1 Activated b 2 -adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. 2 The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. 3 The phosphorylation of the GRK site Ser355,356 during a 10 mM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. 4 In contrast, phosphorylation of the PKA site Ser262 during a 10 mM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. 5 The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. 6 The results indicate that the kinetics of b 2 -adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.

Each of the three members of the p160 steroid receptor coactivator (SRC) family of coactivators (SRC-1, SRC-2 and SRC-3) stimulates estrogen receptor (ER)-alpha function in trans-activation assays. Consequently, we sought to elucidate their contributions to the ER-regulated processes of cell proliferation, apoptosis, and the expression of ERalpha target genes in MCF-7 breast cancer cells. The small interfering RNA depletion of SRC-2 or SRC-3 but not SRC-1 inhibited growth of MCF-7 cells, and this was reflected in decreased cell cycle progression and increased apoptosis in SRC-2- or SRC-3-depleted cells as well as a reduction in ERalpha transcriptional activity measured on a synthetic reporter gene. However, only SRC-3 depletion blocked estradiol stimulated cell proliferation. Depletion of SRC-1 did not affect these events, and together this reveals functional differences between each of the three SRC family coactivators. Regulation of the endogenous ERalpha target gene, c-myc was not affected by depletion of any of the p160 coactivators although depletion of each of them decreased pS2 mRNA expression in estradiol-treated MCF-7 cells. Moreover, progesterone receptor and cyclin D1 gene expression were decreased in SRC-3 small interfering RNA-treated cells. Expression of mRNA and protein levels for the antiapoptotic gene, Bcl-2 was dependent on SRC-3 expression, whereas Bcl-2 protein but not mRNA expression also was sensitive to SRC-1 depletion. Together these data indicate that the closely related p160 coactivators are not functionally redundant in breast cancer cells because they play gene-specific roles in regulating mRNA and protein expression, and they therefore are likely to make unique contributions to breast tumorigenesis.

B-cell Translocation Gene 2 (BTG2/TIS21/PC3) is an anti-proliferative tumor suppressor gene whose expression is significantly reduced in breast carcinomas, and in MCF-7 and T-47D breast cancer cell lines treated with estradiol (E2). In this study the mechanisms involved in E2 down regulation of BTG2 gene expression were examined. Depletion of ERa by siRNA indicated that the receptor is required for E2 down regulation of BTG2 mRNA levels, and cycloheximide experiments indicated that the effect of E2 on BTG2 expression was independent of intermediary protein synthesis. Chromatin immunoprecipitation analyses revealed that ERa interacts with the BTG2 promoter in a ligand-independent fashion whereas transfection experiments indicated that ERa's DNA and ligand binding domains are required for E2 repression of BTG promoter activity. Surprisingly, histone deacetylase (HDACs) activity is essential for basal expression as evidenced by trichostatin A inhibition of BTG2 mRNA levels. Estradiol treatment did not alter histone H3 acetylation although it did induce displacement of RNA polymerase II from the BTG2 gene. Depletion of the ER specific corepressor REA (Repressor of Estrogen Receptor Activity) significantly abrogated E2-mediated BTG2 repression. Taken together, our results reveal a requirement of HDAC activity for basal BTG2 expression and the ERa-REA interaction for estrogen repression of the BTG2 gene. The ability of E2-bound ERa and REA to suppress BTG2 expression indicates a positive role for this corepressor in regulation of breast cancer cell proliferation. ' 2008 Wiley-Liss, Inc.Key words: ERa; BTG2; estrogen-mediated repression; breast cancer cell proliferation B-cell translocation gene 2 (BTG2) is an antiproliferative (ARPO) tumor suppressor protein, because its overexpression leads to blockade of the cells at the G 1 phase of the cell cycle.1,2 It was originally identified in the rat pheochromocytoma PC-12 cell line undergoing neuronal differentiation induced by nerve growth factor, where it was classified as an immediate early gene whose expression could be controlled by mitogenic, differentiative and antiproliferative factors. 3,4 Later, it was demonstrated that BTG2 expression is induced by cytotoxic and genotoxic stress through a p53-dependent mechanism.2,5 Binding sites for p53 and NF-jB are located within the BTG2 promoter, and the latter correlates well to the regulation of BTG2 expression by NF-jB activators such as tumor necrosis factor-a (TNFa) and some members of the transforming growth factor-b (TGFb) family.6,7 Induction of BTG2 expression inhibits S-phase entry via either retinoblastoma (Rb)-dependent 8 or independent pathways, 9,10 and this is attributed, at least in part, to transcriptional suppression of the cyclin D1 gene whose product is required for the G 1 to S phase transition. 8 A loss of nuclear BTG2 expression is observed in ERa positive human breast tumor samples, 11 and a correlation between breast tumor size and BTG2 expression has also been demonstrated. 12 In normal mammary gla...

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The effects of hyperinsulinaemia which promote neointimal hyperplasia after balloon injury of rat carotid artery predominate over the effects which inhibit it even in normal animals.