Abstract
The cyclin-dependent kinase inhibitor p27Kip1 is frequently inactivated in human cancers. Glucocorticoids, acting through the glucocorticoid receptor (GR), are frequently used to treat certain malignancies and are growth inhibitive, but the relationship between GR activity and p27 status has not been explored. We have therefore examined GR-dependent transcriptional activation, receptor phosphorylation, and glucocorticoid-dependent growth inhibition in p27-deficient (p27-/-) murine embryonic fibroblasts (MEFs). We find that GR transcriptional enhancement as well as receptor phosphorylation at two putative cyclin-dependent kinase sites are elevated in p27-/- MEFs, relative to control cells. This increased GR transcriptional activation appears to be mediated through the GR N terminus, and coexpression of the GR N-terminal coactivator, DRIP150, further enhanced GR-dependent transcriptional activation. Furthermore, p27-/- MEFs are partially resistant to the growth inhibitory effects of glucocorticoids. Thus, p27 appears to be an important element in the GR transcription and growth inhibitory responses.
Highlights
Glucocorticoids are a class of steroid hormones that govern metabolism and development and are used clinically for the treatment of inflammatory diseases as well as certain types of malignancies
We find that glucocorticoid receptor (GR) transcriptional enhancement as well as receptor phosphorylation at two putative cyclin-dependent kinase sites are elevated in p27؊/؊ murine embryonic fibroblasts (MEFs), relative to control cells
To this end we examined GR transcriptional regulation, receptor phosphorylation, and gluc ocorticoid-dependent growth inhibition using primary embryonic fibroblasts derived from p27-deficient mice
Summary
Murine Embryo Fibroblast (MEF) Generation and Cell Cultivation— MEFs were generated from 14-day postcoitum mouse embryos of p27Ϫ/ϩ heterozygote mating. BuGR2-coated beads were incubated with cell extracts (1 mg of total protein in lysis buffer per sample) at 4 °C for 3 h on a rocking platform, pelleted by centrifugation, and washed five times in PBS buffer and twice in 50 mM Tris buffer, pH 7.5. Cdk Activity Assay—Protein extracts were prepared from a subconfluent 10-cm plate of wild-type and p27Ϫ/Ϫ MEFs. Cells were washed twice with PBS and lysed for 10 min, on ice, in 200 l of lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.8% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol (DTT), supplemented with protease and phosphatase inhibitors: 0.1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, and 1 g/ml each of aprotinin, pepstatin A, and leupeptin). Transfected cells were washed once in PBS and harvested in 1ϫ reporter lysis buffer (Promega) according to the manufacturer’s instructions. Nuclear-emitted fluorescence was measured under a FACScan flow cytometer (BD Biosciences), and the percentage of cells in each phase of the cell cycle was determined
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