Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.
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ABSTRACT:
SUBMITTER: Weigel C
PROVIDER: S-EPMC4792958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.
| S-EPMC8158145 | biostudies-literature
Project description:Radiotherapy is an efficient tool in cancer treatment, but it brings along the risk of side effects such as fibrosis in the irradiated healthy tissue thus limiting tumor control and impairing quality of life of cancer survivors. Knowledge on radiation-related fibrosis risk and therapeutic options is still limited and requires further research. Recent studies demonstrated that epigenetic regulation of diacylglycerol kinase alpha (DGKA) is associated with radiation-induced fibrosis. However, the specific mechanisms are still unknown. In this review, we scrutinized the role of DGKA in the radiation response and in further cellular functions to show the potential of DGKA as a predictive marker or a novel target in fibrosis treatment. DGKA was reported to participate in immune response, lipid signaling, exosome production, and migration as well as cell proliferation, all processes which are suggested to be critical steps in fibrogenesis. Most of these functions are based on the conversion of diacylglycerol (DAG) to phosphatidic acid (PA) at plasma membranes, but DGKA might have also other, yet not well-known functions in the nucleus. Current evidence summarized here underlines that DGKA activation may play a central role in fibrosis formation post-irradiation and shows a potential of direct DGKA inhibitors or epigenetic modulators to attenuate pro-fibrotic reactions, thus providing novel therapeutic choices.
| S-EPMC7235333 | biostudies-literature
Project description:Diacylglycerol kinase α (DGKα) regulates diacylglycerol levels, catalyzing its conversion into phosphatidic acid. The α isoform is central to immune response regulation; it downmodulates Ras-dependent pathways and is necessary for establishment of the unresponsive state termed anergy. DGKα functions are regulated in part at the transcriptional level although the mechanisms involved remain poorly understood. Here, we analyzed the 5' end structure of the mouse DGKα gene and detected three binding sites for forkhead box O (FoxO) transcription factors, whose function was confirmed using luciferase reporter constructs. FoxO1 and FoxO3 bound to the 5' regulatory region of DGKα in quiescent T cells, as well as after interleukin-2 (IL-2) withdrawal in activated T cells. FoxO binding to this region was lost after complete T cell activation or IL-2 addition, events that correlated with FoxO phosphorylation and a sustained decrease in DGKα gene expression. These data strongly support a role for FoxO proteins in promoting high DGKα levels and indicate a mechanism by which DGKα function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGKα downregulation, IL-2, and anergy avoidance.
| S-EPMC3457341 | biostudies-literature
Project description:Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.
| S-EPMC3221890 | biostudies-literature
Project description:We have studied the relationship between diacylglycerol kinase delta (DGK?) and lipogenesis. There is a marked increase in the expression of DGK? during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGK?, there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGK? promotes lipogenesis. Lipid synthesis is decreased in DGK? knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids, which is higher in the wild type cells than in the DGK? knockouts. In comparison, there was no change in lipid synthesis in DGK? knockout mouse embryo fibroblasts. We also demonstrate that the DGK? knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGK? markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.
| S-EPMC3864584 | biostudies-literature
Project description:Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ? (DGK?), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGK? is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGK? was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGK?-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGK?-null cells were smaller than those found in wild type cells. Rescue of this defect required DGK? catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGK? mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGK? functions in concert with Rac1 to regulate macropinocytosis.
| S-EPMC4689489 | biostudies-literature
Project description:Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.
| S-EPMC8292318 | biostudies-literature
Project description:The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.
| S-EPMC8603347 | biostudies-literature
Project description:Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+ -triggered activation remains unclear. Here we report the first crystal structure of Ca2+ -bound DGKα-EF and analyze the structural changes upon binding to Ca2+ . DGKα-EF adopts a canonical EF-hand fold, but unexpectedly, has an additional α-helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα-EF adopts a protease-susceptible "open" conformation without Ca2+ that tends to form a dimer. Cooperative binding of two Ca2+ ions dissociates the dimer into a well-folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca2+ binding induces substantial conformational changes in DGKα-EF, which likely regulates intra-molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca2+ -induced conformational changes. SIGNIFICANCE STATEMENT: Diacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF-hand domains of diacylglycerol kinase α in its Ca2+ bound form and characterize Ca2+ -induced conformational changes, which likely regulates intra-molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.
| S-EPMC6423725 | biostudies-literature
Project description:Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.
| S-EPMC4259432 | biostudies-literature