Hiroaki Honda

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.

Requirements for localization of p130cas to focal adhesions.

p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.

Acetylation of GATA‐3 affects T‐cell survival and homing to secondary lymphoid organs

Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA‐3 is acetylated in T cells and that a mutation introduced into amino acids 305–307 (KRR‐GATA3) creates local hypoacetylation in GATA‐3. Remarkably, KRR‐GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T‐cell homing to systemic lymphnodes, and prolonged T‐cell survival after activation. These findings have significant implications in that the acetylation state of GATA‐3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA‐3 in T‐cell survival and homing to secondary lymphoid organs.

Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

Antisense repression of proto-oncogene c-Cbl enhances activation of the JAK-STAT pathway but not the ras pathway in epidermal growth factor receptor signaling.

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.

Increased soluble Fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a major complication following allogeneic bone marrow transplantation (BMT). Recently, accumulating evidence indicates that the Fas/Fas ligand (FasL) system is implicated in the pathogenesis of aGVHD in murine models. We determined the serum levels of soluble FasL (sFasL) in BMT recipients using an enzyme-linked immunosorbent assay. The serum sFasL was suppressed during the period of myelosuppression following the preparative regimen and subsequently increased with hematopoietic reconstitution after BMT. In patients with aGVHD, the serum sFasL level was significantly higher than in those without aGVHD. In the mixed lymphocyte reaction assay, sFasL in the supernatants was increased with a significant correlation to the level of 3H-thymidine uptake. Our findings suggest that the Fas/FasL system is activated by allogeneic stimulation and may have close correlation to the development of aGVHD in human BMT.

Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.

Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines.

Aspergillus tracheobronchitis after allogeneic bone marrow transplantation.

Recently Machidaet al presented in this journal a case of a patient with anAspergillus tracheobronchitis after allogeneic BMT together with a review of the literature of this serious complication after BMT. We would like to add our recent experience and comment on the role of galactomannan antigen testing in establishing the diagnosis of this Aspergillusinfection. A 56-year-old female patient underwent allogeneic BMT in our center after induction of first complete morphological and cytogenetic remission of acute myeloid leukemia. After conditioning with idarubicin, cyclophosphamide and busulfan, bone marrow-derived stem cells from her histocompatible sibling were infused. She developed grade II acute GVHD of the skin, for which she was treated with prednisone. Later on she experienced grade IV chronic GVHD of the gastro-intestinal tract and three episodes of CMVcolitis. Treatment with high-dose corticosteroids, cyclosporin, ganciclovir and CMV-hyperimmunoglobulins was given with limited success. On day 193 after BMT she was admitted to our hospital because of severe dyspnea, inspiratory wheezing and hoarseness. Temperature was not raised and persistent severe diarrhea was still present. Physical examination showed severe respiratory distress with use of accessory respiratory muscles and extreme inspiratory wheezing, and on auscultation her breath sounds were hardly audible. Chest X-ray and high-resolution CT scan were unremarkable. Lung function examination demonstrated extreme inspiratory and expiratory obstruction. Yellowish plaques, ulcerations and pseudomembranes were seen on bronchoscopy performed immediately and there were mucus plugs that almost completely obstructed the trachea and bronchi. Microscopic examination of the obstructing material showed hyphae and cultures yielded Aspergillus fumigatus . Biopsy was considered impossible because of the low platelet count. Daily bronchial flushing and treatment with high-dose amphotericin B (1 mg/kg/day, 45 mg daily) and nebulized amphotericin B was started with only temporary improvement. Liposomal amphotericin B 200 mg daily and itraconazole (200 mg, twice daily for 2 days, followed by 200 mg daily) as well as G-CSF (300 mg daily) were added without any improvement. Our patient became ventilator-dependent and could no longer cope with this regime. For this reason it was decided to abstain from further treatment and she died of respiratory failure the same day, 37 days after admission. Permission for autopsy was denied. Like Machidaet al we measured serum galactomannan antigen levels by ELISA (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). In a series of six consecutive serum samples, the highest galactomannan serum ratio found was 0.6 which is below the recommended cut-off level of 1.0. This observation might have several explanations. Firstly, Clarke et al divided fungal tracheobronchitis into two different morphological types. The first type is characterized by intraluminal growth involving the entire circumference of the airway with only superficial invasion and ulceration; tenacious mucus or

Characterization of the mouse prostaglandin F receptor gene: a transgenic mouse study of a regulatory region that controls its expression in the stomach and kidney but not in the ovary.

The actions of prostaglandin F2α are mediated by a cell‐surface receptor (FP), but little is known about the regulation of FP gene expression. To clarify the mechanisms underlying tissue specific transcription of the mouse FP gene, we isolated and characterized mouse genomic DNA clones encoding FP.

Extramedullary relapse in the so-called ‘sanctuary’ sites for chemotherapy after donor lymphocyte infusion

Extramedullary relapse in the so-called ‘sanctuary’ sites for chemotherapy after donor lymphocyte infusion