Kyosuke Hatsushika

Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck.

Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer stem cells (CSCs), are responsible for maintenance and growth of the tumor. CD44 is currently used to identify CSCs as one of the cell surface markers for solid tumors. Here we report the identification, expansion, and characterization of CD44+ cancer stem-like cells from a permanent squamous cell carcinoma of the head and neck (SCCHN) cell line. Under serum-free medium culture conditions, a small population (less than 3%) of CD44+ cells in a permanent cancer cell line was dramatically increased up to around 40%. The CD44+ cell population also showed higher expression of CD133 and ABCG2 as compared with the CD44- cell population. Moreover, CD44+ cells possess not only a marked capacity for forming tumor spheres, proliferation, migration, and invasion in vitro, but also resistance to chemotherapeutic agents. Four genes related to chemoresistance, ABCB1, ABCG2, CYP2C8, and TERT, were up-regulated in a CD44+ cell population. Our findings indicate that a subpopulation of CSCs is maintained in the SCCHN cell line, and the presence of such CSCs has an important clinical implication for head and neck cancer treatment. Further characterization of CSCs may provide new insights for novel therapeutic targets and prognostic markers.

Mast cell regulation of epithelial TSLP expression plays an important role in the development of allergic rhinitis.

Epithelial cell‐derived thymic stromal lymphopoietin (TSLP) is a master switch for asthma or atopic dermatitis by inducing a dendritic cell‐mediated Th2‐type allergic inflammation. Allergic rhinitis is also pathologically characterized by Th2‐type allergic inflammation. This study demonstrates that mast cells regulate the epithelial TSLP expression in allergic rhinitis. TSLP expression was found to be up‐regulated predominantly in the nasal epithelium in the ovalbumin (OVA)‐sensitized and ‐nasally challenged mouse model of allergic rhinitis, which was abolished in mast cell‐deficient WBB6F1‐W/Wv in comparison with control WBB6F1‐+/+ mice. Similarly, the epithelial TSLP expression was reduced in Fc receptor γ chain (FcγR)‐deficient mice, where the high‐affinity IgE receptor (FcϵRI) is not expressed on mast cells, in comparison with control C57BL/6 mice. Furthermore, the administration of neutralizing TSLP antibody during the challenge phase of OVA inhibited the development of allergic rhinitis. These results suggest that the direct stimulation of epithelial cells by antigens alone may not be sufficient to induce TSLP expression in the nasal epithelium, and that mast cell regulation of epithelial TSLP expression, possibly via FcϵRI, plays an important role in the development of allergic rhinitis.

Cigarette smoke extract induces thymic stromal lymphopoietin expression, leading to TH2-type immune responses and airway inflammation

BACKGROUND Both active and passive smoking are considered to be risk factors for asthma development. However, the precise mechanisms involved remain elusive. Recently, thymic stromal lymphopoietin (TSLP) has been shown to play a key role in the development of T(H)2-type allergic inflammation in patients with asthma. OBJECTIVE The aim of this study was to investigate whether there was a causal relationship between cigarette smoke exposure and TSLP expression in the lung. METHODS We examined the effects of repeated intranasal exposure of cigarette smoke extract (CSE) on TSLP mRNA and protein expression in the mouse lung by means of real-time PCR, Western blotting, and immunohistochemistry. We also examined the effects of intranasal exposure of CSE plus ovalbumin (OVA) on T(H)2-type immune responses and lung pathology. RESULTS Repeated exposure of CSE induced TSLP mRNA and protein expression, which was inhibited by treatment with antioxidative N-acetylcysteine and by TNF-alpha receptor I deficiency. In addition, the intranasal exposure of CSE simultaneously with OVA induced OVA-specific T(H)2-type immune responses and airway inflammation, which were inhibited by the blockade of the TSLP activity. CONCLUSION CSE induced TSLP expression in the mouse lung in an oxidative stress-dependent and TNF-alpha receptor I-dependent manner, and when challenged simultaneously with an antigen, CSE promoted the development of airway inflammation in association with T(H)2-type immune responses.

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-κB (NF-κB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-κB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast – and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.

A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: Implications for herniated discs

OBJECTIVE To determine whether thymic stromal lymphopoietin (TSLP) plays a role in the resorption of herniated disc tissue. METHODS The expression of TSLP messenger RNA (mRNA) and protein in mouse intervertebral disc cells was assessed by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analysis. The ability of mouse intervertebral disc cells to respond to TSLP stimulation was examined by Western blot analysis, ELISA, and protein array analysis. Intracellular signaling pathways involved in TSLP signaling in mouse intervertebral disc cells were investigated using several chemical inhibitors. The role of TSLP in macrophage migration into the intervertebral disc was assessed by in vitro migration assay. Finally, TSLP expression in clinical specimens derived from patients with a herniated disc was examined by immunohistochemistry. RESULTS Mouse intervertebral disc cells expressed TSLP mRNA and protein upon stimulation with NF-kappaB-activating ligands such as tumor necrosis factor alpha. In addition, the mouse intervertebral disc cells expressed the TSLP receptor and produced monocyte chemotactic protein 1 (MCP-1; CCL2) and macrophage colony-stimulating factor in response to TSLP stimulation. Both anulus fibrosus and nucleus pulposus intervertebral disc cells expressed MCP-1 upon TSLP stimulation, which was mediated via the phosphatidylinositol 3-kinase/Akt pathway. Consistently, the supernatants of TSLP-activated intervertebral disc cultures had the capacity to induce macrophage migration in an MCP-1-dependent manner. Finally, TSLP and MCP-1 were coexpressed in human herniated disc specimens in which macrophage infiltration into the tissue was observed. CONCLUSION TSLP induced by NF-kappaB-activating ligands in intervertebral discs may contribute to the recruitment of macrophages to the intervertebral disc by stimulating MCP-1 production and may be involved in the resorption of herniated disc tissue.

A possible link between resveratrol and TGF-β : Resveratrol induction of TGF-β expression and signaling

Resveratrol, a polyphenolic compound found in the skin of red fruits, exhibits anti‐inflammatory, anti‐oxidative, and anti‐proliferative characteristics. Transforming growth factor‐β (TGF‐β) is a pleiotropic cytokine that also displays such properties. We therefore hypothesized that there might be a functional link between resveratrol and TGF‐β. This study reports that resveratrol increased transcription of the TGF‐β2 gene, enhanced the production of TGF‐β2 protein, and activated Smad signaling in an autocrine manner in A549 human lung epithelial cell line. Thus, some of the beneficial effects of resveratrol on human health might be mediated, in part, through its effects on TGF‐β expression and signaling.

Transforming growth factor‐β2 polymorphisms are associated with childhood atopic asthma

Background Transforming growth factor (TGF)‐β plays an important role in the regulation of airway inflammation and remodelling in asthma. Recent studies suggest that TGF‐β2 is a predominant isoform expressed in severe asthma and it is also associated with airway remodelling.

TGF-β Signaling May Play a Role in the Development of Goblet Cell Hyperplasia in a Mouse Model of Allergic Rhinitis

BACKGROUND Transforming growth factor-beta (TGF-beta) levels are elevated in the nasal mucosa in allergic rhinitis. However, because TGF-beta is secreted extracellulary in latent complexes, it remains unclear whether the local TGF-beta expression actually drives active signaling and affects the pathophysiology of allergic rhinitis. The objective of this study is to investigate whether TGF-beta signaling is activated in allergic rhinitis and plays a role in the pathophysiology of allergic rhinitis. METHODS An ovabumin (OVA)-sensitized and -nasally challenged mouse model of allergic rhinitis was established and phosphorylation of Smad2 in the nasal mucosa was examined by immunohistochemistry. In addition, the effects of the pharmacological inhibition of endogenous TGF-beta signaling on the allergic rhinitis model were histologically examined. Furthermore, phosphorylation of Smad2 in the nasal mucosa samples obtained from patients with allergic rhinitis was also evaluated. RESULTS In the mouse model of allergic rhinitis, OVA challenge induced phosphorylation of Smad2 predominantly in epithelial cells in the nasal mucosa. In addition, the administration of an inhibitor of TGF-beta type I receptor kinase activity during OVA challenge suppressed goblet cell hyperplasia in the nasal mucosa. Furthermore, phosphorylated Smad2 expression increased in nasal epithelial cells in patients with allergic rhinitis. CONCLUSIONS These results suggest that TGF-beta signaling is activated in epithelial cells in the nasal mucosa in allergic rhinitis and may contribute to the development of goblet cell hyperplasia.

Lung Functions of Japanese Patients with Chronic Rhinosinusitis Who Underwent Endoscopic Sinus Surgery

BACKGROUND Chronic rhinosinusitis (CRS), which is clinically classified into CRS without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP), shows considerable geographic differences and heterogeneity. Eosinophilic (E) CRS with nasal polyps (ECRSwNP) has a higher degree of disease severity and higher frequency of comorbid asthma. Epidemiologic studies in different ethnic populations have improved understanding of the pathophysiology of the disease. Here we report the clinical characteristics of Japanese patients with medically refractory CRS undergoing endoscopic sinus surgery (ESS). METHODS We recruited a total of 210 CRS patients and assessed them by nasal endoscopy, the Lund-Mackay score using computed tomography (CT), peripheral eosinophilia and smoking status. We also examined the comorbidity of asthma, effects of age and lung functions in the patients. RESULTS In this study, 13% of CRSwNP patients and 20% of CRSwNP patients with peripheral blood eosinophilia exhibited obstructive lung dysfunction (FEV1/FVC <70%) despite the absence of an asthma diagnosis. Among elderly nonsmoker patients (≥ 60 years) who had never been diagnosed with asthma, 50% of CRSwNP patients with peripheral blood eosinophilia showed decreased FEV1/FVC <70%. CONCLUSIONS Our findings suggest that asthma is under-diagnosed in CRS patients who undergo ESS, especially the elderly. Although the association between CRS and asthma has been recognized, increased attention to the comorbidity of obstructive airway diseases such as asthma is still needed for management of medically refractory CRS.

Human Eosinophils Have an Intact Smad Signaling Pathway Leading to a Major Transforming Growth Factor-β Target Gene Expression

Background: There is a paradoxical finding that eosinophils are frequently accumulated at the sites of allergic inflammation where transforming growth factor (TGF)-β, a negative regulator of eosinophil survival, is upregulated; however, eosinophil accumulation is persistent. We thus hypothesized that eosinophils might have aberrant TGF-β signaling and be unresponsive to TGF-β. To test the hypothesis, we examined the expression and function of Smad proteins, which are central mediators for TGF-β signaling, in human eosinophils. Methods: Eosinophils were isolated from the peripheral blood of normal donors, and the expression and activation of endogenous Smad proteins were examined by reverse transcription polymerase chain reaction and Western blotting. The Smad function in the transcription of the major TGF-β target gene Smad7 was investigated using a dominant negative form of Smad3. The effect of TGF-β on eosinophil survival was then evaluated by a cell viability assay using normal and asthmatic eosinophils. Results: Human eosinophils expressed mRNAs and proteins of TGF-β typeI and type II receptors, Smad2, Smad3 and Smad4. TGF-β induced the phosphorylation of Smad2 in eosinophils, which was blocked by SB431542, an inhibitor of TGF-β type I receptor kinase. A dominant negative Smad3 protein suppressed TGF-β-induced Smad7 mRNA expression in eosinophils. Finally, TGF-β prevented granulocyte macrophage colony-stimulating factor- or interferon-γ-mediated survival of eosinophils obtained from asthmatic patients as well as normal subjects. Conclusion: Human eosinophils have an intact Smad signaling pathway leading to a major TGF-β target gene expression. Thus, eosinophils might become resistant to TGF-β only in in vivo circumstances.