Rat aortic smooth muscle cell density affects activation of MAP kinase andAkt by menadione and PDGF homodimer BB

Mitogen-activated protein kinases (MAPK) and protein kinase B (PKB or Akt) are major signal transduction molecules regulating cell proliferation, diff erentiation, and apoptosis. We examined how cultured rat aortic vascular sm ooth muscle cells (VSMC at different cell densities respond to selected sti muli and how this is reflected in the two distinct (MAPK and Akt) and yet c ross-talking signaling pathways. VSMC were cultured to 100% confluence, rea ching contact inhibition, and to 60-70% confluence, as sparse, proliferatin g cells. They were treated with menadione (an intracellular generator of O- 2(-)) and/or platelet-derived growth factor homodimer BB (PDGF). In sparse cells, menadione or PDGF alone activated ERK, and together the effect was s ynergistic, whereas in confluent cells menadione's and PDGF's activations o f ERK were, at most, additive. Activation of the upstream ERK kinase (MEK-1 ) paralleled ERK activation except in sparse cells in which the synergistic effects of menadione and PDGF on ERK could not be fully accounted for by M EK-1 activation. Another member of the MAPK family, p38, did not show signi ficant changes. Akt activation by PDGF alone was present under both cell cu lture conditions; Akt activation is blocked by menadione. Co-incubation wit h the reducing agent dithiothreitol or calcium chelators (EDTA/EGTA) inhibi ted partially or completely menadione's effects on MEK/ERK and Akt pathways , as well as menadione's effects on PDGF-induced ERK and Akt activations. T hese data suggest that in VSMC, the state of cell confluence determines how distinct pathways of MAPK activation cross talk. In addition while PDGF ma y function as a survival factor by inducing Akt activation, menadione could promote apoptosis by inhibiting PDGF-induced AM activation independent of cell density. The effects of menadione, but not those of PDGF, are more dep endent on the cellular redox status and extracellular calcium. (C) 2001 Aca demic Press.