Anergic T cells have altered diacylglycerol metabolism, but whether that altered metabolism has a causative function in the induction of T cell anergy is not apparent. To test the importance of diacylglycerol metabolism in T cell anergy, we manipulated diacylglycerol kinases (DGKs), which are enzymes that terminate diacylglycerol-dependent signaling. Overexpression of DGK-alpha resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-alpha-deficient mice and found that DGK-alpha-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-alpha-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-alpha or DGK-zeta proliferated and produced interleukin 2. Pharmacological inhibition of DGK-alpha activity in DGK-zeta-deficient T cells that received an anergizing stimulus proliferated similarly to wild-type T cells that received CD28 costimulation and prevented anergy induction. Our findings suggest that regulation of diacylglycerol metabolism is critical in determining whether activation or anergy ensues after T cell receptor stimulation.

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK ␣ and synergistically promote T cell maturation in the thymus. Absence of both DGK␣ and (DGK␣ ؊/؊ ؊/؊ ) results in a severe decrease in the number of CD4 ؉ CD8 ؊ and CD4 ؊ CD8 ؉ single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGK␣ ؊/؊ ؊/؊ mice. The developmental blockage in DGK␣ ؊/؊ ؊/؊ mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.phosphatidic acid ͉ signaling ͉ tumorigenesis D iacylglycerol (DAG) kinases (DGKs) are a family of enzymes that catalyze phosphorylation of DAG, converting it to phosphatidic acid (PA). Ten DGK isoforms have been identified in mammals and are divided into 5 subtypes based on unique structural features (1, 2). Within a single tissue, multiple DGK isoforms can be expressed. A notable feature of the DGK-mediated reaction is that both the substrate, DAG, and the product, PA, can be important second messengers. DAG can associate with Ras guanyl nucleotide-releasing proteins (RasGRPs), protein kinase Cs (PKCs), protein kinase Ds, chimaerins, and Munc-13s through their cysteine-rich (C1) domains (3). PA has been reported to bind to the SH-2 domain containing tyrosine phosphatase-1 (SHP-1), mammalian target of Rapamycin (mTOR), cAMP-specific phosphodiesterase 4 (PDE4), protein phosphatase-1 (PP-1), son of sevenless (Sos), and type I phosphatidylinositol 4-phosphate 5-kinase (PI5K) (4 -8). Through the regulation of the activities and subcellular localizations of these signaling molecules, DAG and PA play critical roles in signaling from many cell surface receptors (3, 4). It has been hypothesized that DGKs act as crucial regulators of receptor signaling and cellular function by modulating DAG and PA concentrations. However, the physiologic importance of most DGKs and the role of DGK-derived PA in receptor signaling have been poorly defined.T cell maturation in the thymus occurs through CD4 Ϫ CD8 Ϫ double-negative (DN) to CD4 ϩ CD8 ϩ double-positive (DP) and finally to the CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ single-positive (SP) stage (9). Engagement of TCR expressed on DP thymocytes with self-peptide presented by MHCs on thymic stromal cells and bone marrow-derived dendritic cells induces intracellular signaling that can lead to either maturation (positive selection) or cell death (negative selection). In general, TCRs with high affinity to self-antigens elicit strong signals directing negative selection, whereas TCRs with low affinity to self-antigens induce...

. In peripheral lymphoid organs, engagement of the TCR by antigenic peptide presented by major histocompatibility complex molecules on antigen presenting cells initiates a signaling cascade that is required for T cell proliferation and effector function (1, 5). Abnormalities in signals delivered by the TCR may result in either hypo-or hyperactivation leading to undesirable outcomes such as immune deficiency (6 -9) or autoimmunity (10 -12). Engagement of the TCR initiates numerous second messenger cascades. The most proximal known biochemical signal is stimulation of protein tyrosine kinases with subsequent tyrosine phosphorylation of multiple substrates (5,13,14). Among these substrates are phospholipase C␥1 (PLC␥1) (15-17) and critical adapters including linker of activated T cells (18) and SLP-76 (SH2 domain containing leukocyte phosphoprotein of 76 kDa) (19). These proteins in association with another adapter protein, Grb2-related adapter molecule downstream of , are part of a larger multimolecular complex required for PLC␥1 to act efficiently on its substrate (23-28). Activated PLC␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (29). Inositol 1,4,5-trisphosphate induces an influx of Ca 2ϩ that activates the calcineurinnuclear factor of activated T cells (NFAT) signaling pathway (30). DAG allosterically activates both RasGRP (31, 32), a nucleotide exchange factor for Ras, and PKC (33), a serine (threonine) kinase, through associating with the C1 domains of both molecules. Activated RasGRP (34, 35) and PKC (36, 37) in turn stimulate the Ras-ERK-AP-1 and NFB pathways, respectively. Propagation of these signaling cascades promotes the transcription of numerous genes including those encoding cytokines critical for T cell development, activation, and proliferation. Deficiencies of RasGRP (35) and PKC (37) cause defects in positive selection in developing thymocytes and inefficient activation of peripheral T cells. Similarly, PLC␥1 deficiency (with consequent defects in phosphatidylinositol 4,5-bisphosphate-derived second messenger production) abrogates TCR-mediated cellular activation in the Jurkat T cell

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGK initiates RhoGDI release and Rac1 activation. In DGK-deficient fibroblasts PAK1 phosphorylation and Rac1-RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGK, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGK stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGK, and PA to the activation of Rac1: the PA generated by DGK activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility. INTRODUCTIONRho GTPases regulate gene transcription, cell cycle progression, vesicular traffic, and cell polarity (Jaffe and Hall, 2005;Ridley, 2006) but are best known for their ability to coordinate alterations in cellular actin networks that regulate cell morphology. Such changes are necessary for directed cell migration during embryogenesis, inflammation, wound healing, and tumor metastasis (Burridge and Wennerberg, 2004). In mammalian cells, Rac1 promotes actin polymerization and focal complex assembly, leading to lamellipodia protrusion and membrane ruffle formation; Cdc42 regulates filopodial extension; and Rho promotes the assembly of actin stress fibers and focal adhesions (Ridley et al., 1992;Nobes and Hall, 1995).All Rho GTPases cycle between inactive GDP-bound and active GTP-bound conformations; the active versions interact with specific downstream effectors to elicit distinct biological responses. This cycle is tightly regulated by guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), and by GTPaseactivating proteins (GAPs), which inactivate Rho proteins by enhancing their intrinsic GTPase activity. A third class of proteins, guanine nucleotide dissociation inhibitors (GDIs), regulates Rho GTPase function in as many as three distinct ways. RhoGDI, the best characterized member, 1) prevents GDP dissociation, 2) inhibits intrinsic or GAP-stimulated GTP hydrolysis (Chuang et al., 1993b), and 3) regulates GTPase partitioning between the cytosol and plasma membrane (Olofsson, 1999). The latter task is accomplished by sequestering the GTPases as soluble cytosolic complexes in which the C-terminal membrane-tar...

BackgroundOur previous study found that suppression of TRPM7 reduced neuronal death in adult rat ischemic brain injury. It was reported that carvacrol blocked TRPM7 and attenuated brain injury in an adult rat MCAO model. The effects of carvacrol on neonatal stroke remain unknown. This study investigated the effects of carvacrol on neuronal injury and behavioral impairment after hypoxia-ischemia in neonatal mice and the potential signaling pathway underlying these effects.ResultsCarvacrol inhibited TRPM7 current in HEK293 cells over-expressing TRPM7 and TRPM7-like current in hippocampal neurons in a dose-dependent manner. Carvacrol (>200 μM) reduced OGD-induced neuronal injury in cortical neurons. 24 hours after HI, TRPM7 protein level in the ipsilateral hemisphere was significantly higher than in the contralateral hemisphere. Carvacrol (30 and 50 mg/kg) pre-treatment reduced brain infarct volume 24 hours after HI in a dose-dependent manner. Carvacrol pre-treatment also improved neurobehavioral outcomes. Furthermore, animals pre-treated with carvacrol had fewer TUNEL-positive cells in the brain compared to vehicle-treated animals 3 days after HI. Carvacrol pre-treatment also increased Bcl-2/Bax and p-Akt/t-Akt protein ratios and decreased cleaved caspase-3 protein expression 24 hours after HI.ConclusionsCarvacrol pre-treatment protects against neonatal hypoxic-ischemic brain injury by reducing brain infarct volume, promoting pro-survival signaling and inhibiting pro-apoptotic signaling, as well as improving behavioral outcomes. The neuroprotective effect may be mediated by the inhibition of TRPM7 channel function. Carvacrol is a potential drug development target for the treatment of neonatal stroke.

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IKKα is a critical negative regulator of ASC-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKKi facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

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Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKf is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKf in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKf knockdown reduces spine density. Mice deficient in DGKf expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKf is required for spine maintenance but not formation. We propose that PSD-95 targets DGKf to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.