36P Oral Communication - Cellular Signalling C64 The effect of cholesterol depletion on insulin-like growth factor signalling in caveolin-positive and caveolin- negative cells

Caveolae are cholesterol-enriched invaginations of the plasma membrane suggested to be important for many aspects of cellular signalling. Caveolin, a marker protein for caveolae, binds to and modulates the function of various signalling molecules including the tyrosine kinase receptor for insulin (Yamamoto et al, 1998). Components of the closely related insulin-like growth factor (IGF) system are important regulators of cellular function, with effects on growth, survival and metabolism mediated primarily through the type 1 IGF receptor (IGFIR). Recent work proposed that IGFIR associated with caveolin, but the impact of lipid rafts/caveolae on IGF signalling remains controversial (Hong et al, 2004). Consequently, we investigated whether these membrane domains were important for IGFmediated cell proliferation (thymidine uptake) and survival (nuclear morphology) in caveolin-positive (NWTb3) and caveolin-negative (HepG2) cells. Subcellular fractionation and co-immunoprecipitation studies (n=3) in NWTb3 fibroblasts demonstrated that IGFIR associated with caveolin. 5nM IGF-I enhanced proliferation by 2.1±0.1-fold (mean±SEM; Mann-Whitney test; p<0.05; n=4) in NWTb3 cells and 1.4±0.0-fold (n=4) in HepG2 cells. When cellular cholesterol was depleted using 5mM methylcyclodextrin (MCD), which disrupts caveolae (Hailstones et al, 1998), IGF-I-stimulated cell proliferation was not impaired in either NWTb3 (IGF-I 2.1±0.1-fold; IGF-I+MCD 2.1±0.2fold; n=4) or HepG2 cells (IGF-I 1.4±0.0-fold; IGF-I+MCD 1.2±0.1-fold; n=4). 5nM IGF-I protected NWTb3 and HepG2 cells from apoptosis induced by 24hr serum withdrawal (control 41.6±1.6% and 4.8±0.0%; IGF-I 6.1±0.4% and 1.3±0.2% apoptosis respectively; n=4). After MCD treatment, IGF-I-stimulated cell survival was significantly impaired in both NWTb3 (IGF-I 6.1±0.4%; IGFI+MCD 27.1±1.0% apoptosis; n=4) and HepG2 cells (IGF-I 1.3±0.2%; IGF-I+MCD 2.6±0.2% apoptosis; n=4), although there was no effect from MCD alone (MCD 45.4±0.9% and 5.0±0.0% apoptosis respectively; n=4). MCD treatment therefore induced the same effect on IGFinduced proliferation and survival in caveolin-positive NWTb3 and caveolin-negative HepG2 cells. This suggests that whilst IGFIR associates with caveolin by co-immunoprecipitation and subcellular fractionation, the interaction between IGFIR and caveolin may not be obligatory for IGF-mediated cell proliferation or survival.