor p27. EGF reduced the expression of p27. Inhibition of PI 3 kinase action or MAPK activity abolished the reduction in p27 expression induced by EGF. These data provide the evidence that a