Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis.

Tumor-associated macrophages (TAMs) are often associated with chemoresistance and resultant poor clinical outcome in solid tumors. Here, we demonstrated that TAMs-released chemokine-C-C motif chemokine 22 (CCL22) in esophageal squamous cell carcinoma (ESCC) stroma was tightly correlated with the chemoresistance of ESCC patients. TAMs-secreted CCL22 was able to block the growth inhibitory and apoptosis-promoting effects of cisplatin on ESCC cells. Mechanistically, CCL22 stimulated intratumoral diacylglycerol kinase α (DGKα) to produce phosphatidic acid (PA), which suppressed the activity of NADPH oxidase 4 (NOX4) and then blocked the overproduction of intratumoral reactive species oxygen (ROS) induced by cisplatin. CCL22 activated DGKα/nuclear factor-κB (NF-κB) axis to upregulate the level of several members of ATP binding cassette (ABC) transporter superfamily, including ABC sub-family G member 4 (ABCG4), ABC sub-family A member 3 (ABCA3), and ABC sub-family A member 5 (ABCA5), to lower the intratumoral concentration of cisplatin. Consequently, these processes induced the cisplatin resistance in ESCC cells. In xenografted models, targeting DGKα with 5'-cholesterol-conjugated small-interfering (si) RNA enhanced the chemosensitivity of cisplatin in ESCC treatment, especially in the context of TAMs. Our data establish the correlation between the TAMs-induced intratumoral metabolic product/ROS axis and chemotherapy efficacy in ESCC treatment and reveal relevant molecular mechanisms.

Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

BACKGROUND: Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I). METHODS: We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E2 (PGE2) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed. RESULTS: DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE2 secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE2-mediated activation of PKA signaling by DGK inhibition. CONCLUSIONS: Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE2-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.

Myristic acid selectively augments ß-tubulin levels in C2C12 myotubes via diacylglycerol kinase δ.

Effective amelioration of type II diabetes requires therapies that increase both glucose uptake activity per cell and skeletal muscle mass. Myristic acid (14:0) increases diacylglycerol kinase (DGK) δ protein levels and enhances glucose uptake in myotubes in a DGKδ-dependent manner. However, it is still unclear whether myristic acid treatment affects skeletal muscle mass. In this study, we found that myristic acid treatment increased the protein level of ß-tubulin, which constitutes microtubules and is closely related to muscle mass, in C2C12 myotubes but not in the proliferation stage in C2C12 myoblasts. However, lauric (12:0), palmitic (16:0) and oleic (18:1) acids failed to affect DGKδ and ß-tubulin protein levels in C2C12 myotubes. Moreover, knockdown of DGKδ by siRNA significantly inhibited the increased protein level of ß-tubulin in the presence of myristic acid, suggesting that the increase in ß-tubulin protein by myristic acid depends on DGKδ. These results indicate that myristic acid selectively affects ß-tubulin protein levels in C2C12 myotubes via DGKδ, suggesting that this fatty acid improves skeletal muscle mass in addition to increasing glucose uptake activity per cell.

mTORC1 is involved in DGKß-induced neurite outgrowth and spinogenesis.

Diacylglycerol kinase ß (DGKß) is an enzyme converting DG to phosphatidic acid (PA) and is specifically expressed in neurons, especially those in the cerebral cortex, hippocampus and striatum. We previously reported that DGKß induces neurite outgrowth and spinogenesis, contributing to higher brain function including emotion and memory, and plasma membrane localization of DGKß via the C1 domain and a cluster of basic amino acids at the C-terminus is necessary for its function. To clarify the mechanisms involved in neuronal development by DGKß, we investigated whether DGKß activity induces neurite outgrowth using human neuroblastoma SH-SY5Y cells. DGKß induced neurite outgrowth by activation of mammalian target of rapamycin complex 1 (mTORC1) through a kinase-dependent pathway. In addition, in primary cultured cortical and hippocampal neurons, inhibition of mTORC1 abolished DGKß induced-neurite outgrowth, branching and spinogenesis. These results indicated that DGKß induces neurite outgrowth and spinogenesis by activating mTORC1 in a kinase-dependent pathway.

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

Diacylglycerol 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.

Diacylglycerol kinase zeta inhibits G(alpha)q-induced atrial remodeling in transgenic mice.

BACKGROUND: Our previous study showed that diacylglycerol kinase zeta (DGKzeta), which degenerates diacylglycerol (DAG), inhibits ventricular structural remodeling and rescues activated G protein (alpha)q (G(alpha)q)-induced heart failure. However, whether DGKzeta inhibits atrial remodeling is still unknown. OBJECTIVE: This study aimed to elucidate the effects of DGKzeta on atrial remodeling. METHODS: A transgenic mouse (G(alpha)q-TG) with cardiac expression of activated G(alpha)q and a double transgenic mouse (G(alpha)q/DGKzeta-TG) with cardiac overexpression of DGKzeta and activated G(alpha)q were created. RESULTS: During electrocardiogram (ECG) recording for 10 min, atrial fibrillation was observed in 5 of 11 anesthetized G(alpha)q-TG mice but not in any wild-type (WT) and G(alpha)q/DGKzeta-TG mice (P <.05). All of the ECG parameters measured were prolonged in the G(alpha)q-TG compared with WT mice. Interestingly, in G(alpha)q/DGKzeta-TG mice, although the PR and RR intervals were still prolonged, the P interval, QRS complex, and QT interval were not different from those in WT mice. In Langendorff-perfused hearts, the incidence of atrial tachyarrhythmia induced by rapid atrial pacing was greater in G(alpha)q-TG hearts than in G(alpha)q/DGKzeta-TG hearts (P <.05). Action potential duration prolongation and impulse conduction slowing were observed in G(alpha)q-TG atria compared with G(alpha)q/DGKzeta-TG atria. Dilatation of the left atrium with thrombus formation was observed in 9 G(alpha)q-TG hearts but not in any G(alpha)q/DGKzeta-TG hearts. Moreover, the degree of extensive interstitial fibrosis in the left atrium was greater in G(alpha)q-TG hearts than that in G(alpha)q/DGKzeta-TG hearts (P <.05). CONCLUSION: These results show that DGKzeta inhibits G(alpha)q-induced atrial remodeling and suggest that DGKzeta is a novel therapeutic target for atrial fibrillation.

Receptores de feromonas de mamíferos: supervivencia y sexualidad / Mammalian pheromone receptors: survival and sexuality

En 1995 Catherine Dulac y Richard Axel publicaron la existencia de una nueva familia de genes que codificaban los posibles receptores de feromonas, pertenecientes a la amplia familia de los de siete hélices transmembranares y acoplados a proteínas G. Desde entonces se han clonado nuevos genes que han sido agrupados en dos familias, los receptores vomeronasales tipo 1 y 2, V1R y V2R, con diferente estructura y situados con diferente distribución en el órgano vomeronasal. La naturaleza química de las feromonas y de las proteínas que las asocian y transportan conocidas como lipocalinas es otro de los aspectos de los que se dispone de abundante información. Los mecanismos de transducción de la señal mediada por feromonas sobre los receptores V1R y V2R implican la activación de la fosfolipasa C tipo β2, PLCβ2, generando el fosfatidilinositol trifosfato y el diacilglicerol en la cara interna de la membrana neuronal. El diacilglicerol es un ligando endógeno, que permite la apertura del canal de la familia TRPC (Transient Receptor Potential Channel) denominado TRPC2 que se abre y deja pasar iones Ca2+ y Na+ al interior de la neurona sensorial, iniciando la despolarización de la membrana y originando el potencial de acción. La señal eléctrica es conducida al bulbo olfativo auxiliar por axones que llegan de modo disperso y establecen conexión con las células mitrales, las cuales envían sus prolongaciones hasta el sistema límbico y otras estructuras cerebrales, donde influencian o provocan las respuestas de supervivencia de la especie, entre ellas las de apareamiento y agresividad. Un aspecto relevante desde el punto de vista evolutivo es que en primates el gen TRPC2 es un pseudogen sin funcionalidad y por lo tanto el órgano vomeronasal es un vestigio carente de función. Recientes estudios indican que la captación de feromonas en primates se realiza a través del epitelio olfativo y el bulbo olfativo principal e incluso en otros mamíferos esta estructura parece mediar en algunas respuestas especie específicas

In 1995 Catherine Dulac and Richard Axel discovered a new gene family corresponding to the pheromone receptors. They were members of the seven transmembrane helix coupled to G proteins. Since then, new genes have been clonned and grouped according their sequence homology in two main families of vomeronasal receptors the V1R and the V2R. They exhibit different distribution pattern at the vomeronasal epithelium, where they are coupled to different G proteins. The chemical nature of the mammalian pheromones is very diverse and can associate with proteins called lipocalins to reach the vomeronasal organ. The transduction mechanisms of pheromone receptors, V1R and V2R, require respectively a Gi and a Go proteins, to further activate a phospholipase C, the PLCβ2. This enzyme hydrolyses the phosphatidyl inositol located at the plasma membrane originating phosphatidylinositol triphosphate and diacylglycerol. Diacylglycerol is an endogenous ligand that opens the TRPC2 channel (Transient Receptor Potential Channel), allowing the entrance of cations, mostly Ca2+ y Na+. The membrane depolarisation at the vomeronasal neuron originates the action potential that is sent to the accessory olfactory bulb by the axon, which in a different way as those from the main olfactory epithelia, do not organise the axonal prolongations and reach the mitral neurones in a disperse way, without forming a glomerular structure, afterwards the mitral cells send their axons to the limbic system and other cerebral structures related to aggressive behaviour and mating. It is relevant to underline that in monkeys from the old world and primates including humans, the vomeronasal organ is only a vestigial structure without function. The reason relies on the TRPC2 gene, which is a pseudo gene, without physiological function. Recent experimental approaches have demonstrated that the sensing of some pheromonal signals in these species, and also in mammals with a functional vomeronasal organ, can be carried out by the main olfactory epithelia through the main olfactory bulb. This structure being also connected to the hypothalamus, where neurones releasing LHRH can control sexual behaviour. These data confirm the broad possibilities of signalling through pheromones and that much effort is still required to fully understand their possibilities

Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice.

BACKGROUND: Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. METHODS AND RESULTS: To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. CONCLUSIONS: These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

A traditional herbal medicine, rikkunshi-to (TJ-43), prevents intracellular signaling disorders in gastric smooth muscle of diabetic rats.

Prevention of diabetic gastrointestinal dysfunction is of utmost importance. The present study demonstrated that diacylglycerol kinase (DGK) activity in diabetic gastric smooth muscle in the resting state was approximately 3.5-fold greater than that in controls. However, oral administration of TJ-43 (1% of food intake) or subcutaneous insulin injection (12 units/kg/day) in streptozotocin-induced diabetic rats (DM) for 2 weeks prevented DGK abnormalities based on the control level. Increased DGK activity in the resting state of DM was inhibited significantly by R59022, neomycin or staurosporine; in contrast, these drugs did not affect DGK activity in controls, insulin-treated DM or TJ-43-treated DM. In controls, the endogenous phosphatidic acid (PA) level was inhibited significantly by R59022 or neomycin but not affected by staurosporine. On the other hand, these three drugs significantly inhibited endogenous PA levels in DM, and neomycin significantly inhibited endogenous PA levels in insulin-treated and TJ-43-treated DM. This suggests that TJ-43 could prevent alteration of DGK activity and PA formation without reduction of blood glucose levels. Moreover, these effects were greater than those of insulin treatment. Results suggested that TJ-43 treatment influenced the hyperreactivity of DGK and DAG formation via phospholipase C activity. In conclusion, TJ-43 can be recommended with respect to enhancement of the quality of life in patients displaying diabetic gastrointestinal complications.

Do phosphatidylinositides modulate vertebrate phototransduction?

Mammalian rod cyclic nucleotide gated (CNG) channels (i.e., alpha plus beta subunits) are strongly inhibited by phosphatidylinositol 4, 5-bisphosphate (PIP(2)) when they are expressed in Xenopus oocytes and studied in giant membrane patches. Cytoplasmic Mg-ATP inhibits CNG currents similarly, and monoclonal antibodies to PIP(2) reverse the effect and hyperactivate currents. When alpha subunits are expressed alone, PIP(2) inhibition is less strong; olfactory CNG channels are not inhibited. In giant patches from rod outer segments, inhibition by PIP(2) is intermediate. Other anionic lipids (e.g., phosphatidyl serine and phosphatidic acid), a phosphatidylinositol-specific phospholipase C, and full-length diacylglycerol have stimulatory effects. Although ATP also potently inhibits cGMP-activated currents in rod patches, the following findings indicate that ATP is used to transphosphorylate GMP, generated from cGMP, to GTP. First, a phosphodiesterase (PDE) inhibitor, Zaprinast, blocks inhibition by ATP. Second, inhibition can be rapidly reversed by exogenous regulator of G-protein signaling 9, suggesting G-protein activation by ATP. Third, the reversal of ATP effects is greatly slowed when cyclic inosine 5'-monophosphate is used to activate currents, as expected for slow inosine 5' triphosphate hydrolysis by G-proteins. Still, other results remain suggestive of regulatory roles for PIP(2). First, the cGMP concentration producing half-maximal CNG channel activity (K(1/2)) is decreased by PIP(2) antibody in the presence of PDE inhibitors. Second, the activation of PDE activity by several nucleotides, monitored electrophysiologically and biochemically, is reversed by PIP(2) antibody. Third, exogenous PIP(2) can enhance PDE activation by nucleotides.