Role of Snai2 and Notch signaling in salivary gland myoepithelial cell fate.

Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.

Induction of salivary gland-like cells from epithelial tissues transdifferentiated from mouse embryonic fibroblasts.

Salivary gland hypofunction due to radiation therapy for head and neck cancer or Sjögren syndrome may cause various oral diseases, which can lead to a decline in the quality of life. Cell therapy using salivary gland stem cells is a promising method for restoring hypofunction. Herein, we show that salivary gland-like cells can be induced from epithelial tissues that were transdifferentiated from mouse embryonic fibroblasts (MEFs). We introduced four genes, Dnp63a, Tfap2a, Grhl2, and Myc (PTMG) that are known to transdifferentiate fibroblasts into oral mucosa-like epithelium in vivo into MEFs. MEFs overexpressing these genes showed epithelial cell characteristics, such as cobblestone appearance and E-cadherin positivity, and formed oral epithelial-like tissue under air-liquid interface culture conditions. The epithelial sheet detached from the culture dish was infected with adenoviruses encoding Sox9 and Foxc1, which we previously identified as essential factors to induce salivary gland formation. The cells detached from the cell sheet formed spheres 10 days after infection and showed a branching morphology. The spheres expressed genes encoding basal/myoepithelial markers, cytokeratin 5, cytokeratin 14, acinar cell marker, aquaporin 5, and the myoepithelial marker α-smooth muscle actin. The dissociated cells of these primary spheres had the ability to form secondary spheres. Taken together, our results provide a new strategy for cell therapy of salivary glands and hold implications in treating patients with dry mouth.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands.

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

Cytokine profiles in maternal serum are candidates for predicting an optimal timing for the delivery in early-onset fetal growth restriction.

OBJECTIVE: We examined whether maternal serum cytokine profiles of mothers with early-onset fetal growth restriction (FGR) were associated with delivery within 2 weeks after sampling during the third trimester. STUDY DESIGN: This exploratory prospective cross-sectional study included a total of 20 singleton fetuses with early-onset FGR and 31 healthy controls. Maternal serum samples during the early third trimester were analyzed for 23 cytokines. RESULTS: Of 20 fetuses with early-onset FGR, 14 had delivery within 2 weeks after sampling. Multivariate analysis revealed that maternal serum concentrations of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) and soluble CD40 ligand (sCD40L) were independently associated with delivery within 2 weeks in early-onset FGR. Among cases of early-onset FGR, concentrations of almost all maternal serum cytokines were similar. Maternal serum sVEGFR-1 concentrations were high when delivery occurred within 2 weeks. Maternal serum sCD40L concentrations were elicited only in cases in which delivery within 2 weeks occurred due to fetal deterioration. CONCLUSION: We identified two biomarkers, one specific for FGR and the other dependent on severity, that were significant components of angiogenic activities and inflammation factors. Imbalances in serum protein expression may have a substantial effect on the pathogenesis of FGR.

Extracellular matrix loss in chondrocytes after exposure to interleukin-1ß in NADPH oxidase-dependent manner.

Osteoarthritis is a degenerative joint disease caused by excessive death of chondrocytes and loss of the extracellular matrix (ECM) in articular cartilage. We previously reported that reactive oxygen species (ROS) generated by the NADPH oxidase (NOX) isoform NOX-2 are involved in chondrocyte death induced by interleukin-1ß (IL-1ß). In this study, we investigate the role of NOX-2 in the production and degradation of ECM by chondrocytes. Although IL-1ß lowered the mRNA expression of type II collagen (Col2a1) and aggrecan (Acan) in mouse chondrocyte-like ATDC5 cells, RNA silencing of Nox2 did not change the mRNA expression of these major components of the ECM of cartilage. Hence, NOX-2 is not involved in the IL-1ß-induced suppression of ECM production. On the other hand, the NOX inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), the ROS scavenger N-acetylcysteine and an antisense oligodeoxynucleotide for Nox2 prevented the loss of proteoglycan induced by IL-1ß in highly differentiated ATDC5 cells. Furthermore, AEBSF did not affect the expression of hyaluronidase-1 and -2, whereas it suppressed hyaluronidase activity in culture medium. IL-1ß-induced intra- and extracellular acidification was also suppressed by AEBSF, as was the antisense oligodeoxynucleotide for Nox2. Since hyaluronidase activity is known to be higher under acidic conditions, NOX-2 probably contributes to ECM loss by the activation of hyaluronidase through acidification.

The ß-catenin signaling pathway induces aggressive potential in breast cancer by up-regulating the chemokine CCL5.

ß-Catenin signaling plays a pivotal role in the genesis of a variety of malignant tumors, but its role in breast cancer has not been fully elucidated. Here, we examined whether deregulation of ß-catenin signaling is related to the aggressive characteristics of certain types of breast cancers. Analysis of cytokine levels in MDA-MB-231 cells overexpressing a constitutively active form of ß-catenin (CAß-catenin) revealed a higher level of CCL5 expression. Cells transfected with CAß-catenin or stimulated with recombinant CCL5 exhibited increased cell invasion activity and spheroid formation in vitro. Furthermore, CAß-catenin-transfected MDA-MB-231 cells formed larger tumor masses that contained more Ki-67-positive cells and infiltrating lymphocytes than did the control cells. An inhibitor of CCR5 and a pan-CXCR neutralizing antibody dramatically reduced CAß-catenin-promoted activities. In addition to CCL5, 6-BIO, a chemical activator of ß-catenin, induced cell invasion and spheroid formation in MDA-MB-231 cells. Furthermore, high levels of nuclear ß-catenin accumulation were detected in breast cancer in patients with metastasis but not in those without metastasis. Nuclear ß-catenin localization is related to increased CCL5 production in breast cancer. These findings suggest that ß-catenin expression enhances tumor progression via chemokine production in breast cancers and that ß-catenin signaling is a critical regulator of the aggressive traits of breast cancers.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1ß but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1ß in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1ß were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

ANGPTL4 regulates the metastatic potential of oral squamous cell carcinoma.

Lymph node metastasis is a major factor for poor prognosis in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms of lymph node metastasis are unclear. We determined that angiopoietin-like protein 4 (ANGPTL4) mRNA and protein expression were increased in OSCC cells established from the primary site in metastatic cases. In addition, ANGPTL4 expression in biopsy specimens was correlated with the presence of lymph node metastasis. Therefore, our initial findings suggest that OSCC cells expressing ANGPTL4 may possess metastatic ability. Furthermore, cell culture supernatants from OSCC cells that metastasized to the lymph node contain ANGPTL4 and promote invasive ability. These findings suggest that secreted ANGPTL4 may affect the invasive ability of OSCC. Moreover, the rates of positive ANGPTL4 expression at the primary site were significantly higher in the lymph node metastasis group. These results demonstrate that ANGPTL4 contributes to OSCC metastasis by stimulating cell invasion. Therefore, ANGPTL4 is a potential therapeutic target for preventing cancer metastasis.

Potential role of hematopoietic pre-B-cell leukemia transcription factor-interacting protein in oral carcinogenesis.

BACKGROUND: Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes. MATERIALS AND METHODS: Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted. RESULTS: HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation. CONCLUSION: HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.

Loss of ß-catenin induces multifocal periosteal chondroma-like masses in mice.

Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of ß-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of ß-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after ß-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of ß-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a ß-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable ß-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of ß-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.