Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.

Platelet-activating factor (PAF), an alkylether phospholipid, is produced in the brain when it is subjected to various stimuli. Using a Xenopus oocyte expression system, we obtained evidence for functional PAF receptor mRNA expression in rat brain. The presence of the PAF receptor was confirmed and shown to be quite ubiquitous in the CNS by RNA blot and radioligand binding studies. To investigate the neuronal functions of PAF, intracellular Ca2+ increase elicited by nanomolar PAF application was analyzed in cultured rat hippocampal cells. Fractions of NMDA-responsive cells and non-NMDA-responsive cells were shown to respond to PAF, suggesting a potential role for PAF in the Ca2+ signaling pathway in the hippocampus.

Sequential requirements of the N-terminal palmitoylation site and SH2 domain of Src family kinases in the initiation and progression of FcepsilonRI signaling.

Initial biochemical signaling originating from high-affinity immunoglobulin E receptor (FcepsilonRI) has been ascribed to Src family kinases. To understand the mechanisms by which individual kinases drive the signaling, we conducted reconstitution experiments: FcepsilonRI signaling in RBL2H3 cells was first suppressed by a membrane-anchored, gain-of-function C-terminal Src kinase and then reconstructed with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-myc epitope. Those constructs derived from Lyn and Fyn, which are associated with detergent-resistant membranes (DRMs), physically interacted with resting FcepsilonRI and reconstructed clustering-induced signaling that leads to calcium mobilization and ERK1 and -2 activation. c-Src-derived construct, which was excluded from DRMs, failed to interact with FcepsilonRI and to restore the signaling, whereas creation of palmitoylatable Cys3 enabled it to interact with DRMs and with FcepsilonRI and to restore the signaling. Deletion of Src homology 3 (SH3) domain from the Lyn-derived construct did not alter its ability to transduce the series of signaling. Deletion of SH2 domain did not affect its association with DRMs and with FcepsilonRI nor clustering-induced tyrosine phosphorylation of FcepsilonRI beta and gamma subunits, but it almost abrogated the next step of tyrosine phosphorylation of Syk and its recruitment to FcepsilonRI. These findings suggest that Lyn and Fyn could, but c-Src could not, drive FcepsilonRI signaling and that N-terminal palmitoylation and SH2 domain are required in sequence for the initial interaction with FcepsilonRI and for the signal progression to the molecular assembly.

ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Differential composition of cytosol 5'-nucleotidases between T and B lymphoblasts.

WI-L2 cells (a B-lymphoblastoid cell line) were more resistant than CEM cells (a T-lymphoblastoid cell line) to deoxyadenosine, ara-A (9-beta-D-arabinofuranosyladenine), or ara-C (1-beta-D-arabinofuranosylcytosine) inhibition. This was caused by a difference in the composition of cytosol 5'-nucleotidases between WI-L2 and CEM cells. In intact cells, the endogenous production of deoxyadenosine from WI-L2 cells deficient in adenosine kinase (EC 2.7.1.20) and deoxycytidine kinase (EC 2.7.1.74) was consistently high, despite changes in endogenous adenosine production. Endogenous production of deoxyadenosine from CEM cells deficient in adenosine kinase and deoxycytidine kinase was, however, coordinated with endogenous adenosine production. In broken cells, cytosol dAMPase (2'-deoxyadenosine 5'-monophosphate 5'-nucleotidase) activity of WI-L2 cells was 3-5-fold higher than that of CEM cells. dAMPase activity could be separated from ATP-activated IMPase (inosine 5'-monophosphate 5'-nucleotidase) by gel filtration (molecular weight: dAMPase; 39,000-46,000; ATP-activated IMPase, greater than 150,000). Cytosol ATP-activated IMPase and dAMPase were isolated by phosphocellulose or DEAE-Bio-Gel A chromatography from non-specific phosphatases. The ATP-activated IMPase showed only marginal activity towards dAMP (2'-deoxyadenosine 5'-monophosphate), ara-AMP (9-beta-D-arabinofuranosyladenine 5'-monophosphate), or ara-CMP (cytosine-beta-D-arabinofuranoside 5'-monophosphate), even in the presence of ATP. The activity of ATP-activated IMPase was similar in WI-L2 and CEM cells. dAMPase was separated into two peaks by DEAE-Bio-Gel A chromatography; one of these peaks degraded ara-AMP and ara-CMP. The activities of both peaks from WI-L2 cells were higher than those from CEM cells. These results show that the degradation of dAMP, ara-AMP or ara-CMP was more specific and rapid in WI-L2 than in CEM cells.

Solubilization and partial purification of leukotriene C4 synthase from guinea-pig lung: a microsomal enzyme with high specificity towards 5,6-epoxide leukotriene A4.

Enzymic activities catalyzing allylic epoxide, leukotriene A4, to leukotriene C4 by conjugation with glutathione were present mainly in microsomal fractions of spleens and lungs of guinea pigs and rats. Leukotriene C4 (LTC4) synthase was solubilized from the microsomes of guinea-pig lung by the new procedures of a combination of 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), digitonin and KCl. The enzyme was partially purified by two steps of column chromatography which resulted in a complete resolution of the enzyme from glutathione S-transferases (EC 2.5.1.18). The partially purified LTC4 synthase showed a Vmax value of 40 nmol/min per mg, and the apparent Km values for LTA4 and glutathione were 36 microM and 1.6 mM, respectively. The enzyme was unstable, and half of the activity was lost by incubation at 37 degrees C for 3 min. Glutathione at 10 mM completely protected the enzyme against this inactivation, while other sulfhydryl-group-reducing reagents were ineffective. The partially purified enzyme revealed a high specificity towards 5,6-epoxide leukotrienes (LTA4 and its methyl ester), while rat cytosolic glutathione S-transferases catalyzed conjugation of glutathione to various positional isomers of epoxide leukotrienes.

Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase.

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.

Endotoxin transduces Ca2+ signaling via platelet-activating factor receptor.

Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality. Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS-induced Ca2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (PAF) receptor antagonists, thus suggesting a cross-talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342-346; (1991) J. Biol. Chem. 266, 20400-20405] expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor.

The human platelet-activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5'-noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF-kappa B and Sp-1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP-1, AP-2, and Sp-1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL-1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides.

We designed a microplate-based assay method for mitogen-activated protein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in heretofore used assays. The micro-trap phosphorylation assay has advantages over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.

Differential inhibitory effects of auranofin on leukotriene B4 and leukotriene C4 formation by human polymorphonuclear leukocytes.

Auranofin (AF) is a newly introduced oral gold compound having antirheumatic properties, and its efficacy in the treatment of bronchial asthma is now under investigation. In this study, we examined the effects of AF on leukotriene (LT) formation by human polymorphonuclear leukocytes (PMNs) stimulated with the calcium ionophore A23187. AF inhibited LTC4 formation in a dose-dependent manner with an IC50 (concentration required to produce 50% inhibition of control) of 3.2 microM. In contrast, LTB4 formation was not prevented by AF at concentrations up to 6 microM, but it was reduced to 59 +/- 4% (mean +/- SE, N = 3) of control by an 8 microM concentration. As a next step, we explored the mechanisms of the differential inhibitory effects of AF using cell-free systems. When arachidonic acid (AA) and reduced glutathione (GSH) were used as substrates, AF inhibited LTC4 synthesis more effectively (IC50 = 14 microM) than LTB4 synthesis (IC50 = 100 microM). However, LTB4 and LTC4 syntheses from LTA4 were affected only slightly by AF within the concentrations tested (3-100 microM). These results in the cell-free systems indicate that the inhibition of LT formation was caused by a reduction of LTA4 synthesis and that the differential inhibitory effects can be ascribed to the higher Km value of glutathione S-transferase for LTA4 than that of LTA4 hydrolase in PMNs. In accordance with this hypothesis, LTC4 synthesis was more dependent than LTB4 synthesis on LTA4 concentrations within 25-100 microM, and AA-861, a 5-lipoxygenase inhibitor, caused similar differential inhibitory effects on the formation of LTs by intact PMNs. The inhibitory effect of AF on LT formation at physiological concentrations may play some role in the efficacy of this drug.

Activation of mitogen-activated protein kinase and arachidonate release via two G protein-coupled receptors expressed in the rat hippocampus.

Platelet-activating factor and somatostatin receptors, two G protein-coupled receptors expressed in the rat hippocampus, were analyzed for the downstream signaling pathways in Chinese hamster ovary cells stably expressing each receptor. Ligand stimulation to each CHO cell line induced (1) inhibition of forskolin-induced accumulation of cAMP, (2) arachidonate release, and (3) activation of mitogen-activated protein kinase and MAP kinase kinase. In contrast, inositol phosphate breakdown was seen only in the PAF-stimulated CHO cells. The induction of these signals accompanied no detectable Ras activation. Suppression of the signals by pertussis toxin was almost complete for the somatostatin receptor but partial for the PAF receptor, suggesting that the somatostatin receptor couples only with PTX-sensitive G protein, while the PAF receptor couples with both PTX-sensitive and -insensitive G proteins. A model of G protein-mediated signaling pathways was proposed in which the signals from Gi and those from Gq converge at MAP kinase kinase and lead to arachidonate release. The present system using CHO cells is useful for analyzing signaling pathways from G proteins to MAP kinase kinase and will thereby provide clues for understanding the mechanisms underlying the physiological and pathological events mediated by PAF, somatostatin, and other G protein-coupled receptors in the central nervous system and other tissues.

Suppression of human polymorphonuclear leukocyte phagocytosis by adenosine analogs.

The effects of adenosine analogs on human polymorphonuclear leukocyte (PMN) phagocytosis to latex beads or sheep red blood cells were investigated in an in vitro experiment. The purine compounds such as N6-phenylisopropyladenosine (PIA), 5'-N-ethylcarboxamido-adenosine (NECA), an A2 adenosine receptor agonist, and adenosine as high as 2 mM had no effect on PMN phagocytosis. In contrast, 2',5'-dideoxyadenosine (DDA), a P-site adenosine receptor agonist, and 5'-methylthioadenosine (MTA), a naturally occurring purine nucleoside, caused profound inhibition of phagocytic function of PMNs in a dose-dependent manner. Dipyridamole, which blocks purine nucleoside uptake, reversed the suppression due to MTA. Theophylline, an R-site receptor antagonists did not prevent the effect of MTA. These results suggested that phagocytosis of PMNs is suppressed by stimulation of the P-site adenosine receptors.

Identification of Zfp521/ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia.

E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: growth factor independent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.

Overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induced acute leukemia in p210BCR/ABL transgenic mice.

Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1DeltaC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICD Delta C). In addition, generation of Tg for both p210BCR/ABL and Notch1DeltaC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1DeltaC/NICD Delta C with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.