BBA - Molecular Cell Research (v.1640, #1)

Over the past dozen years, the majority of clinical gene therapy trials for inherited genetic diseases and cancer therapy have been performed using murine onco-retrovirus as the gene delivery vector. The earliest systems used were relatively inefficient in both the rates of transduction and expression of the transgene. Formidable obstacles inherent in the cell biology and/or the immunology of the target cell systems limited the efficacy of gene therapy for many target diseases. Development of novel retrovirus gene transfer systems that are in progress have begun to overcome these obstacles. Evidence of this progress is the recent successful functional correction of the immune T and B lymphocyte deficiency in patients with X-linked severe combined immunodeficiency (X-SCID) and adenosine deaminase (ADA)-deficient SCID following onco-retrovirus vector ex vivo transduction of autologous marrow stem cells [Science 296 (2002) 2410; Science 288 (2000) 669; N. Engl. J. Med. 346 (2002) 1185]. These achievements of prolonged clinical benefit from gene therapy were tempered by the finding of insertional mutageneses in two of the treated X-SCID patients [N. Engl. J. Med. 348 (2003) 255].
Keywords: Onco-retrovirus; Gene therapy; Lentivirus;

Quantifying the syncytialisation of human placental trophoblast BeWo cells grown in vitro by Yoshiki Kudo; C.A.R. Boyd; Hiroshi Kimura; P.R. Cook; C.W.G. Redman; I.L. Sargent (25-31).
We have generated lines of BeWo cells that constitutively and stably express either histone H2B tagged with the green fluorescent protein (GFP), or the mitochondrial targeting sequence of subunit VIII of cytochrome c oxidase fused with a red fluorescent protein; one line has nuclei that fluoresce green, the other mitochondria that fluoresce red. Expression of these tagged proteins has no effect on the rates of DNA, RNA and protein synthesis, or on the amounts of human chorionic gonadotropin (hCG) secreted after treatment with forskolin. We used fluorescence-activated cell sorting (FACS) to monitor the extent of cell fusion (syncytialisation) between these two lines; fused cells are readily and accurately detected by their green/red fluorescence. This assay should prove useful in the investigation of the molecular mechanisms involved in trophoblast syncytialisation.
Keywords: Trophoblast; Syncytialisation; Cell fusion; FACS; BeWo cell;

The YIPP (tyrosine-isoleucine-proline-proline, amino acids 319–322) motif within the C-terminal part of the human AT1 receptor is associated with angiotensin II (AII)-induced activation of the Jak-STAT pathway and phospholipase Cγ1 phosphorylation. We report here that mutations of the YIPP motif strongly affect ligand-binding to the receptor. We analysed AT1 receptors of the wild type (WT) and 11 mutants with a FLAG-epitope-tag within their C-terminal portion. Mutations of the “P–P” amino acid sequence of this motif decreased both AII binding and the AII-induced intracellular Ca2+ transients. Mutant and WT receptors were expressed equally in the cell membrane and were localized within the plasma membrane. These results suggest that the “P–P” amino acid sequence within the YIPP motif is important for AII binding to the AT1 receptor.
Keywords: AT1 receptor; YIPP motif; Radioligand-binding assay; Confocal laser-scanning microscopy; Western blot; Intracellular Ca2+ mobilization;

Platelet interaction with CNBr peptides from type II collagen via integrin α2β1 by Gianni F. Guidetti; Fabio Greco; Alessandra Bertoni; Camilla Giudici; Manuela Viola; Ruggero Tenni; Enrica M. Tira; Cesare Balduini; Mauro Torti (43-51).
Adhesion of blood platelets to fibrillar collagens plays a crucial role in haemostasis. Collagen type II is a homotrimeric member of the fibrillar collagen family, whose ability to interact with platelets has been poorly investigated. In this work, we analysed platelet adhesion to the whole collagen type II molecule, as well as to its CNBr peptides. We found that collagen type II is as efficient as collagen type I in supporting platelet adhesion. Platelet binding sites on collagen type II were identified in two different CNBr-derived peptides, CB8 and CB11. The ability of these peptides to support platelet adhesion required the triple helical conformation. Interaction of platelets with CB8 and CB11 peptides was totally dependent on the presence of Mg2+ ions, and was completely inhibited by the anti-integrin α2β1 antibody P1E6. Upon adhesion to CB8 and CB11, a significant increase in intracellular protein tyrosine phosphorylation was observed. The pattern of tyrosine phosphorylated proteins in CB8- and CB11-adherent platelets was very similar to that observed in platelets adherent to the whole collagen molecule. By immunoprecipitation experiments, we identified two substrates that were tyrosine phosphorylated in adherent platelets as the tyrosine kinase Syk and the PLCγ2 isozyme. By contrast, platelet adhesion to CB8 and CB11 did not promote tyrosine phosphorylation of FcR γ-chain. Finally, we found that collagen type II, but not the CNBr-derived peptides, was able to induce cell aggregation associated to protein tyrosine phosphorylation when added to a platelet suspension. These results identify the CNBr peptides from collagen type II CB8 and CB11 as ligands for platelet integrin α2β1, and recognise their ability to support platelet adhesion and activation.
Keywords: Platelet; Collagen type II; Integrin α2β1; CNBr peptide; Tyrosine phosphorylation;

Expression of adenylyl cyclase isoforms in neutrophils by Ling-Chu Chang; Chung-Jieh Wang; Yi-Lee Lin; Jih-Pyang Wang (53-60).
In the present study, we have identified the expression of adenylyl cyclase (AC) isoforms in rat neutrophils according to the mRNA analysis and the distinct mode of regulation of isoform activity. Agarose gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR)-amplified products resulted in a single band of the expected size for each product with nucleotide sequences corresponding to AC1 to AC9. AC1 was abundant, while AC2, 6 and 9 were of moderate expression among the AC isoforms in neutrophils based on the quantitative real-time RT-PCR analysis. Exposure of neutrophils to Ca2+ ionophore A23187, isoproterenol and forskolin stimulated cellular cyclic AMP accumulation. EDTA and the calmodulin (CaM) antagonist, trifluoperazine, prevented the A23187-induced response. Pretreatment with pertussis toxin (PTX) inhibited the α2-adrenergic agonist, UK14304-induced cellular cyclic AMP elevation. In addition, UK14304 augmented the cyclic AMP elevation when cells were stimulated by isoproterenol. Phorbol 12-myristate 13-acetate (PMA) attenuated the augmentation response of UK14304 and isoproterenol. Treatment of the membrane preparations from rat neutrophils with Ca2+/CaM, forskolin, isoproterenol, GTPγS or Gβγ all increased cyclic AMP production. The addition of protein kinase C (PKC) catalytic fragment and Gβγ augmented the Ca2+/CaM- and isoproterenol-stimulated AC activity, respectively. However, forskolin and the activated protein kinase A (PKA) attenuated the GTPγS- and isoproterenol-stimulated AC activity, respectively. KT5720, a PKA inhibitor, reversed the inhibition by PKA. Taken together, these data suggest the presence of four groups of AC isoforms in rat neutrophils.
Keywords: Adenylyl cyclase; Neutrophil; Revese transcription-polymerase chain reaction;

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling by Wendy M. Pruitt; Antoine E. Karnoub; A.Corinne Rakauskas; Michel Guipponi; Stylianos E. Antonarakis; Alexei Kurakin; Brian K. Kay; John Sondek; David P. Siderovski; Channing J. Der (61-68).
Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.
Keywords: Dbl family protein; Phosphatidylinositol 3-kinase; Dbl homology domain; Pleckstrin homology domain; Cdc42;

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A2 (TXA2), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-β (IL1-β)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI2, as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF. In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF formation with I50's of 2.6 and 5.5 μM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 μM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI2 generation was determined. An average 8-fold stimulation of 6-ketoPGF formation was obtained with quiescent IL1-β-exposed HUVECs pretreated for 18 h with 25 μM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-β-treated HUVECs prelabeled with 25 μM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis.
Keywords: Conjugated linoleic acid; Endothelial prostacyclin; Cyclooxygenase enzyme;

Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling by Pradeep Sathyanarayana; Manoj K. Barthwal; Mary Ellen Lane; Summer F. Acevedo; Efthimios M.C. Skoulakis; Andreas Bergmann; Ajay Rana (77-84).
Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine–threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper.
Keywords: MLK; dMLK; slipper; Hep; dMKK4; dJNK;

The G protein-coupled 5-HT1A receptor causes suppression of caspase-3 through MAPK and protein kinase Cα by Tatyana Adayev; Indrani Ray; Rachna Sondhi; Tomasz Sobocki; Probal Banerjee (85-96).
The 5-HT1A agonist 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) causes inhibition of caspase-3 and apoptosis via the extracellular signal-regulated kinases (ERK1/2) in hippocampal HN2–5 cells. Two 5-HT1A agonists, Repinotan hydrochloride (BAY x 3702) and 8-OH-DPAT, block caspase-3 activation and apoptosis caused by anoxia/reoxygenation and H2O2 treatment. This is reversed upon transient expression of dominant negative Ras (N17Ras) and Raf-1 (Raf301), confirming the involvement of Ras and Raf-1 in this 5-HT1A-R→ERK1/2→caspase-3 pathway. A selective inhibitor of phospholipase Cβ (PLCβ) (U73122) but not a general protein kinase C (PKC) inhibitor (GFX) reversed the 5-HT1A-R-mediated ERK1/2 stimulation. However, both GFX and the PKCα and PKCβ1 inhibitor Gö6976 reversed the ERK1/2-mediated inhibition of caspase-3. ERK-dependent activation of only PKCα was observed in immunoprecipitates obtained from 5-HT1A agonist-treated HN2–5 cells. Finally, transient expression of kinase-negative PKCα eliminated the 8-OH-DPAT-evoked block on the H2O2-triggered caspase-3 stimulation, establishing PKCα as a link between ERK and caspase-3 (5-HT1A-R→PLC→ERK1/2→PKCα→caspase-3). Our results elucidate a novel yet general, neuroprotective pathway through which G protein-coupled receptors could cause inhibition of effector caspases, such as caspase-3.
Keywords: 5-HT1A receptor; BAY x 3702; ERK1/2; PKC; Caspase-3;