Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells

We investigated the ability of EGF to stimulate the phosphorylation (i.e. a ctivation) df extracellular signal-regulated kinases (ERKs) in freshly isol ated porcine granulosa cells (pGC) held in suspension. pGCs were isolated f rom the ovaries of prepubertal gigs at slaughter, and equilibrated for 24 h at 37 degrees C in 12 x 75 mm culture tubes. The cells were then treated w ith 0-10 ng/ml GF for 1-240 min. Treatments were terminated, and the total cell lysates were subjected to SDS-PAGE and Western analysis. The Westerns were blotted with anti-panERK and with anti-phosphoERK, antibodies that rec ognize all forms of ERKs and the phosphorylated (i.e. activated) forms of E RKs, respectively. Western blot analysis with the panERK antibody revealed a gel shift of ERKs, suggesting hyperphosphorylation after treatment with a s little as 0.1 ng/ml of EGF. Phosphorylation of the ERKs was confirmed by using the phosphoERK antibody, which indicated increased phosphorylation of ERKs above control with 0.1 ng/ml EGF and maximal phosphorylation of ERK w ith 5-10 ng/ml EGF. Activation of ERK by EGF, as measured by both gel shift analysis and active ERK blotting, in the freshly isolated pGC was rapid, i ncreasing above controls after I min of treatment, maintaining high levels through 40 min, and declining from 60 to 240 min. These data indicate that EGF stimulates active ERK in a time- and concentration-dependent manner in freshly isolated pGCs and that this experimental approach represents an eff ective manner with which to evaluate the role of EGF and the ERK signal tra nsduction pathway in Freshly harvested pGC. (C) 1999 Elsevier Science Inc. All rights reserved.