Effects of prolonged stimulation with heated tobacco products (Ploom TECH+ ) on gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Heated tobacco products have recently become commercially available. These products, as well as combustible cigarettes, produce aerosols; the risk of various diseases associated with heated tobacco products may be the same or higher than that with combustible cigarettes. In this study, we examined the effect of Ploom TECH+ extract on gingival epithelial cells. METHODS: Tobacco leaves from Ploom TECH+ tobacco capsules and water were mixed and heated; the supernatant subsequently collected was the heated tobacco product (HTP; control: HTP not added). Normal human gingival epithelial progenitors were cultured alternately with or without HTP for a total of 1 month. Subsequently, RNA, DNA, and proteins were isolated from these samples and comprehensively analyzed using RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and western blotting, respectively. RESULTS: RNA-seq revealed that 284 genes showed a twofold increase and 145 genes showed a twofold decrease in gene expression. A heat map showed genetic differences between the control and HTP groups. A principal component analysis plot showed a clear genetic distribution between the control and HTP. Gene Ontology (GO) analysis showed that genes related to seven GO terms, including cornification and keratinization, were induced by long-term HTP stimulation. By contrast, GO pathways with a significant decrease in component expression were not detected. RRBS revealed that CpG island methylation increased more than twofold in 158 genes and decreased to less than twofold in 171 genes. Methylation of these CpG islands was not correlated with changes in gene expression levels. HTP treatment increased S100A7 expression. CONCLUSION: Long-term HTP stimulation affected epithelial differentiation and keratinization of gingival epithelial cells. Thus, habitual use of Ploom TECH+ may be a risk factor for tobacco-related oral mucosal diseases.

The Role of Hedgehog Signaling in the Melanoma Tumor Bone Microenvironment.

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion.

High-mobility group box 1 induces bone destruction associated with advanced oral squamous cancer via RAGE and TLR4.

Bone destruction of maxillary and mandibular bone by invasive oral squamous cell cancer (OSCC) raises various problems in the management of patients, resulting in poor outcomes and survival. However, the mechanism behind bone destruction by OSCC remains unclear. High-mobility group box 1 (HMGB1), a highly conserved ubiquitous nuclear non-histone DNA-binding protein, has been demonstrated to be secreted by aggressive cancers and regulate osteoclastogenesis, a central player during bone destruction. We therefore reasoned that HMGB1 secreted by OSCCs contributes to bone destruction. Our results showed that HMGB1 is produced by human cell lines of OSCC and promotes osteoclastogenesis via up-regulation of the expression of receptor activator of nuclear factor kappa-Β ligand in osteoblasts and osteocytes, and consequently osteoclastic bone destruction in mice. Further, we found that these actions of HMGB1 are mediated via the receptor for advanced glycation end products and toll-like receptors. These findings suggest that HMGB1 of OSCC and its down-stream signal pathways are potential targets for the treatment of bone destruction associated with advanced OSCC.

Hedgehog Signaling in Organogenesis and the Tumor Microenvironment.

The Hedgehog signaling pathway was first discovered in 1980 during a large-scale genetic screening seeking to find mutations that affect larval body segment development in the fruit fly, Drosophila melanogaster [...].

Sonic Hedgehog Regulates Bone Fracture Healing.

Bone fracture healing involves the combination of intramembranous and endochondral ossification. It is known that Indian hedgehog (Ihh) promotes chondrogenesis during fracture healing. Meanwhile, Sonic hedgehog (Shh), which is involved in ontogeny, has been reported to be involved in fracture healing, but the details had not been clarified. In this study, we demonstrated that Shh participated in fracture healing. Six-week-old Sprague-Dawley rats and Gli-CreERT2; tdTomato mice were used in this study. The right rib bones of experimental animals were fractured. The localization of Shh and Gli1 during fracture healing was examined. The localization of Gli1 progeny cells and osterix (Osx)-positive cells was similar during fracture healing. Runt-related transcription factor 2 (Runx2) and Osx, both of which are osteoblast markers, were observed on the surface of the new bone matrix and chondrocytes on day seven after fracture. Shh and Gli1 were co-localized with Runx2 and Osx. These findings suggest that Shh is involved in intramembranous and endochondral ossification during fracture healing.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

Expression and Role of IL-1ß Signaling in Chondrocytes Associated with Retinoid Signaling during Fracture Healing.

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1ß (IL-1ß) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1ß and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1ß in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1ß and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1ß decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1ß-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1ß treatment, and the IL-1ß-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1ß-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1ß via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process.

The Role of Sonic Hedgehog Signaling in the Tumor Microenvironment of Oral Squamous Cell Carcinoma.

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of SHH expression appear to correlate with cancer progression. However, the role of SHH in the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC) is still unclear. No studies have compared the expression of SHH in different subtypes of OSCC and focused on the relationship between the tumor parenchyma and stroma. In this study, we analyzed SHH and expression of its receptor, Patched-1 (PTCH), in the TME of different subtypes of OSCC. Fifteen endophytic-type cases (ED type) and 15 exophytic-type cases (EX type) of OSCC were used. H&E staining, immunohistochemistry (IHC), double IHC, and double-fluorescent IHC were performed on these samples. ED-type parenchyma more strongly expressed both SHH and PTCH than EX-type parenchyma. In OSCC stroma, CD31-positive cancer blood vessels, CD68- and CD11b-positive macrophages, and α-smooth muscle actin-positive cancer-associated fibroblasts partially expressed PTCH. On the other hand, in EX-type stroma, almost no double-positive cells were observed. These results suggest that autocrine effects of SHH induce cancer invasion, and paracrine effects of SHH govern parenchyma-stromal interactions of OSCC. The role of the SHH pathway is to promote growth and invasion.

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling.

Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically, as it can invade facial bones and cause bone pain that is undertreated and poorly understood. Here we studied HNSCC bone pain (HNSCC-BP) in an intratibial mouse xenograft model that uses a human HNSCC cell line (SAS cells). These mice develop HNSCC-BP associated with an upregulation of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in the dorsal root ganglia (DRGs) of sensory nerve cell bodies. Our experiments demonstrated that the inhibition of monocarboxylate transporter 4 (MCT4) by short hairpin (shRNA) transduction suppressed the HNSCC-BP, the lactate level in bone marrow, and the pERK1/2 expression in DRG. The sensory nerves also expressed increased levels of the acid-sensing receptor TRPV1. DRG neurons co-cultured with SAS cells showed increased neurite outgrowth, and were inhibited by MCT4 silencing with shRNA. Collectively, our results show that HNSCC induced an acidic bone microenvironment that evokes HNSCC-BP via MCT4 expression.

A novel PI3K inhibitor iMDK suppresses non-small cell lung Cancer cooperatively with A MEK inhibitor.

The PI3K-AKT pathway is expected to be a therapeutic target for non-small cell lung cancer (NSCLC) treatment. We previously reported that a novel PI3K inhibitor iMDK suppressed NSCLC cells in vitro and in vivo without harming normal cells and mice. Unexpectedly, iMDK activated the MAPK pathway, including ERK, in the NSCLC cells. Since iMDK did not eradicate such NSCLC cells completely, it is possible that the activated MAPK pathway confers resistance to the NSCLC cells against cell death induced by iMDK. In the present study, we assessed whether suppressing of iMDK-mediated activation of the MAPK pathway would enhance anti-tumorigenic activity of iMDK. PD0325901, a MAPK inhibitor, suppressed the MAPK pathway induced by iMDK and cooperatively inhibited cell viability and colony formation of NSCLC cells by inducing apoptosis in vitro. HUVEC tube formation, representing angiogenic processes in vitro, was also cooperatively inhibited by the combinatorial treatment of iMDK and PD0325901. The combinatorial treatment of iMDK with PD0325901 cooperatively suppressed tumor growth and tumor-associated angiogenesis in a lung cancer xenograft model in vivo. Here, we demonstrate a novel treatment strategy using iMDK and PD0325901 to eradicate NSCLC.

Autologous Blood Injection for the Treatment of Recurrent Temporomandibular Joint Dislocation.

Temporomandibular joint (TMJ) dislocation can occur during daily activities and negatively affect a patient's quality of life. Although both nonsurgical and surgical techniques have been used to treat recurrent TMJ dislocation, the former is not always successful and the latter, although having a high success rate, is invasive and requires hospitalization. Recently, autologous blood injection has been used to treat recurrent TMJ dislocation. However, this technique is not yet widely used in clinical practice. We designed this study to obtain further information as to efficacy, safety and stability of autologous blood injection for recurrent TMJ dislocation.

Expression pattern of sonic hedgehog signaling and calcitonin gene-related peptide in the socket healing process after tooth extraction.

Sonic Hedgehog (SHH), a neural development inducer, plays a significant role in the bone healing process. Calcitonin gene-related peptide (CGRP), a neuropeptide marker of sensory nerves, has been demonstrated to affect bone formation. The roles of SHH signaling and CGRP-positive sensory nerves in the alveolar bone formation process have been unknown. Here we examined the expression patterns of SHH signaling and CGRP in mouse socket by immunohistochemistry and immunofluorescence analysis. We found that the expression level of SHH peaked at day 3 and was then decreased at 5 days after tooth extraction. CGRP, PTCH1 and GLI2 were each expressed in a similar pattern with their highest expression levels at day 5 and day 7 after tooth extraction. CGRP and GLI2 were co-expressed in some inflammatory cells and bone forming cells. In some areas, CGRP-positive neurons expressed GLI2. In conclusion, SHH may affect alveolar bone healing by interacting with CGRP-positive sensory neurons and thus regulate the socket's healing process after tooth extraction.

Correction: Inhibition of the Growth Factor MDK/Midkine by a Novel Small Molecule Compound to Treat Non-Small Cell Lung Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0071093.].

Novel role of miR-181a in cartilage metabolism.

Micro RNA (miRNA) is a small non-coding post-transcriptional RNA regulator that is involved in a variety of biological events. In order to specify the role of miRNAs in cartilage metabolism, we comparatively analyzed the expression profile of known miRNAs in chicken sternum chondrocytes representing early and late differentiation stages. Interestingly, none of the miRNAs displaying strong expression levels showed remarkable changes along with differentiation, suggesting their roles in maintaining the homeostasis rather than cytodifferentiation of chondrocytes. Among these miRNAs, miR-181a, which is known to play critical roles in a number of tissues, was selected and was further characterized. Human microarray analysis revealed remarkably stronger expression of miR-181a in human HCS-2/8 cells, which strongly maintained a chondrocytic phenotype, than in HeLa cells, indicating its significant role in chondrocytes. Indeed, subsequent investigation indicated that miR-181a repressed the expression of two genes involved in cartilage development. One was CCN family member 1 (CCN1), which promotes chondrogenesis; and the other, the gene encoding the core protein of aggrecan, a major cartilaginous proteoglycan, aggrecan. Based on these findings, negative feedback system via miR-181a to conserve the integrity of the cartilaginous phenotype may be proposed.

Severe gummy smile with class II malocclusion treated with LeFort I osteotomy combined with horseshoe osteotomy and intraoral vertical ramus osteotomy.

In this article, we report the successful surgical treatment of a patient, 34 years of age, who had a severe gummy smile and a class II malocclusion. The patient had an 11-mm gingival exposure during full smile and a convex profile. A LeFort I osteotomy combined with a horseshoe osteotomy was used for the superior repositioning of the maxilla;then, an intraoral vertical ramus osteotomy (IVRO) and genioplasty were performed for mandibular advancement. The maxilla was acceptably impacted 8mm at the first incisor and 5mm at the first molar. Both the occlusion and facial appearance were significantly improved by this surgical-orthodontic treatment. Our results suggest that the combination of a horseshoe osteotomy with a LeFort I osteotomy is a useful technique for reliable superior repositioning of the maxilla.

Angiogenesis Assays for the Analysis of CCN Proteins.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the actin of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) and CCN2-neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM). In addition, we introduce an autofluorescence imaging of blood vessels in the subcutaneous tumor xenograft mouse model. These assays can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Immunohistochemical Analysis of CCN2 in Experimental Fracture Healing Models.

Skeletal fractures are most common large-organ traumatic injuries that impact the functions and esthetic outcomes and quality of life. Unfortunately, infection during the fracture healing process and inadequate blood supply to the bone impede reduced ability to produce cartilage and effective bone callus formation, leading to nonunion or delayed union fracture. Therefore, studying the mechanism of fracture healing is an important task in solving the problem of fracture healing failure. Animal models of bone fracture healing are important tools to investigate the pathogenesis and develop treatment strategies. This protocol introduces researchers to a bone repair model utilizing the ribs of rats and the immunohistological expression of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) during the fracture healing processes.

Mouse Models of Tumor Bone Metastasis and Invasion for Studying CCN Proteins.

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

Bone formation ability of Gli1+ cells in the periodontal ligament after tooth extraction.

During the process of socket healing after tooth extraction, osteoblasts appear in the tooth socket and form alveolar bone; however, the source of these osteoblasts is still uncertain. Recently, it has been demonstrated that cells expressing Gli1, a downstream factor of sonic hedgehog signaling, exhibit stem cell properties in the periodontal ligament (PDL). Therefore, in the present study, the differentiation ability of Gli1+-PDL cells after tooth extraction was analyzed using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. After the final administration of tamoxifen to iGli1/Tomato mice, Gli1/Tomato+ cells were rarely detected in the PDL. One day after the tooth extraction, although inflammatory cells appeared in the tooth socket, Periostin+ PDL-like tissues having a few Gli1/Tomato+ cells remained near the alveolar bone. Three days after the extraction, the number of Gli1/Tomato+ cells increased as evidenced by numerous PCNA+ cells in the socket. Some of these Gli1/Tomato+ cells expressed BMP4 and Phosphorylated (P)-Smad1/5/8. After seven days, the Osteopontin+ bone matrix was formed in the tooth socket apart from the alveolar bone. Many Gli1/Tomato+ osteoblasts that were positive for Runx2+ were arranged on the surface of the newly formed bone matrix. In the absence of Gli1+-PDL cells in Gli1-CreERT2/Rosa26-loxP-stop-loxP-tdDTA (iGli1/DTA) mice, the amount of newly formed bone matrix was significantly reduced in the tooth socket. Therefore, these results collectively suggest that Gli1+-PDL cells differentiate into osteoblasts to form the bone matrix in the tooth socket; thus, this differentiation might be regulated, at least in part, by bone morphogenetic protein (BMP) signaling.