Toshihide Iwashita

Galanin and galanin receptor type 1 suppress proliferation in squamous carcinoma cells: activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors

Galanin receptor 1 (GALR1) maps to a common region of 18q loss in head and neck squamous cell carcinomas and is frequently inactivated by methylation. To investigate effects of GALR1 and its signaling pathways, we stably expressed hemaglutinin-tagged GALR1 in a human oral carcinoma cell line (UM-SCC-1-GALR1) that expresses no endogenous GALR1. In transfected cells, galanin induced activation of the extracellular-regulated protein kinase-1/2 (ERK1/2) and suppressed proliferation. Galanin stimulation mediated decreased expression of cyclin D1 and increased expression of the cyclin-dependent kinase inhibitors (CKI), p27Kip1 and p57Kip2. Pretreatment with the ERK1/2-specific inhibitor U0126 prevented these galanin-induced effects. Phosphatidylinositol 3-kinase (PI3K) pathway activation did not differ in UM-SCC-1-GALR1 and UM-SCC-1-mock cells after galanin treatment. Pertussis toxin and LY294002 inhibition demonstrated that galanin and GALR1 induce ERK1/2 activation via Gαi, not the PI3K pathway-linked to the Gβγ subunit. Galanin and GALR1 also inhibit colony formation and tumor growth in vivo. Our results implicate GALR1, a Gi protein-coupled receptor, as a tumor suppressor gene that inhibits cell proliferation via ERK1/2 activation.

Galanin receptor subtype 2 suppresses cell proliferation and induces apoptosis in p53 mutant head and neck cancer cells.

Purpose: Galanin and its three receptors (GALR1-3) are expressed in many normal tissues, but silenced in some tumors. Contradictory roles for galanin and its receptors in various tumors have been reported. To understand their function, investigations of individual galanin receptors are necessary. In head and neck squamous carcinoma cells (HNSCC) with silenced GALR1 and GALR2, we showed that reexpressed GALR1 suppresses tumor cell proliferation via Erk1/2-mediated effects on cdk inhibitors and cyclin D1. Others showed that GALR2 could induce apoptosis in neuroblastoma cells with wild-type p53, whereas GALR2 stimulated proliferation in small cell lung cancer. In this study, we investigated the role of GALR2 in HNSCC cells that have mutant p53 and do not express GALR1. Experimental Design: UM-SCC-1, a human oral carcinoma cell line with a splice site mutation causing a 46-bp p53 off-frame deletion, was stably transfected to express GALR2 (UM-SCC-1-GALR2). Results: Galanin treatment of UM-SCC-1-GALR2 caused morphologic changes and a marked decrease in cell number that were not observed in UM-SCC-1-mock cells. Galanin and GALR2 resulted in decreased bromodeoxyuridine incorporation, p27Kip1 and p57Kip2 up-regulation, and decreased cyclin D1 expression. These effects were similar to GALR1 signaling in HNSCC, but GALR2 also induced caspase-3–dependent apoptosis, which was confirmed by Annexin-V staining and DNA fragmentation analysis. These were not observed with GALR1. Conclusion: This study shows that GALR2 reexpression can inhibit cell proliferation and induce apoptosis in HNSCC cells with mutant p53. GALR2 may be a feasible target for HNSCC therapy.

Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences

BackgroundMyofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions.ResultsWe demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts.ConclusionsThis is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

Mouse embryonic stem cells inhibit murine cytomegalovirus infection through a multi-step process.

In humans, cytomegalovirus (CMV) is the most significant infectious cause of intrauterine infections that cause congenital anomalies of the central nervous system. Currently, it is not known how this process is affected by the timing of infection and the susceptibility of early-gestational-period cells. Embryonic stem (ES) cells are more resistant to CMV than most other cell types, although the mechanism responsible for this resistance is not well understood. Using a plaque assay and evaluation of immediate-early 1 mRNA and protein expression, we found that mouse ES cells were resistant to murine CMV (MCMV) at the point of transcription. In ES cells infected with MCMV, treatment with forskolin and trichostatin A did not confer full permissiveness to MCMV. In ES cultures infected with elongation factor-1α (EF-1α) promoter-green fluorescent protein (GFP) recombinant MCMV at a multiplicity of infection of 10, less than 5% of cells were GFP-positive, despite the fact that ES cells have relatively high EF-1α promoter activity. Quantitative PCR analysis of the MCMV genome showed that ES cells allow approximately 20-fold less MCMV DNA to enter the nucleus than mouse embryonic fibroblasts (MEFs) do, and that this inhibition occurs in a multi-step manner. In situ hybridization revealed that ES cell nuclei have significantly less MCMV DNA than MEF nuclei. This appears to be facilitated by the fact that ES cells express less heparan sulfate, β1 integrin, and vimentin, and have fewer nuclear pores, than MEF. This may reduce the ability of MCMV to attach to and enter through the cellular membrane, translocate to the nucleus, and cross the nuclear membrane in pluripotent stem cells (ES/induced pluripotent stem cells). The results presented here provide perspective on the relationship between CMV susceptibility and cell differentiation.

Expression of β-Adrenoceptor Subtypes in Urothelium, Interstitial Cells and Detrusor of the Human Urinary Bladder

Objective: We examined whether interstitial cells (ICs) of the human urinary bladder expressed β‐adrenoceptor (AR) subtypes, and semiquantitatively compared the staining intensity among urothelium, ICs and detrusor muscles.

Cytomegalovirus Initiates Infection Selectively from High-Level β1 Integrin–Expressing Cells in the Brain

Cytomegalovirus (CMV) is a prevalent pathogen in intrauterine infections that causes congenital anomalies such as CMV encephalitis, which is characterized by the focal areas of reactive gliosis, reactive mononuclear cells, microglial nodules, and ventriculoencephalitis. To elucidate the mechanisms of CMV susceptibility in the developing brain, cell tropism and the infectious dynamics of CMV infection were investigated. We evaluated intraventricular and intravascular infections from the perspective of the distribution of CMV and its receptor (β1 integrin) in the earliest phase of infection. Murine CMV (MCMV) immediate early 1-positive cells were colocalized mainly with meninges and choroid plexus (after intraventricular infection) or with endothelial cells and pericytes (after intravascular infection). Using green fluorescent protein-expressing recombinant MCMV particles and fluorescent microbeads (100 to 300 nm), we revealed that CMV particle size is the primary factor determining the initial CMV distribution. β1 Integrin inhibition using a shRNA and functional blocking antibody significantly reduced MCMV infection. IHC analysis, flow cytometric, and brain slice analyses strongly support that high-level β1 integrin-expressing cells (eg, endothelial cells, pericytes, meninges, choroid plexus, and neural stem progenitor cells) are the first targets of MCMV. Therefore, our data demonstrate that the initial distributions of MCMV particles and β1 integrin determine the distinct pattern of infection in the brain in the acute phase.

Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection.

Congenital cytomegalovirus (CMV) infection is the leading viral cause of neurodevelopmental disorders in humans, with the most severe and permanent sequelae being those affecting the cerebrum. As the fetal immune reactions to congenital CMV infection in the brain and their effects on cerebral development remain elusive, our aim was to investigate primitive innate immunity to CMV infection and its effects on cerebral corticogenesis in a mouse model for congenital CMV infection using a precise intraplacental inoculation method.

Radiologic pleuroparenchymal fibroelastosis-like lesion in connective tissue disease-related interstitial lung disease

Background Radiologic pleuroparenchymal fibroelastosis (PPFE)-like lesion including pulmonary apical cap can be occasionally observed in clinical settings. However, the significance of radiologic PPFE-like lesion is unclear in connective tissue disease (CTD)-related interstitial lung disease (ILD). Materials and methods A total of 113 patients with CTD-related ILD were enrolled and assessed for radiologic PPFE-like lesion, which was defined as bilateral, upper lobe, and subpleural dense consolidations with or without pleural thickening on chest high-resolution computed tomography. The clinical, radiologic, and pathologic characteristics were evaluated. Results Radiologic PPFE-like lesion was found in 21 patients (19%) and were relatively frequent in those with systemic sclerosis (6/14: 43%) and primary Sjögrens syndrome (4/14: 29%). Patients with PPFE-like lesion were significantly older, had lower body mass index, higher ratio of residual volume to total lung capacity, and higher complication rate of pneumothorax and/or pneumomediastinum than those without. Twelve of the 21 patients were diagnosed radiologically as usual interstitial pneumonia (UIP) or possible UIP pattern. Two of three patients who underwent surgical lung biopsy of the upper lobes showed UIP on histopathology. Another patient was confirmed to have upper lobe PPFE on autopsy. During the clinical course, progression of the radiologic PPFE-like lesions was observed in 13 of 21 patients. Six patients died (mortality rate: 29%) and their PPFE-like lesions were commonly progressive. In the total cohort, our multivariate analysis identified the presence of PPFE-like lesion as a significant risk factor for respiratory death (hazard ratio: 4.10, 95% confidence interval: 1.33–12.65, p = 0.01). Conclusion In patients with CTD-related ILD, radiologic PPFE-like lesion, which may present as not only PPFE but also apical cap and upper lobe subpleural fibrosis commonly due to UIP, was not uncommon and was associated with poor prognosis. Clinicians should be cautious with this radiologic finding, particularly when it is progressive.

Effects of intrapulmonary viral tropism and cytokine expression on the histological patterns of cytomegalovirus pneumonia

Pulmonary cytomegalovirus (CMV) infection causes fatal CMV pneumonia (CMVp) in immunocompromised patients; however, the mechanisms underlying CMV‐infection‐induced pulmonary lesion development remain largely unknown. We examined the relationship between CMVp patterns and intrapulmonary viral tropism, including expression of inflammatory cytokines and related molecules. Double immunohistochemistry of CMV antigen and cellular markers showed that epithelial tropism was associated with a diffuse alveolar damage (DAD) pattern (CMVp‐DAD) while stromal tropism was associated with a predominantly interstitial inflammation/fibrosis (IIF) (CMVp‐IIF) or a combination of DAD and IIF (CMVp‐complex). Transforming growth factor (TGF)‐β1 expression was relevant to CMV‐induced tissue injury, and its expression was higher in CMVp‐complex and CMVp‐IIF than in CMVp‐DAD. Expression of integrin β6 (ITGB6), an adhesion molecule and important activator of TGF‐β1 in interstitial pneumonia, was lost in CMV‐infected pneumocytes, especially CMVp‐DAD, whereas CMV‐negative pneumocytes in CMVp‐complex and CMVp‐IIF showed overexpression. Diffuse interleukin (IL)‐8 up‐regulation and strong expression were present in both CMV‐infected pneumocytes and stromal cells only in CMVp‐IIF cases with marked interstitial neutrophilic infiltration. On the basis of viral tropism and the expression of TGF‐β1, ITGB6, and IL‐8, we conclude that CMV‐infected pulmonary cells play an important role in the development of diverse CMVp patterns.

Lung cancer development in patients with connective tissue disease-related interstitial lung disease: A retrospective observational study.

AbstractPrevious studies have reported that patients with idiopathic pulmonary fibrosis occasionally develop lung cancer (LC). However, in connective tissue disease (CTD)-related interstitial lung disease (ILD), there are few data regarding the LC development. The aim of the present study was to evaluate the clinical significance of LC development in patients with CTD-ILD. A retrospective review of our database of 562 patients with ILD between 2000 and 2014 identified 127 patients diagnosed with CTD-ILD. The overall and cumulative incidences of LC were calculated. In addition, the risk factors and prognostic impact of LC development were evaluated. The median age at the ILD diagnosis was 63 years (range 37–84 years), and 73 patients (57.5%) were female. The median follow-up period from the ILD diagnosis was 67.4 months (range 10.4–322.1 months). During the period, 7 out of the 127 patients developed LC (overall incidence 5.5%). The cumulative incidences at 1, 3, and 5 years were 0.0%, 1.8%, and 2.9%, respectively. The risk of LC development was significantly higher in patients with higher smoking pack-year (odds ratio [OR] 1.028; 95% confidence interval [CI] 1.008–1.049; P = 0.007) and emphysema on chest high-resolution computed tomography (OR 14.667; 95% CI 2.871–74.926; P = 0.001). The median overall survival time after developing LC was 7.0 months (95% CI 4.9–9.1 months), and the most common cause of death was LC, not ILD. According to the Cox proportional hazard model analysis with time-dependent covariates, patients who developed LC showed significantly poorer prognosis than those who did not (hazard ratio 87.86; 95% CI 19.56–394.67; P < 0.001). In CTD-ILD, clinicians should be careful with the risk of LC development in patients with a heavy smoking history and subsequent emphysema. Although not so frequent, the complication could be a poor prognostic determinant.