Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

Short-term physical inactivity induces diacylglycerol accumulation and insulin resistance in muscle via lipin1 activation.

Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization (HCI) to mice with normal or high-fat diet (HFD) and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. Although 2-wk HFD alone did not alter intramyocellular diacylglycerol (IMDG) accumulation, HCI alone increased it by 1.9-fold and HCI after HFD further increased it by 3.3-fold. Parallel to this, we found increased protein kinase C ε (PKCε) activity, reduced insulin-induced 2-deoxyglucose (2-DOG) uptake, and reduced phosphorylation of insulin receptor ß (IRß) and Akt, key molecules for insulin signaling pathway. Lipin1, which converts phosphatidic acid to diacylglycerol, showed increase of its activity by HCI, and dominant-negative lipin1 expression in muscle prevented HCI-induced IMDG accumulation and impaired insulin-induced 2-DOG uptake. Furthermore, 24-h leg cast immobilization in human increased lipin1 expression. Thus, even short-term immobilization increases IMDG and impairs insulin sensitivity in muscle via enhanced lipin1 activity.NEW & NOTEWORTHY Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. To model physical inactivity, we applied 24-h hind-limb cast immobilization to mice with normal or high-fat diet and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. We found that even short-term immobilization increases intramyocellular diacylglycerol and impairs insulin sensitivity in muscle via enhanced lipin1 activity.

Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), a common cause of heart failure, is characterized by cardiac dilation and reduced left ventricular ejection fraction, but the underlying mechanisms remain unclear. To investigate the mechanistic basis, we performed global metabolomic analysis of myocardial tissues from the left ventricles of J2N-k cardiomyopathic hamsters. This model exhibits symptoms similar to those of human DCM, owing to the deletion of the δ-sarcoglycan gene. Charged and lipid metabolites were measured by capillary electrophoresis mass spectrometry (MS) and liquid chromatography MS(/MS), respectively, and J2N-k hamsters were compared with J2N-n healthy controls at 4 (presymptomatic phase) and 16weeks (symptomatic) of age. Disturbances in membrane phospholipid homeostasis were initiated during the presymptomatic phase. Significantly different levels of charged metabolites, occurring mainly in the symptomatic phase, were mapped to primary metabolic pathways. Reduced levels of metabolites in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, together with large decreases in major triacylglycerol levels, suggested that decreased energy production leads to cardiac contractile dysfunction in the symptomatic phase. A mild reduction in glutathione and a compensatory increase in ophthalmate levels suggest increased oxidative stress in diseased tissues, which was confirmed by histochemical staining. Increased levels of 4 eicosanoids, including prostaglandin (PG) E2 and 6-keto-PGF1α, in the symptomatic phase suggested activation of the protective response pathways. These results provide mechanistic insights into DCM pathogenesis and may help identify new targets for therapeutic intervention and diagnosis.

Remitting Seronegative Symmetrical Synovitis with Pitting Edema Syndrome as a Manifestation of Recurrent Ovarian Cancer.

We herein report a case of ovarian cancer recurrence detected every time with symptoms of remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome. A 46-year-old woman who had a history of ovarian cancer 9 months earlier developed joint pain along with pitting edema in both hands and was diagnosed with RS3PE syndrome. Two and four years after initial surgery for ovarian cancer, symptoms of RS3PE syndrome appeared, and a recurrent site was detected. With resection of the relapsed sites and increased maintenance dose of methylprednisolone, these symptoms improved within a month.

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)ß, to lipid mobilization in proximal mast cells and distal fibroblasts.

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)ß), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)ß (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)ß, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)ß is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.

Seronegative Full-house Nephropathy with Crohn's Disease.

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease. Lupus nephritis (LN) is a major risk factor for mortality in SLE, and glomerular "full-house" immunofluorescence staining is a well-known characteristic of LN. However, some cases of non-lupus glomerulonephritis can also present with a "full-house" immunofluorescence pattern. We recently encountered a patient with full-house nephropathy (FHN) during adalimumab administration for Crohn's disease. IgA nephropathy or idiopathic FHN was diagnosed, and treatment with steroids was started, after which there was improvement in proteinuria. The prognosis of FHN has been reported to be poor; therefore, aggressive treatment is required for such patients.

A negative regulator of delayed prostaglandin D2 production in mouse mast cells.

We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.

Using gene expression profiling to identify a prognostic molecular spectrum in gliomas.

Histopathological classification of gliomas is often clinically inadequate due to the diversity of tumors that fall within the same class. The goal of the present study was to identify prognostic molecular features in diffusely infiltrating gliomas using gene expression profiling. We selected 3456 genes expressed in gliomas, including 3012 genes found in a gliomal expressed sequence tag collection. The expression levels of these genes in 152 gliomas (100 glioblastomas, 21 anaplastic astrocytomas, 19 diffuse astrocytomas, and 12 anaplastic oligodendrogliomas) were measured using adapter-tagged competitive polymerase chain reaction, a high-throughput reverse transcription-polymerase chain reaction technique. We applied unsupervised and supervised principal component analyses to elucidate the prognostic molecular features of the gliomas. The gene expression data matrix was significantly correlated with the histological grades, oligo-astro histology, and prognosis. Using 110 gliomas, we constructed a prediction model based on the expression profile of 58 genes, resulting in a scheme that reliably classified the glioblastomas into two distinct prognostic subgroups. The model was then tested with another 42 tissues. Multivariate Cox analysis of the glioblastoma patients using other clinical prognostic factors, including age and the extent of surgical resection, indicated that the gene expression profile was a strong and independent prognostic parameter. The gene expression profiling identified clinically informative prognostic molecular features in astrocytic and oligodendroglial tumors that were more reliable than the traditional histological classification scheme.

The lack of the C-terminal domain of adipose triglyceride lipase causes neutral lipid storage disease through impaired interactions with lipid droplets.

CONTEXT: The molecular mechanisms by which triglycerides in lipid droplets (LDs) are synthesized, stored, and degraded need to be elucidated. OBJECTIVE: The objectives were to report siblings with neutral lipid storage disease with myopathy (NLSDM) with a novel mutation of adipose triglyceride lipase (ATGL) and determine whether the C-terminal part of ATGL containing the hydrophobic region plays a role in the interaction with LDs. DESIGN AND PATIENTS: Skin fibroblasts and peripheral blood leukocytes were obtained from NLSDM patients. In vitro experiments were performed with fibroblasts and COS7 cells. MAIN OUTCOME MEASURES: Transfection studies were used to assess the effects of various recombinant ATGL proteins on lipase activities and lipid contents. Fluorescence microscopy were used for determination of intracellular distribution of ATGL proteins. RESULTS: The direct sequence of ATGL cDNA reveals that a patient is a homozygote for the 4-bp deletion, leading to a premature stop codon and causes the lack of the C terminus of the protein including the hydrophobic domain. Overexpressed control ATGL in NLSDM fibroblasts was found around the rims of LDs and caused significantly reduced cellular lipid accumulation. In contrast, NLSDM ATGL was homogeneously located in the cytoplasm despite the presence of LDs and had almost no effect on LD degradation despite its similar lipase activity. A series of C-terminal truncated ATGLs without the intact hydrophobic domain failed to localize around and degrade LDs. CONCLUSIONS: These findings indicate that the domain including the hydrophobic region of ATGL was essential for association with LDs.

Gene expression-based molecular diagnostic system for malignant gliomas is superior to histological diagnosis.

PURPOSE: Current morphology-based glioma classification methods do not adequately reflect the complex biology of gliomas, thus limiting their prognostic ability. In this study, we focused on anaplastic oligodendroglioma and glioblastoma, which typically follow distinct clinical courses. Our goal was to construct a clinically useful molecular diagnostic system based on gene expression profiling. EXPERIMENTAL DESIGN: The expression of 3,456 genes in 32 patients, 12 and 20 of whom had prognostically distinct anaplastic oligodendroglioma and glioblastoma, respectively, was measured by PCR array. Next to unsupervised methods, we did supervised analysis using a weighted voting algorithm to construct a diagnostic system discriminating anaplastic oligodendroglioma from glioblastoma. The diagnostic accuracy of this system was evaluated by leave-one-out cross-validation. The clinical utility was tested on a microarray-based data set of 50 malignant gliomas from a previous study. RESULTS: Unsupervised analysis showed divergent global gene expression patterns between the two tumor classes. A supervised binary classification model showed 100% (95% confidence interval, 89.4-100%) diagnostic accuracy by leave-one-out cross-validation using 168 diagnostic genes. Applied to a gene expression data set from a previous study, our model correlated better with outcome than histologic diagnosis, and also displayed 96.6% (28 of 29) consistency with the molecular classification scheme used for these histologically controversial gliomas in the original article. Furthermore, we observed that histologically diagnosed glioblastoma samples that shared anaplastic oligodendroglioma molecular characteristics tended to be associated with longer survival. CONCLUSIONS: Our molecular diagnostic system showed reproducible clinical utility and prognostic ability superior to traditional histopathologic diagnosis for malignant glioma.

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox.

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

Prediction of docetaxel response in human breast cancer by gene expression profiling.

PURPOSE: Docetaxel is one of the most effective anticancer drugs available in the treatment of breast cancer. Nearly half of the treated patients, however, do not respond to chemotherapy and suffer from side effects. The ability to reliably predict a patient's response based on tumor gene expression will improve therapeutic decision making and save patients from unnecessary side effects. PATIENTS AND METHODS: A total of 44 breast tumor tissues were sampled by biopsy before treatment with docetaxel, and the response to therapy was clinically evaluated by the degree of reduction in tumor size. Gene expression profiling of the biopsy samples was performed with 2,453 genes using a high-throughput reverse transcriptase polymerase chain reaction technique. Using genes differentially expressed between responders and nonresponders, a diagnostic system based on the weighted-voting algorithm was constructed. RESULTS: This system predicted the clinical response of 26 previously unanalyzed samples with over 80% accuracy, a level promising for clinical applications. Diagnostic profiles in nonresponders were characterized by elevated expression of genes controlling the cellular redox environment (ie, redox genes, such as thioredoxin, glutathione-S-transferase, and peroxiredoxin). Overexpression of these genes protected cultured mammary tumor cells from docetaxel-induced cell death, suggesting that enhancement of the redox system plays a major role in docetaxel resistance. CONCLUSION: These results suggest that the clinical response to docetaxel can be predicted by gene expression patterns in biopsy samples. The results also suggest that one of the molecular mechanisms of the resistance is activation of a group of redox genes.

Prediction of peritoneal metastasis in advanced gastric cancer by gene expression profiling of the primary site.

Peritoneal metastasis is the most common cause of tumour progression in advanced gastric cancer. Clinicopathological findings including cytologic examination of peritoneal lavage have been applied to assess the risk of peritoneal metastasis, but are sometimes inadequate for predicting peritoneal metastasis in individuals. Hence, we tried to construct a new prediction system for peritoneal metastasis by using a PCR-based high throughput array with 2304 genes. The prediction system, constructed from the learning set comprised of 30 patients with the most informative 18 genes, classified each case into a 'good signature group' or 'poor signature group'. Then, we confirmed the predictive performance in an additional validation set comprised of 24 patients, and the prediction accuracy for peritoneal metastasis was 75%. Kaplan-Meier analysis with peritoneal metastasis revealed significant difference between these two groups (P=0.0225). By combining our system with conventional clinicopathological factors, we can identify high risk cases for peritoneal metastasis more accurately.

Regulation of superoxide-producing NADPH oxidases in nonphagocytic cells.

The membrane-integrated protein gp91phox functions as the catalytic center of the superoxide-producing phagocyte NADPH oxidase. Recent studies have identified homologs of gp91phox in nonphagocytic cells, which constitute the NADPH oxidase (Nox) family. Activation of the Nox oxidases leads to production of reactive oxygen species (ROS), thereby participating in a variety of biological events, such as host defense, hormone biosynthesis, and signal transduction. The activity of the Nox enzymes is regulated by various proteins, including the small GTPase Rac; regulatory mechanisms differ dependent on the type of the Nox proteins. For example, an oxidase activator (p47phox or Noxo1) and an oxidase activator (p67phox or Noxa1) are absolutely required for superoxide production by gp91phox and Nox1, but not by Nox3. Rac, albeit probably dispensable to the Nox3 activity, plays an essential role in activation of gp91phox. Thus, functional reconstitution of Nox systems is crucial for the study of Nox regulation. Here we describe a basic method for the reconstitution of Nox systems by expression of oxidase proteins in transfectable cells.

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F(2alpha)-synthetic activity.

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.

Molecular prediction of response to 5-fluorouracil and interferon-alpha combination chemotherapy in advanced hepatocellular carcinoma.

PURPOSE: The prognosis of hepatocellular carcinoma (HCC) is very poor, particularly in patients with tumors that have invaded the major branches of the portal vein. Combination chemotherapy with intra-arterial 5-fluorouracil and subcutaneous interferon-alpha has shown promising results for such advanced HCC, but it is important to develop the ability to accurately predict chemotherapeutic responses. EXPERIMENTAL DESIGN: We analyzed the expression of 3,080 genes using a polymerase chain reaction-based array in 20 HCC patients who were treated with combination chemotherapy after reduction surgery. After unsupervised analyses, a supervised classification method for predicting chemotherapeutic responses was constructed. To minimize the number of predictive genes, we used a random permutation test to select only significant (P < 0.01) genes. A leave-one-out cross-validation confirmed the gene selection. We also prepared an additional 11 cases for validation of predictive performance. RESULTS: Hierarchical clustering analysis and principal component analysis with all 3,080 genes revealed distinct gene expression patterns in responders (those with complete response or partial response) and nonresponders (those with stable disease or progressive disease) to the combination chemotherapy. Using a weighted-voting classification method with either all genes or only significant genes as assessed by permutation testing, the objective responses to treatment were correctly predicted in 17 of 20 cases (accuracy, 85%; positive predictive value, 100%; negative predictive value, 80%). Moreover, patients in the validation dataset could be classified into two distinct prognostic groups using 63 predictive genes. CONCLUSIONS: Molecular analysis of 63 genes can predict the response of patients with advanced HCC and major portal vein tumor thrombi to combination chemotherapy with 5-fluorouracil and interferon-alpha.

INPP4B Is a PtdIns(3,4,5)P3 Phosphatase That Can Act as a Tumor Suppressor.

UNLABELLED: Inositol polyphosphate 4-phosphatase B (INPP4B) has been identified as a tumor suppressor mutated in human breast, ovary, and prostate cancers. The molecular mechanism underlying INPP4B's tumor-suppressive role is currently unknown. Here, we demonstrate that INPP4B restrains tumor development by dephosphorylating the PtdIns(3,4,5)P3 that accumulates in situations of PTEN deficiency. In vitro, INPP4B directly dephosphorylates PtdIns(3,4,5)P3. In vivo, neither inactivation of Inpp4b (Inpp4b(Δ/Δ)) nor heterozygous deletion of Pten (Pten(+/-)) in mice causes thyroid abnormalities, but a combination of these mutations induces malignant thyroid cancers with lung metastases. At the molecular level, simultaneous deletion of Inpp4b and Pten synergistically increases PtdIns(3,4,5)P3 levels and activates AKT downstream signaling proteins in thyroid cells. We propose that the PtdIns(3,4,5)P3 phosphatase activity of INPP4B can function as a "back-up" mechanism when PTEN is deficient, making INPP4B a potential novel therapeutic target for PTEN-deficient or PIK3CA-activated cancers. SIGNIFICANCE: Although INPP4B expression is reduced in several types of human cancers, our work on Inpp4B-deficient mice provides the first evidence that INPP4B is a bona fide tumor suppressor whose function is particularly important in situations of PTEN deficiency. Our biochemical data demonstrate that INPP4B directly dephosphorylates PtdIns(3,4,5)P3.

Comparison of gene expression profiling during postnatal development of mouse dentate gyrus and cerebellum.

Both the dentate gyrus of the hippocampus and the cerebellar cortex consist mainly of granule cells and develop postnatally. The granule cells in both tissues are presumed to be similar. Changes in gene expression were analyzed during the postnatal development of the dentate gyrus. Altogether, expression patterns of 1,937 genes were determined by adaptor-tagged competitive PCR. More than 90% of the genes belong to groups characterized by elevated expression either at earlier or later stages of development. A majority of the genes expressed showed marked changes during the developmental process, but there was little correlation between gene function and expression, unlike that observed during mouse postnatal cerebellar development. Despite anatomical and physiological similarities between these two processes, the gene expression profiles are completely different.