FIG. 5.
Involvement of the PI3K-AKT-FRAP pathway in heregulin-induced HIF-1α expression. (A) Stimulation of AKT activity and HIF-1α expression by heregulin. Serum-starved MCF-7 cells were exposed to vehicle or heregulin for 6 h and analyzed for phospho-AKT (top), total AKT (middle), or HIF-1α protein (bottom) by immunoblot assay. (B) Effect of serum and heregulin stimulation on S6 kinase activity. Serum-starved cells were treated with serum or heregulin for 6 h prior to immunoblot assay with antibodies specific for phosphorylated p70s6k and its p85 isoform (top) or total p70s6k protein (bottom). (C) Effect of kinase inhibitors on heregulin-stimulated HIF-1α expression. Serum-starved cells were pretreated for 30 min with vehicle or inhibitor (AG825, PD98059, rapamycin, or LY294002) and then exposed to no treatment, 10% FBS (Serum), or 100 ng of heregulin/ml for 6 h prior to HIF-1α immunoblot assay of whole-cell lysates. (D) Analysis of HIF-1α mRNA expression. MCF-7 cells were serum starved and exposed to heregulin in the absence or presence of rapamycin for 6 h. Total RNA was isolated, and blot hybridization was performed with a HIF-1α cDNA probe (top). A photograph of the ethidium bromide-stained gel demonstrates equal loading of RNA as determined by the intensity of the 18S and 28S rRNA bands (bottom).