Abstract:
Mitogen-activated protein kinase (MAPK) signaling pathways also known as extracellular signalregulated kinase (ERK1/2), are among the most important pathways a cell can use to respond to changes in their extracellular environments. The ERK1/2 pathway is known to play a key role in cell survival, cell death, cell differentiation, and cell proliferation in response to a variety of stresses. Reducing
apoptotic cell death during myocardial hypoxia and/or ischemia has substantial clinical significance. Interestingly, activation of the ERK1/2 pathway has been shown to protect a rat cardiomyoblast cell line (H9c2) from apoptosis following nutrient/energy deprivation. Placenta growth factor (PlGF), a key molecule in angiogenesis and member of the VEGF family, is a ligand for flt-1. Although we have shown that cardiomyocytes upregulate PlGF mRNA expression during hypoxia, its function is not understood. In order to assess PlGF activation of ERK1/2, cells will be made quiescent by serum starving with 1% FCS media for 20 hours, and then treated with 10% FCS or 50ng/mL PlGF for 10 minutes. Cell cultures will then be lysed and harvested using a TPER:protease inhibitor
lysis buffer. SDS-Page, and Western blotting techniques utilizing antibodies specific for phospho-ERK1/2 and total ERK1/2 will be used to determine flt-1 activation. These techniques will determine if PlGF can induce ERK1/2 activation, and ultimately cell survival in H9c2 cells.
