Gene expression after HOS cells treated with DGKZ (diacylglycerol kinase zeta)-silencing by a microarray profiling (Affymetrix)
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PROVIDER: PRJNA475546 | ENA |
REPOSITORIES: ENA
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Project description:DGKZ could promote proliferation and inhibit apoptosis in osteosarcoma,both in vivo and in vitro. To identify downstream molecular mechanism of DGKZ in proliferation of osteosarcoma, we performed a microarray profiling (Affymetrix) in human HOS cells treated with DGKZ knockdown.
2018-06-12 | GSE115590 | GEO
Project description:In addition to lipid second messengers derived from the plasma membrane, increasing evidence supports the existence of nuclear lipid-dependent signaling networks. Diacylglycerol is a key second messenger, generated at the nuclear level, which is metabolized by diacylglycerol kinases (DGKs). It has been demonstrated that nuclear DGK-ζ negatively regulates cell cycle progression. The aim of this study was to identify key determinants of nuclear DGK-ζ-dependent cell cycle arrest in C2C12 mouse myoblasts. Using DNA microarrays, Real-Time RT-PCR and western blot, we demonstrated that nuclear DGK-ζ downregulated the expression of cyclin D1 and increased the expression of TIS21/BTG2/PC3, a transcriptional regulator of cyclin D1 with a strong anti-proliferative function. Overexpression of TIS21/BTG2/PC3 blocked the cells in G1 phase of the cell cycle and decreased the levels of Ser807/811 phosphorylated retinoblastoma protein, similarly to overexpression of DGK-ζ. Moreover, during myogenic differentiation of C2C12 cells, we showed an increase of TIS21/BTG2/PC3 expression and a decrease in cyclin D1 levels. siRNA downregulation of TIS21/BTG2/PC3 impaired myogenic differentiation by opposing cell cycle arrest. In summary, these data identify TIS21/BTG2/PC3 and cyclin D1 as downstream effectors of the nuclear DGK-ζ and highlight the importance of this DGK isoform in the regulation of myoblast proliferation and differentiation.
2009-09-30 | GSE11770 | GEO
Project description:Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) α and ζ act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGKα and DGKζ to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGKζ, indicating functional differences between these proteins. DGKζ kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGKζ responsiveness. We used GFP-DGKζ and -DGKα chimeras to assess translocation dynamics during IS formation. Only GFP-DGKζ translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGKζ with DAG consumption at the IS. This study highlights a DGKζ-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.
| S-EPMC3216665 | biostudies-literature
Project description:Diacylglycerol kinase ζ (DGKζ) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGKζ has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGKζ, together with DGKα, as a potential target for cancer immunotherapy. However, DGKζ has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGKζ using a baculovirus-insect cell expression system for enzymological and structural studies. Full-length DGKζ remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGKζ is in a fully functional state. The K m values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGKα and DGKε, and its affinities for DG and phosphatidylserine are comparable to those of DGKα/DGKε. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGKζ is comprised of 25% α-helices and 18% β-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGKζ. The purification of DGKζ allows detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.
| S-EPMC6935893 | biostudies-literature
Project description:Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.
| S-EPMC2199204 | biostudies-literature
Project description:This SuperSeries is composed of the following subset Series: GSE38133: TGF-beta1 effect on human osteosarcoma cell line MNNG/HOS GSE38134: Expression analysis of human osteosarcoma cell line MNNG/HOS sarcospheres, hypoxia-induced sarcospheres and TGF-beta1-induced sarcospheres Refer to individual Series
2012-05-22 | E-GEOD-38135 | biostudies-arrayexpress
Project description:Natural regulatory T (nT(reg)) cells are important for maintaining tolerance to self- and foreign antigens, and they are thought to develop from thymocytes that receive strong T cell receptor (TCR)-mediated signals in the thymus. TCR engagement leads to the activation of phospholipase C-γ1, which generates the lipid second messenger diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. We used mice that lack the ζ isoform of DAG kinase (DGKζ), which metabolizes DAG to terminate its signaling, to enhance TCR-mediated signaling and identify critical signaling events in nT(reg) cell development. Loss of DGKζ resulted in increased numbers of thymic CD25(+)Foxp3(-)CD4(+) nT(reg) cell precursors and Foxp3(+)CD4(+) nT(reg) cells in a cell-autonomous manner. DGKζ-deficient T cells exhibited increased nuclear translocation of the nuclear factor κB subunit c-Rel, as well as enhanced extracellular signal-regulated kinase (ERK) phosphorylation in response to TCR stimulation, suggesting that these downstream pathways may contribute to nT(reg) cell development. Indeed, reducing c-Rel abundance or blocking ERK phosphorylation abrogated the increased generation of nTreg cells by DGKζ-deficient thymocytes. The extent of ERK phosphorylation correlated with TCR-mediated acquisition of Foxp3 in immature thymocytes in vitro. Furthermore, the development of nT(reg) cells was augmented in mice in which ERK activation was selectively enhanced in T cells. Together, these data suggest that DGKζ regulates the development of nT(reg) cells by limiting the extent of activation of the ERK and c-Rel signaling pathways.
| S-EPMC4103616 | biostudies-literature
Project description:Enhancement of NK cell function could be beneficial in treatment of a variety of tumors and infections. However, efforts to improve NK cell function by disrupting negative regulators that target proximal signaling pathways paradoxically results in hyporesponsive rather than hyperresponsive NK cells. In this study, we demonstrate that genetic deletion of diacylglycerol kinase (DGK)ζ, a negative regulator of diacylglycerol-mediated signaling, has the desired effect of enhancing NK cell function due to its distal position in the activating receptor-mediated signaling cascade. Upon stimulation through multiple activating receptors, NK cells from mice lacking DGKζ display increased cytokine production and degranulation in an ERK-dependent manner. Additionally, they have improved cytotoxic functions against tumor cell lines. The enhancement of NK cell function by DGKζ deficiency is NK cell-intrinsic and developmentally independent. Importantly, DGKζ deficiency does not affect inhibitory NK cell receptor expression or function. Thus, DGKζ knockout mice display improved missing self recognition, as evidenced by enhanced rejection of a TAP-deficient tumor in vivo. We propose that enzymes that negatively regulate distal activating receptor signaling pathways such as DGKζ represent novel targets for augmenting the therapeutic potential of NK cells.
| S-EPMC4935923 | biostudies-literature
Project description:Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well.
| S-EPMC20810 | biostudies-literature
Project description:Investigation of whole genome gene expression level changes in human osteosarcoma cell line MNNG/HOS treated by TGF-beta1 for three days (mesophase) and five days (sarcospheres iOSCs), compared to non-treatment cells (residual adherent cells). A three chip study using total RNA cover from three cultures of non-treatment human osteosarcoma cell line MNNG/HOS (residual adherent cells), TGF-beta1 treated three days osteosarcoma cell line MNNG/HOS (mesophase) and TGF-beta1 treated five days osteosarcoma cell line MNNG/HOS ( only collected the suspending sarcospheres iOSCs). Each chip measures the expression level of 45033 genes from osteosarcoma cell line MNNG/HOS.
2012-05-22 | E-GEOD-38133 | biostudies-arrayexpress