Diacylglycerol kinase ε localizes to subsurface cisterns of cerebellar Purkinje cells.

Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.

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.

Diacylglycerol kinase epsilon suppresses expression of p53 and glycerol kinase in mouse embryo fibroblasts.

The incorporation of glycerol into lipid was measured using SV40 transformed mouse embryo fibroblasts (MEFs) from either wild-type (WT) mice or from mice in which the epsilon isoform of diacylglycerol kinase (DGKε) was knocked out (DGKε-/-). We present an explanation for our finding that DGKε-/- MEFs exhibited greater uptake of 3H-glycerol into the cell and a greater incorporation into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε-/- and WT MEFs. Glycerol kinase is more highly expressed in the DGKε-/- cells than in their WT counterparts. In addition, the activity of glycerol kinase is greater in the DGKε-/- cells than in their WT counterparts. Other substrates that enter the cell independent of glycerol kinase, such as pyruvate or acetate, are incorporated into lipid to the same extent between DGKε-/- and WT cell lines. We also show that expression of p53, a transcription factor that increases the synthesis of glycerol kinase, is increased in DGKε-/- MEFs in comparison to WT cells. We conclude that the increased incorporation of glycerol into lipids in DGKε-/- cells is a consequence of up-regulation of glycerol kinase and not a result of an increase in the rate of lipid synthesis. Furthermore, increased expression of the pro-survival gene, p53, in cells knocked out for DGKε suggests that cells over-expressing DGKε would have a greater propensity to become tumorigenic.

Effect of Sulindac and Erlotinib vs Placebo on Duodenal Neoplasia in Familial Adenomatous Polyposis: A Randomized Clinical Trial.

IMPORTANCE: Patients with familial adenomatous polyposis (FAP) are at markedly increased risk for duodenal polyps and cancer. Surgical and endoscopic management of duodenal neoplasia is difficult and chemoprevention has not been successful. OBJECTIVE: To evaluate the effect of a combination of sulindac and erlotinib on duodenal adenoma regression in patients with FAP. DESIGN, SETTING, AND PARTICIPANTS: Double-blind, randomized, placebo-controlled trial, enrolling 92 participants with FAP, conducted from July 2010 through June 2014 at Huntsman Cancer Institute in Salt Lake City, Utah. INTERVENTIONS: Participants with FAP were randomized to sulindac (150 mg) twice daily and erlotinib (75 mg) daily (n = 46) vs placebo (n = 46) for 6 months. MAIN OUTCOMES AND MEASURES: The total number and diameter of polyps in the proximal duodenum were mapped at baseline and 6 months. The primary outcome was change in total polyp burden at 6 months. Polyp burden was calculated as the sum of the diameters of polyps. The secondary outcomes were change in total duodenal polyp count, change in duodenal polyp burden or count stratified by genotype and initial polyp burden, and percentage of change from baseline in duodenal polyp burden. RESULTS: Ninety-two participants (mean age, 41 years [range, 24-55]; women, 56 [61%]) were randomized when the trial was stopped by the external data and safety monitoring board because the second preplanned interim analysis met the prespecified stopping rule for superiority. Grade 1 and 2 adverse events were more common in the sulindac-erlotinib group, with an acne-like rash observed in 87% of participants receiving treatment and 20% of participants receiving placebo (P < .001). Only 2 participants experienced grade 3 adverse events. [table: see text]. CONCLUSIONS AND RELEVANCE: Among participants with FAP, the use of sulindac and erlotinib compared with placebo resulted in a lower duodenal polyp burden after 6 months. Adverse events may limit the use of these medications at the doses used in this study. Further research is necessary to evaluate these preliminary findings in a larger study population with longer follow-up to determine whether the observed effects will result in improved clinical outcomes. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT 01187901.

Loss of diacylglycerol kinase epsilon in mice causes endothelial distress and impairs glomerular Cox-2 and PGE2 production.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε (DGKE) that encodes the lipid kinase DGKε (Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531-536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377-384, 2013). DGKε is unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2 are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2 supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Downregulation of diacylglycerol kinase ζ enhances activation of cytokine-induced NF-κB signaling pathway.

The transcription factor NF-κB family serves as a key component of many pathophysiological events such as innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Various cell signals trigger activation of the regulatory mechanisms of NF-κB, resulting in its nuclear translocation and transcriptional initiation. The diacylglycerol kinase (DGK) family, a lipid second messenger-metabolizing enzyme in phosphoinositide signaling, is shown to regulate widely various cellular processes. Results of recent studies suggest that one family member, DGKζ, is closely involved in immune and inflammatory responses. Nevertheless, little is known about the regulatory mechanism of DGKζ on NF-κB pathway in cytokine-induced inflammatory signaling. This study shows that siRNA-mediated DGKζ knockdown in HeLa cells facilitates degradation of IκB, followed by nuclear translocation of NF-κB p65 subunit. In addition, DGKζ-deficient MEFs show upregulation of p65 subunit phosphorylation at Serine 468 and 536 and its interaction with CBP transcriptional coactivator upon TNF-α stimulation. These modifications of p65 subunit might engender enhanced NF-κB transcriptional reporter assay of DGKζ knockdown cells. These findings provide further insight into the regulatory mechanisms of cytokine-induced NF-κB activation.

Regulation of sonic hedgehog expression by integrin ß1 and epidermal growth factor receptor in intestinal epithelium.

We previously found that conditional deletion of integrin ß1 in intestinal epithelium of mice caused early postnatal lethality and intestinal phenotypic changes including excessive proliferation and defective differentiation of intestinal epithelium due to loss of Hedgehog expression. Here, we link these defects to the Hedgehog (Hh) signaling pathway and show that loss of integrin ß1 leads to excessive phosphorylation of MEK-1 and increased expression of ErbB receptors, including the epidermal growth factor receptor (EGFR). We show that increased EGFR signaling attenuates Hh abundance and that an EGFR inhibitor rescues conditional ß1 integrin null pups from postnatal lethality. These studies link the loss of Hh expression in the intestinal epithelium of integrin ß1-deficient mice to excessive EGFR/MAPK signaling, and identify a unique mechanism for crosstalk between stromal and epithelial signaling pathways that is critical for intestinal epithelial differentiation and function.

DGKζ under stress conditions: "to be nuclear or cytoplasmic, that is the question".

Eukaryotic cells have evolved to possess a distinct subcellular compartment, the nucleus, separated from the cytoplasm in a manner that allows the precise operation of the chromatin, thereby permitting controlled access to the regulatory elements in the DNA for transcription and replication. In the cytoplasm, genetic information contained in the DNA sequence is translated into proteins, including enzymes that catalyze various reactions, such as metabolic processes, energy control, and responses to changing environments. One mechanism that regulates these events involves phosphoinositide turnover signaling, which generates a lipid second messenger, diacylglycerol (DG). Since DG acts as a potent activator of several signaling molecules, it should be tightly regulated to keep cellular responsiveness within a physiological range. DG kinase (DGK) metabolizes DG by phosphorylating it to generate phosphatidic acid, thus serving as a critical regulator of DG signaling. Phosphoinositide turnover is employed differentially in the nucleus and the cytoplasm. A member of the DGK family, DGKζ, localizes to the nucleus in various cell types and is considered to regulate nuclear DG signaling. Recent studies have provided evidence that DGKζ shuttles between the nucleus and the cytoplasm in neurons under pathophysiological conditions. Transport of a signal regulator between the nucleus and the cytoplasm should be a critical function for maintaining basic processes in the nucleus, such as cell cycle regulation and gene expression, and to ensure communication between nuclear processes and cytoplasmic functions. In this review, a series of studies on nucleocytoplasmic translocation of DGKζ have been summarized, and the functional implications of this phenomenon in postmitotic neurons and cancer cells under stress conditions are discussed.

Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-ε expression.

IFN-ε is a unique type I IFN whose constitutive expression in lung, brain, small intestine, and reproductive tissues is only partially understood. Our previous observation that posttranscriptional events participate in the regulation of IFN-ε mRNA expression led us to investigate whether the 5' and/or 3' untranslated regions (UTR) have regulatory functions. Surprisingly, we found that full-length IFN-ε 5'UTR markedly suppressed mRNA expression under basal conditions. Analysis of the secondary structure of this region predicted formation of two stable stem-loop structures, loops 1 and 2. Studies using luciferase constructs harboring various stretches of IFN-ε 5'UTR and mutant constructs in which the conformation of loop structures was disrupted showed that loop 1 is essential for regulation of mRNA expression. Incubation of HeLa cell extracts with agarose-bound RNAs harboring IFN-ε loop structures identified importin 9 (IPO9), a molecular transporter and chaperone, as a candidate that associates with these regions of the 5'UTR. IPO9 overexpression decreased, and IPO9 silencing increased basal IFN-ε expression. Our studies uncover a previously undescribed function for IPO9 as a specific, and negative, posttranscriptional regulator of IFN-ε expression, and they identify key roles for IFN-ε stem-loop structure 1 in this process. IPO9-mediated effects on 5'UTRs appear to extend to additional mRNAs, including hypoxia-inducible factor-1α, that can form specific loop structures.

Cytoplasmic localization of DGKζ exerts a protective effect against p53-mediated cytotoxicity.

The transcription factor p53 plays a crucial role in coordinating the cellular response to various stresses. Therefore, p53 protein levels and activity need to be kept under tight control. We report here that diacylglycerol kinase ζ (DGKζ) binds to p53 and modulates its function both in the cytoplasm and nucleus. DGKζ, a member of the DGK family that metabolizes a lipid second messenger diacylglycerol, localizes primarily to the nucleus in various cell types. Recently, reports have described that excitotoxic stress induces DGKζ nucleocytoplasmic translocation in hippocampal neurons. In the study reported here we found that cytoplasmic DGKζ attenuates p53-mediated cytotoxicity against doxorubicin-induced DNA damage by facilitating cytoplasmic anchoring and degradation of p53 through a ubiquitin-proteasome system. Concomitantly, decreased levels of nuclear DGKζ engender downregulation of p53 transcriptional activity. Consistent with these in vitro cellular experiments, DGKζ-deficient brain exhibits high levels of p53 protein after kainate-induced seizures and even under normal conditions. These findings provide novel insights into the regulation of p53 function and suggest that DGKζ serves as a sentinel to control p53 function both during normal homeostasis and in stress responses.

Deficiency of phospholipase A2 group 7 decreases intestinal polyposis and colon tumorigenesis in Apc(Min/+) mice.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that mediates diverse effects such as physiological and pathological inflammation, immunosuppression, and cancer. Several lines of evidence support both positive and negative roles for PAF in carcinogenesis. PAF stimulates cell growth, oncogenic transformation, and metastasis, but can also limit proliferation and induce apoptosis. The biological context and microenvironment seem to define whether PAF has pro- or anticarcinogenic effects. To investigate the role of exacerbated PAF signaling in colon cancer, we conducted cell-based and in vivo studies using genetically engineered mice lacking expression of phospholipase A2 group 7 (PLA2G7), an enzyme that specifically metabolizes PAF and structurally related glycerophospholipids. Absence of Pla2g7 robustly decreased intestinal polyposis and colon tumor formation in Apc(Min)(/+) mice, suggesting an antitumorigenic role for PAF in settings characterized by aberrant function of the tumor suppressor Adenomatous polyposis coli (Apc). In colonic epithelial cells, exposure to a PAF analog led to dephosphorylation of Akt at serine-473 and induction of apoptosis. The mechanism of this response involved formation of a complex between ß-arrestin 1 and the Akt phosphatase PHLPP2, and activation of the intrinsic pathway of apoptosis. Our results suggest that strategies based on inhibiting PLA2G7 activity or increasing PAF-mediated signaling hold promise for the treatment of intestinal malignancies that harbor mutations in APC.

The basis of the substrate specificity of the epsilon isoform of human diacylglycerol kinase is not a consequence of competing hydrolysis of ATP.

The diacylglycerol kinase from E. coli transfers some of the γ-phosphate of ATP to water as well as to diacylglycerol. We also demonstrate that glycerol can act as an acceptor for the phosphate of ATP. We have compared this behavior with that of the only mammalian isoform of diacylglycerol kinase that exhibits acyl chain specificity, i.e. DGKɛ. The purpose of the study was to determine if differences in the competition between ATPase activity and lipid phosphorylation could contribute to the observed acyl chain specificity with different diacylglycerols. Neither with the highly specific substrate of DGKɛ, 1-stearoyl-2-arachidonoyl glycerol, nor with a less specific substrate, 1-stearoyl-2-linoleoyl glycerol, is there any evidence for ATP hydrolysis accompanying substrate phosphorylation. Thus, at least for this isoform of diacylglycerol kinase, water does not compete with diacylglycerol as an acceptor of the γ-phosphate of ATP. The results demonstrate that the substrate specificity of mammalian DGKɛ is not a consequence of different degrees of ATP hydrolysis in the presence of different species of diacylglycerol.

Diacylglycerol kinase δ modulates Akt phosphorylation through pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2).

Discovering proteins that modulate Akt signaling has become a critical task, given the oncogenic role of Akt in a wide variety of cancers. We have discovered a novel diacylglycerol signaling pathway that promotes dephosphorylation of Akt. This pathway is regulated by diacylglycerol kinase δ (DGKδ). In DGKδ-deficient cells, we found reduced Akt phosphorylation downstream of three receptor tyrosine kinases. Phosphorylation upstream of Akt was not affected. Our data indicate that PKCα, which is excessively active in DGKδ-deficient cells, promotes dephosphorylation of Akt through pleckstrin homology domain leucine-rich repeats protein phosphatase (PHLPP) 2. Depletion of either PKCα or PHLPP2 rescued Akt phosphorylation in DGKδ-deficient cells. In contrast, depletion of PHLPP1, another Akt phosphatase, failed to rescue Akt phosphorylation. Other PHLPP substrates were not affected by DGKδ deficiency, suggesting mechanisms allowing specific modulation of Akt dephosphorylation. We found that ß-arrestin 1 acted as a scaffold for PHLPP2 and Akt1, providing a mechanism for specificity. Because of its ability to reduce Akt phosphorylation, we tested whether depletion of DGKδ could attenuate tumorigenic properties of cultured cells and found that DGKδ deficiency reduced cell proliferation and migration and enhanced apoptosis. We have, thus, discovered a novel pathway in which diacylglycerol signaling negatively regulates Akt activity. Our collective data indicate that DGKδ is a pertinent cancer target, and our studies could lay the groundwork for development of novel cancer therapeutics.

Phosphatidylinositol-4-phosphate 5-kinase isoforms exhibit acyl chain selectivity for both substrate and lipid activator.

Phosphatidylinositol 4,5-bisphosphate is mostly produced in the cell by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) and has a crucial role in numerous signaling events. Here we demonstrate that in vitro all three isoforms of PIP5K, α, ß, and γ, discriminate among substrates with different acyl chains for both the substrates phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) although to different extents, with isoform γ being the most selective. Fully saturated dipalmitoyl-PtdIns4P was a poor substrate for all three isoforms, but both the 1-stearoyl-2-arachidonoyl and the 1-stearoyl-2-oleoyl forms of PtdIns4P were good substrates. V(max) was greater for the 1-stearoyl-2-arachidonoyl form compared with the 1-stearoyl-2-oleoyl form, although for PIP5Kß the difference was small. For the α and γ isoforms, K(m) was much lower for 1-stearoyl-2-oleoyl PtdIns4P, making this lipid the better substrate of the two under most conditions. Activation of PIP5K by phosphatidic acid is also acyl chain-dependent. Species of phosphatidic acid with two unsaturated acyl chains are much better activators of PIP5K than those containing one saturated and one unsaturated acyl chain. PtdIns is a poor substrate for PIP5K, but it also shows acyl chain selectivity. Curiously, there is no acyl chain discrimination among species of phosphatidic acid in the activation of the phosphorylation of PtdIns. Together, our findings indicate that PIP5K isoforms α, ß, and γ act selectively on substrates and activators with different acyl chains. This could be a tightly regulated mechanism of producing physiologically active unsaturated phosphatidylinositol 4,5-bisphosphate species in the cell.

DGKζ is degraded through the cytoplasmic ubiquitin-proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry.

Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin-proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity.

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.

Novel mechanism for regulation of plasma platelet-activating factor acetylhydrolase expression in mammalian cells.

The plasma form of PAF-AH [PAF (platelet-activating factor) acetylhydrolase; also known as LpPLA(2) (lipopoprotein-associated phospholipase A(2)), PLA(2)G7] catalyses the release of sn-2 fatty acyl residues from PAF, oxidatively fragmented phospholipids, and esterified isoprostanes. The plasma levels of this enzyme vary widely among mammalian species, including mice and humans, but the mechanisms that account for these differences are largely unknown. We investigated the basis for these variations using molecular and biochemical approaches. We identified an N-terminal domain that played key roles in the determination of steady-state expression levels. The mouse N-terminal domain robustly enhanced protein expression levels, possibly owing to its ability to adopt a globular conformation that is absent in the human protein. We investigated the mechanism(s) whereby the N-terminal stretch modulated PAF-AH levels and found that differential expression was not due to variations in the efficiency of transcription, translation, or mRNA stability. Studies designed to evaluate the ability of precursor forms of PAF-AH to mature to fully active proteins indicated that the N-terminal end of human and mouse PAF-AH played important and opposite roles in this process. These domains also modulated the levels of expression of an unrelated polypeptide by affecting the stability of precursor forms of the protein. These studies provide insights that contribute to our understanding of the molecular features and mechanisms that contribute to differential expression of plasma PAF-AH in mammals.

Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal growth factor receptor abundance and degradation through ubiquitin-specific protease 8.

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

The levels of retinoic acid-inducible gene I are regulated by heat shock protein 90-alpha.

Retinoic acid-inducible gene I (RIG-I) is an intracellular pattern recognition receptor that plays important roles during innate immune responses to viral dsRNAs. The mechanisms and signaling molecules that participate in the downstream events that follow activation of RIG-I are incompletely characterized. In addition, the factors that define intracellular availability of RIG-I and determine its steady-state levels are only partially understood but are likely to play a major role during innate immune responses. It was recently reported that the antiviral activity of RIG-I is negatively regulated by specific E3 ubiquitin ligases, suggesting participation of the proteasome in the regulation of RIG-I levels. In this study, we used immunoprecipitation combined with mass spectrometry to identify RIG-I-interacting proteins and found that RIG-I forms part of a protein complex that includes heat shock protein 90-alpha (HSP90-alpha), a molecular chaperone. Biochemical studies using purified systems demonstrated that the association between RIG-I and HSP90-alpha is direct but does not involve participation of the CARD domain. Inhibition of HSP90 activity leads to the dissociation of the RIG-I-HSP90 complex, followed by ubiquitination and proteasomal degradation of RIG-I. In contrast, the levels of RIG-I mRNA are unaffected. Our studies also show that the ability of RIG-I to respond to stimulation with polyinosinic:polycytidylic acid is abolished when its interaction with HSP90 is inhibited. These novel findings point to HSP90-alpha as a chaperone that shields RIG-I from proteasomal degradation and modulates its activity. These studies identify a new mechanism whose dysregulation may seriously compromise innate antiviral responses in mammals.