PARENT SESSION
TRAINEE RESEARCH COMPETITION
67
INSULIN-LIKE GROWTH FACTOR-I PREVENTS FAS LIGAND-INDUCED APOPTOSIS OF GRANULOSA CELLS VIA THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT KINASE PATHWAY.
Hu, Che-Lin1, Cowan, Robert1, Harman, Rebecca1, Roberson, Mark2, Quirk, Susan1, 1 2
ABSTRACT- Fas antigen (Fas) is a cell-surface receptor that triggers apoptosis when bound by Fas ligand (FasL). Bovine granulosa cells (GC) express Fas and FasL and are susceptible to FasL-induced apoptosis when cultured in the absence of growth factors. Several growth factors, including insulin-like growth factor-I (IGF), protect GC from FasL-induced killing. IGF activates both the phosphatidylinositol 3-kinase (PI3K)/Akt kinase and the mitogen-activated protein kinase (MAPK) signaling pathways. We sought to determine which pathway mediates the inhibition of FasL-induced killing by IGF. Bovine GC were collected from 2-4 mm follicles and cultured in DMEM-F12 containing 10% FBS. Media was then changed to DMEM-F12 supplemented with BSA, 100 ng/ml insulin, transferrin and selenium (ITS) on day 1. On day 2, GC were treated with 0 or 100 ng/ml IGF at t=0 h and with 0 or 100 ng/ml FasL at t=4 h. The number of viable cells was assessed at 24 h by trypan blue exclusion. Parallel cultures were treated similarly and pretreated at -0.5 h with a PI3K inhibitor, LY294002, or a MAPK pathway inhibitor, PD98059. Treatment with IGF decreased the percentage of cells killed by FasL (29±8% in controls vs 14±5% with IGF, p<0.05). In the presence of LY294002, IGF had no effect on FasL-induced killing (37±5% in controls vs 35±5% with IGF, p>0.05). In the presence of PD98059, IGF inhibited FasL-induced killing (31±6% in controls vs 16±2% with IGF, p<0.05). Immunoblot analysis of parallel cultures showed rapid phosphorylation of Akt which became significant 2 h after IGF treatment (1.6-fold increase, p<0.05) and remained elevated at 24 h. Similar increases in Akt phosphorylation were observed following treatment with IGF in the presence of PD98059, but not in the presence of LY294002. Phosphorylation of ERK1/2 was increased by IGF, and this was prevented by PD98059 but not by LY294002. These results suggest that IGF protects granulosa cells from FasL-induced killing through activation of the PI3K/Akt kinase pathway, but not through the MAPK pathway.
KEY WORDS: IGF-I, granulosa cells, Fas, apoptosis