Tetherin/BST2, a physiologically and therapeutically relevant regulator of platelet receptor signalling.

The reactivity of platelets, which play a key role in the pathogenesis of atherothrombosis, is tightly regulated. The integral membrane protein tetherin/bone marrow stromal antigen-2 (BST-2) regulates membrane organization, altering both lipid and protein distribution within the plasma membrane. Because membrane microdomains have an established role in platelet receptor biology, we sought to characterize the physiological relevance of tetherin/BST-2 in those cells. To characterize the potential importance of tetherin/BST-2 to platelet function, we used tetherin/BST-2-/- murine platelets. In the mice, we found enhanced function and signaling downstream of a subset of membrane microdomain-expressing receptors, including the P2Y12, TP thromboxane, thrombin, and GPVI receptors. Preliminary studies in humans have revealed that treatment with interferon-α (IFN-α), which upregulates platelet tetherin/BST-2 expression, also reduces adenosine diphosphate-stimulated platelet receptor function and reactivity. A more comprehensive understanding of how tetherin/BST-2 negatively regulates receptor function was provided in cell line experiments, where we focused on the therapeutically relevant P2Y12 receptor (P2Y12R). Tetherin/BST-2 expression reduced both P2Y12R activation and trafficking, which was accompanied by reduced receptor lateral mobility specifically within membrane microdomains. In fluorescence lifetime imaging-Förster resonance energy transfer (FLIM-FRET)-based experiments, agonist stimulation reduced basal association between P2Y12R and tetherin/BST-2. Notably, the glycosylphosphatidylinositol (GPI) anchor of tetherin/BST-2 was required for both receptor interaction and observed functional effects. In summary, we established, for the first time, a fundamental role of the ubiquitously expressed protein tetherin/BST-2 in negatively regulating membrane microdomain-expressed platelet receptor function.

The tumor suppressor RhoBTB1 controls Golgi integrity and breast cancer cell invasion through METTL7B.

BACKGROUND: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. METHODS: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RESULTS: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. CONCLUSION: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.

RNA interference approaches to examine Golgi function in animal cell culture.

The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting.

FoxO1 is required for the regulation of preproglucagon gene expression by insulin in pancreatic alphaTC1-9 cells.

Forkhead/winged helix box gene, group O-1 (FoxO1) is a member of a family of nuclear transcription factors regulated by insulin-dependent phosphorylation and implicated in the development of the endocrine pancreas. We show here firstly that FoxO1 protein is expressed in both primary mouse islet alpha and beta cells. Examined in clonal alphaTC1-9 cells, insulin caused endogenous FoxO1 to translocate from the nucleus to the cytoplasm. Demonstrating the importance of nuclear exclusion of FoxO1 for the inhibition of preproglucagon gene expression, FoxO1 silencing by RNA interference reduced preproglucagon mRNA levels by >40% in the absence of insulin and abolished the decrease in mRNA levels elicited by the hormone. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed direct binding of FoxO1 to a forkhead consensus binding site, termed GL3, in the preproglucagon gene promoter region, localized -1798 bp upstream of the transcriptional start site. Deletion or mutation of this site diminished FoxO1 binding and eliminated transcriptional regulation by glucose or insulin. FoxO1 silencing also abolished the acute regulation by insulin, but not glucose, of glucagon secretion, demonstrating the importance of FoxO1 expression in maintaining the alpha-cell phenotype.