Purchase this article with an account.
Marjorie F. Lou, Yin Wang; Mechanism Of PDGF-induced Cell Proliferation: The Role Of Phospholipase A2 alpha (cPLA2) And Calcium In Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3926.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Previously we have found that the mitogenic action of platelet-derived growth factor (PDGF) is mediated by redox signaling using reactive oxygen species (ROS) generated from NADPH oxidase (NOX) and that chemical inhibitor of phospholipase A2 alpha (cPLA2) or NOX could completely eradicate PDGF action. This study is to investigate the molecular mechanism for cPLA2 and the requirement of calcium in regulating PDGF function.
cPLA2 was examined using human lens epithelial (HLE) B3 cells as a model. ROS generation induced by PDGF was analyzed by lucigenin-based luminescence assay. Cell proliferation was measured by cell counting and by BrdU assay. Human cPLA2 alpha gene was cloned via RT-PCR followed by site-directed mutagenesis to construct HLE B3 cells expressing either inactive cPLA2 enzyme with S228A mutation (S228A), or cPLA2 devoid of translocating function with deleted calcium-binding C2 domain (C2D). Activity of cPLA2 was measured by arachidonic acid (AA) released from cell membrane using [3H]-AA pre-labeled cells. Effect of calcium on cPLA2 function was examined by treating cells with calcium ionophore, calcium chelating agent or thapsgargin. Activation of ERK, JNK, p38, or Akt was detected by Western blotting using specific antibodies.
In S228A HLE B3 cells, the PDGF-induced cell proliferation with concomitant ROS generation and activation of JNK and ERK were decreased in comparison with the Vec control. However, P-Akt and P-p38 were not affected. C2D cells also showed similar malfunction as the S228A mutant. Calcium was required for PDGF cell signaling, including membrane translocation of cPLA2, AA release and ROS generation. This Ca2+-dependent process appeared to rely on the Ca2+ store in ER, as thapsigargin, an agent specifically releases Ca2+ from ER store, augmented HLE B3 cellular response to PDGF stimulation.
PDGF mitogenic action is mediated only by an intact cPLA2, and requires calcium released from ER.
This PDF is available to Subscribers Only