DGKζ Deficiency Research Articles

Diacylglycerol kinase zeta deficiency attenuates papain-induced type 2 airway inflammation

Allergic airway diseases are caused by inappropriate immune responses directed against inhaled environmental antigens. We previously reported that the inhibition of diacylglycerol (DAG) kinaseζ (DGKζ),an enzyme that terminates DAG-mediated signaling,protects against T cell-mediated allergic airway inflammation by blocking Th2 cell differentiation.In this study, we tested whether DGKζ deficiency also affects allergic airway disease mediated by type 2 innate lymphoid cells (ILC2)s. DGKζ-deficient mice displayed diminished ILC2 function and reduced papain-induced airway inflammation compared to wildtype mice. Unexpectedly, however, mice with hematopoietic cell-specific deletion ofDGKζ displayed intact airway inflammation upon papain challenge. Rather, bone marrow chimera studies revealed thatDGKζ deficiency in the non-hematopoietic compartment was responsible for the reduction in papain-induced airway inflammation. These data suggest that DGK might represent a novel therapeutic target not only for T cell-dependent but also ILC2-dependent allergic airway inflammation by affecting non-hematopoietic cells.

Manipulation of diacylglycerol and ERK-mediated signaling differentially controls CD8+ T cell responses during chronic viral infection.

Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen exposure is less understood. Here, we investigated the role of diacylglycerol (DAG)-mediated signaling downstream of the TCR during chronic lymphocytic choriomeningitis virus clone 13 (LCMV CL13) infection by blocking DAG kinase zeta (DGKζ), a negative regulator of DAG. We examined the activation, survival, expansion, and phenotype of virus-specific T cell in the acute and chronic phases of LCMV CL13-infected in mice after DGKζ blockade or selective activation of ERK. Upon LCMV CL13 infection, DGKζ deficiency promoted early short-lived effector cell (SLEC) differentiation of LCMV-specific CD8+ T cells, but this was followed by abrupt cell death. Short-term inhibition of DGKζ with ASP1570, a DGKζ-selective pharmacological inhibitor, augmented CD8+ T cell activation without causing cell death, which reduced virus titers both in the acute and chronic phases of LCMV CL13 infection. Unexpectedly, the selective enhancement of ERK, one key signaling pathway downstream of DAG, lowered viral titers and promoted expansion, survival, and a memory phenotype of LCMV-specific CD8+ T cells in the acute phase with fewer exhausted T cells in the chronic phase. The difference seen between DGKζ deficiency and selective ERK enhancement could be potentially explained by the activation of the AKT/mTOR pathway by DGKζ deficiency, since the mTOR inhibitor rapamycin rescued the abrupt cell death seen in virus-specific DGKζ KO CD8+ T cells. Thus, while ERK is downstream of DAG signaling, the two pathways lead to distinct outcomes in the context of chronic CD8+ T cell activation, whereby DAG promotes SLEC differentiation and ERK promotes a memory phenotype.

Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes

BackgroundThe inhibitory functions triggered by the programmed cell death-1 (PD-1) receptor following binding to its ligand (PD-L1) protect healthy organs from cytotoxic T cells, and neutralize antitumor T cell attack....

Diacylglycerol Kinase ζ Regulates Macrophage Responses in Juvenile Arthritis and Cytokine Storm Syndrome Mouse Models.

Dysregulation of monocyte and macrophage responses are often observed in children with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS), a potentially fatal complication of chronic rheumatic diseases. Both conditions are associated with activation of TLR signaling in monocyte and macrophage lineage cells, leading to overwhelming inflammatory responses. Despite the importance of TLR engagement in activating proinflammatory macrophages, relatively little is known about activation of intrinsic negative regulatory pathways to attenuate excessive inflammatory responses. In this study, we demonstrate that loss of diacylglycerol (DAG) kinase (Dgk) ζ, an enzyme which converts DAG into phosphatidic acid, limits inflammatory cytokine production in an arthritic mouse model dependent on TLR2 signaling and in a CSS mouse model dependent on TLR9 signaling. In vitro, Dgkζ deficiency results in reduced production of TNF-α, IL-6, and IL-1β and in limited M1 macrophage polarization. Mechanistically, Dgkζ deficiency decreases STAT1 and STAT3 phosphorylation. Moreover, Dgkζ levels are increased in macrophages derived from mice with CSS or exposed to plasma from sJIA patients with active disease. Our data suggest that Dgkζ induction in arthritic conditions perpetuates systemic inflammatory responses mediated by macrophages and highlight a potential role of Dgkζ-DAG/phosphatidic acid axis as a modulator of inflammatory cytokine production in sJIA and CSS.

Diacylglycerol kinase ζ is a negative regulator of GPVI-mediated platelet activation

AbstractDiacylglycerol kinases (DGKs) are a family of enzymes that convert diacylglycerol (DAG) into phosphatidic acid (PA). The ζ isoform of DGK (DGKζ) has been reported to inhibit T-cell responsiveness by downregulating intracellular levels of DAG. However, its role in platelet function remains undefined. In this study, we show that DGKζ was expressed at significant levels in both platelets and megakaryocytes and that DGKζ-knockout (DGKζ-KO) mouse platelets were hyperreactive to glycoprotein VI (GPVI) agonists, as assessed by aggregation, spreading, granule secretion, and activation of relevant signal transduction molecules. In contrast, they were less responsive to thrombin. Platelets from DGKζ-KO mice accumulated faster on collagen-coated microfluidic surfaces under conditions of arterial shear and stopped blood flow faster after ferric chloride–induced carotid artery injury. Other measures of hemostasis, as measured by tail bleeding time and rotational thromboelastometry analysis, were normal. Interestingly, DGKζ deficiency led to increased GPVI expression on the platelet and megakaryocyte surfaces without affecting the expression of other platelet surface receptors. These results implicate DGKζ as a novel negative regulator of GPVI-mediated platelet activation that plays an important role in regulating thrombus formation in vivo.

DGKζ deficiency protects against peripheral insulin resistance and improves energy metabolism

Diacylglycerol kinases (DGKs) regulate the balance between diacylglycerol (DAG) and phosphatidic acid. DGKζ is highly abundant in skeletal muscle and induces fiber hypertrophy. We hypothesized that DGKζ influences functional and metabolic adaptations in skeletal muscle and whole-body fuel utilization. DAG content was increased in skeletal muscle and adipose tissue, but unaltered in liver of DGKζ KO mice. Linear growth, body weight, fat mass, and lean mass were reduced in DGKζ KO versus wild-type mice. Conversely, male DGKζ KO and wild-type mice displayed a similar robust increase in plantaris weight after functional overload, suggesting that DGKζ is dispensable for muscle hypertrophy. Although glucose tolerance was similar, insulin levels were reduced in high-fat diet (HFD)-fed DGKζ KO versus wild-type mice. Submaximal insulin-stimulated glucose transport and p-Akt Ser473 were increased, suggesting enhanced skeletal muscle insulin sensitivity. Energy homeostasis was altered in DGKζ KO mice, as evidenced by an elevated respiratory exchange ratio, independent of altered physical activity or food intake. In conclusion, DGKζ deficiency increases tissue DAG content and leads to modest growth retardation, reduced adiposity, and protection against insulin resistance. DGKζ plays a role in the control of growth and metabolic processes, further highlighting specialized functions of DGK isoforms in type 2 diabetes pathophysiology.

Diacylglycerol Kinase ζ Is a Target To Enhance NK Cell Function.

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.

DGKζ Downregulation Enhances Osteoclast Differentiation and Bone Resorption Activity Under Inflammatory Conditions.

Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF-κB ligand (RANKL). RANKL-induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow-derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. J. Cell. Physiol. 232: 617-624, 2017. © 2016 Wiley Periodicals, Inc.

Reciprocal regulation of p53 and NF-κB by diacylglycerol kinase ζ

Diacylglycerol kinase (DGK) participates in lipid mediated-signal transduction. It phosphorylates diacylglycerol (DG) to phosphatidic acid (PA), thereby regulating the balanced control of these second messenger actions. Previous reports have described that one DGK family, DGKζ, is closely involved in stress responses under various conditions. Cellular stress response, a physiological process enabling cells to cope with an altered environment, is finely tuned through various signaling cascades and their molecular crosstalk. The major components of stress response are p53 and NF-κB. p53 generally serves as a proapoptotic transcriptional factor, whereas NF-κB promotes resistance to programmed cell death under most circumstances. Recent studies have suggested that DGKζ facilitates p53 degradation in cytoplasm through ubiquitin proteasome system and that DGKζ deletion upregulates p53 protein levels under basal and DNA-damage conditions. Counter-intuitively, however, DGKζ deletion suppresses p53 transcriptional activity despite increased p53 levels. In contrast, DGKζ knockdown engenders enhancement of NF-κB pathway in response to cytokines such as TNF-α and IL-1β. In response to these cytokines, DGKζ downregulation accelerates phosphorylation of the p65 subunit and its nuclear translocation, thereby enhancing NF-κB transcriptional activity. Furthermore, DGKζ deficiency is shown to promote increased association of p65 subunit with the transcriptional cofactor CBP. It is particularly interesting that this association is observed even under basal conditions in the absence of stimulation. These findings suggest that DGKζ plays a role in sequestration of the limiting pool of CBP/p300 between the NF-κB p65 subunit and p53, and that DGKζ downregulation shifts CBP/p300 toward the NF-κB subunit to regulate reciprocally antagonistic phenotypes of these transcription factors.

Diacylglycerol Kinase ζ (DGKζ) Is a Critical Regulator of Bone Homeostasis Via Modulation of c-Fos Levels in Osteoclasts.

Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.

Diacylglycerol Kinase Zeta Positively Controls the Development of iNKT-17 Cells

Invariant natural killer T (iNKT) cells play important roles in bridging innate and adaptive immunity via rapidly producing a variety of cytokines. A small subset of iNKT cells produces IL-17 and is generated in the thymus during iNKT-cell ontogeny. The mechanisms that control the development of these IL-17-producing iNKT-17 cells (iNKT-17) are still not well defined. Diacylglycerol kinase ζ (DGKζ) belongs to a family of enzymes that catalyze the phosphorylation and conversion of diacylglycerol to phosphatidic acid, two important second messengers involved in signaling from numerous receptors. We report here that DGKζ plays an important role in iNKT-17 development. A deficiency of DGKζ in mice causes a significant reduction of iNKT-17 cells, which is correlated with decreased RORγt and IL-23 receptor expression. Interestingly, iNKT-17 defects caused by DGKζ deficiency can be corrected in chimeric mice reconstituted with mixed wild-type and DGKζ-deficient bone marrow cells. Taken together, our data identify DGKζ as an important regulator of iNKT-17 development through iNKT-cell extrinsic mechanisms.

Decreased Diacylglycerol Metabolism Enhances ERK Activation and Augments CD8+ T Cell Functional Responses

Modulation of T cell receptor signal transduction in CD8(+) T cells represents a novel strategy toward enhancing the immune response to tumor. Recently, levels of guanine exchange factors, RasGRP and SOS, within T cells have been shown to represent a key determinant in the regulation of the analog to the digital activation threshold of Ras. One important for regulating activation levels of RasGRP is diacylglycerol (DAG), and its levels are influenced by diacylglycerol kinase-ζ (DGKζ), which metabolizes DAG into phosphatidic acid, terminating DAG-mediated Ras signaling. We sought to determine whether DGKζ-deficient CD8(+) T cells demonstrated enhanced in vitro responses in a manner predicted by the current model of Ras activation and to evaluate whether targeting this threshold confers enhanced CD8(+) T cell responsiveness to tumor. We observed that DGKζ-deficient CD8(+) T cells conform to most predictions of the current model of how RasGRP levels influence Ras activation. But our results differ in that the EC(50) value of stimulation is not altered for any T cell receptor stimulus, a finding that suggests a further degree of complexity to how DGKζ deficiency affects signals important for Ras and ERK activation. Additionally, we found that DGKζ-deficient CD8(+) T cells demonstrate enhanced responsiveness in a subcutaneous lymphoma model, implicating the analog to a digital conversion threshold as a novel target for potential therapeutic manipulation.

Diacylglycerol kinase zeta regulates microbial recognition and host resistance to Toxoplasma gondii (51.16)

Abstract Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting diacylglycerol (DAG) to phosphatidic acid (PA). Here, we demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mϕ) and dendritic cells (DC). DGKζ deficiency results in impaired IL-12 and TNFα production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the PI3K-Akt pathway and that inhibition of PI3K activity can restore LPS-induced IL-12 production by DGKζ deficient macrophages. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.

Diacylglycerol kinase ζ regulates microbial recognition and host resistance to Toxoplasma gondii

Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol (DAG) kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting DAG to phosphatidic acid (PA). We demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mφ) and dendritic cells. DGKζ deficiency results in impaired interleukin (IL) 12 and tumor necrosis factor α production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the phosphatidylinositol 3–kinase (PI3K)–Akt pathway and that inhibition of PI3K activity or treatment with PA can restore lipopolysaccharide-induced IL-12 production by DGKζ-deficient Mφ. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.