Disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells results in tumor progression, possibly through an increase of MMP-2 and MMP-9 expression.

OBJECTIVES: This study aimed to clarify the molecular mechanism underlying the higher invasion and metastasis abilities of LMF4 cells than those of HSC-3 cells by comparing the expression levels of the tumor suppressor factor, cell adhesion molecule 1 (CADM1). METHODS: We explored 1) whether CADM1 expression level was downregulated in LMF4 cells compared with HSC-3 cells, 2) whether CADM1 expression knockdown increased the expression levels of matrix metalloproteinases (MMPs), 3) the exact cellular signaling pathways responsible for increased MMP expression after knockdown of CADM1 expression, and 4) whether disruption of CADM1-dependent HSC-3 cell adhesion increased the migratory and invasive activities of HSC-3 cells. RESULTS: CADM1 expression was lower in the LMF4 than in the HSC-3 cells. The knockdown of CADM1 increased the expression of MMP-2 and MMP-9 in HSC-3 cells. In addition, the upregulation of MMP-2 expression after CADM1 knockdown was abrogated by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 and the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. The upregulation of MMP-9 expression after the knockdown of CADM1 was abrogated by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 MAP kinase (MAPK) inhibitor SB203580 and LY294002. Anti-CADM1 neutralizing antibody evoked migratory and invasive abilities of HSC-3 cells. CONCLUSION: The disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells resulted in tumor progression, possibly through an increase in MMP-2 expression in a MEK/PI3K-dependent manner and an increase in MMP-9 expression in a JNK/p38 MAPK/PI3K-dependent manner.

Receptor tyrosine kinase ligands and inflammatory cytokines cooperatively suppress the fibrogenic activity in temporomandibular-joint-derived fibroblast-like synoviocytes via mitogen-activated protein kinase kinase/extracellular signal-regulated kinase.

Osteoarthritis (OA)-related fibrosis is a possible cause of temporomandibular joint (TMJ) stiffness. However, the molecular mechanisms underlying the fibrogenic activity in fibroblast-like synoviocytes (FLSs) remain to be clarified. The present study examined the effects of receptor tyrosine kinase (RTK) ligands, such as fibroblast growth factor (FGF)-1 and epidermal growth factor (EGF), on myofibroblastic differentiation of the FLS cell line FLS1, which is derived from the mouse TMJ. The present study revealed that both FGF-1 and EGF dose-dependently suppressed the expression of the myofibroblast (MF) markers, including α-smooth muscle actin (α-SMA) and type I collagen, in FLS1 cells. Additionally, both FGF-1 and EGF activated extracellular signal-regulated kinase (ERK) in FLS1 cells. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 abrogated the FGF-1- and EGF-mediated suppression of MF marker expression. On the other hand, inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, also suppressed the expression of MF markers in FLS1 cells. Importantly, U0126 abrogated the inflammatory cytokine-mediated suppression of MF marker expression. Interestingly, RTK ligands and inflammatory cytokines additively suppressed the expression of type I collagen. These results suggested that RTK ligands and inflammatory cytokines cooperatively inhibited the fibrogenic activity in FLSs derived from the TMJ in a MEK/ERK-dependent manner. The present findings partially clarify the molecular mechanisms underlying the development of OA-related fibrosis in the TMJ and may aid in identifying therapeutic targets for this condition. Additionally, FGF-1 and EGF could be therapeutically utilized to prevent OA-related fibrosis around the inflammatory TMJ.

Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition.

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.

IL­1ß and TNF­α suppress TGF­ß­promoted NGF expression in periodontal ligament­derived fibroblasts through inactivation of TGF­ß­induced Smad2/3­ and p38 MAPK­mediated signals.

Mechanosensitive (MS) neurons in the periodontal ligament (PDL) pass information to the trigeminal ganglion when excited by mechanical stimulation of the tooth. During occlusal tooth trauma of PDL tissues, MS neurons are injured, resulting in atrophic neurites and eventual degeneration of MS neurons. Nerve growth factor (NGF), a neurotrophic factor, serves important roles in the regeneration of injured sensory neurons. In the present study, the effect of pro­inflammatory cytokines, including interleukin 1ß (IL­1ß) and tumor necrosis factor α (TNF­α), on transforming growth factor ß1 (TGF­ß1)­induced NGF expression was evaluated in rat PDL­derived SCDC2 cells. It was observed that TGF­ß1 promoted NGF expression via Smad2/3 and p38 mitogen­activated protein kinase (MAPK) activation. IL­1ß and TNF­α suppressed the TGF­ß1­induced activation of Smad2/3 and p38 MAPK, resulting in the abrogation of NGF expression. NGF secreted by TGF­ß1­treated SCDC2 cells promoted neurite extension and the expression of tyrosine hydroxylase, a rate­limiting enzyme in dopamine synthesis in rat pheochromocytoma PC12 cells. These results suggested that pro­inflammatory cytokines suppressed the TGF­ß­mediated expression of NGF in PDL­derived fibroblasts through the inactivation of TGF­ß­induced Smad2/3 and p38 MAPK signaling, possibly resulting in the disturbance of the regeneration of injured PDL neurons.

Cell-cell interactions between monocytes/macrophages and synoviocyte-like cells promote inflammatory cell infiltration mediated by augmentation of MCP-1 production in temporomandibular joint.

Many inflammatory cells are known to be home to inflamed temporomandibular joint (TMJ) tissues by stimulation with cytokines and chemokines produced by inflammatory lesions in the TMJ. However, how the inflammatory cells affect the progression of inflammation in TMJ synovial tissues after their homing to inflamed TMJ site is still uncertain. Here, we isolated and cultured TMJ synoviocyte-like cells (TMJSCs) from murine TMJ tissues. We demonstrated that interleukin 1ß (IL-1ß) up-regulated expression of monocyte chemoattractant protein 1 (MCP-1) in TMJSCs. In addition, we found that IL-1ß-treated TMJSCs strongly promoted migratory activity of mouse monocyte/macrophage RAW264.7 cells through secretion of MCP-1. On the other hand, IL-1ß up-regulated expression levels of intracellular adhesion molecule 1 (ICAM-1), a leukocyte adhesion ligand in TMJSCs. In addition, IL-1ß promoted cell-cell adhesion between TMJSCs and RAW264.7 cells. Intriguingly, we also found that cell-cell interactions mediated through soluble factors other than IL-1ß and cell-cell adhesion molecules between IL-1ß-stimulated TMJSCs and RAW264.7 cells synergistically augmented secretion of MCP-1 from these cells. Therefore, these results suggested that the IL-1ß-induced recruitment of monocyte/macrophage lineage cells to inflamed synovial membranes in TMJ was further augmented by the cell-cell interaction-induced secretion of MCP-1 from the inflammation site, possibly resulting in prolonged inflammatory responses in TMJ synovial tissue.

Water-soluble factors eluated from surface pre-reacted glass-ionomer filler promote osteoblastic differentiation of human mesenchymal stem cells.

Surface pre-reacted glass­ionomer (S­PRG)-containing dental materials, including composite and coating resins have been used for the restoration and/or prevention of dental cavities. S­PRG is known to have the ability to release aluminum, boron, fluorine, silicon, and strontium ions. Aluminum ions are known to be inhibitors whereas boron, fluorine, silicon, and strontium ions are known to be promoters of mineralization, via osteoblasts. However, it remains to be clarified how an aqueous eluate obtained from S­PRG containing these ions affects the ability of mesenchymal stem cells (MSCs), which are known to be present in dental pulp and bone marrow, to differentiate into osteogenic cell types. The present study demonstrated that 200­ to 1,000­fold­diluted aqueous eluates obtained from S­PRG significantly upregulated the mRNA expression level of the osteogenic differentiation marker alkaline phosphatase in human MSCs (hMSCs) without exhibiting the cytotoxic effect. In addition, the 500­ to 1,000­fold­diluted aqueous eluates obtained from S­PRG significantly and clearly promoted mineralization of the extracellular matrix of hMSCs. It was additionally demonstrated that hMSCs cultured on the cured resin composites containing S­PRG fillers exhibited osteogenic differentiation in direct correlation with the weight percent of S­PRG fillers. These results strongly suggested that aqueous eluates of S­PRG fillers promoted hard tissue formation by hMSCs, implicating that resins containing S­PRG may act as a useful biomaterial to cover accidental exposure of dental pulp.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.

Transforming growth factor-ß1 suppresses bone morphogenetic protein-2-induced mesenchymal-epithelial transition in HSC-4 human oral squamous cell carcinoma cells via Smad1/5/9 pathway suppression.

Squamous cell carcinoma is the most common cancer in the oral cavity. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) promotes the epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (hOSCC) cells; however, it remains to be clarified whether the TGF-ß superfamily member bone morphogenetic protein (BMP) affects this process in hOSCC cells. Here, we examined the independent and collective effects of TGF-ß1 and BMP-2 on EMT and mesenchymal­epithelial transition (MET) in a panel of four hOSCC cell lines. Notably, we found that HSC-4 cells were the most responsive to BMP-2 stimulation, which resulted in the upregulation of Smad1/5/9 target genes such as the MET inducers ID1 and cytokeratin 9 (CK9). Furthermore, BMP-2 downregulated the mesenchymal marker N-cadherin and the EMT inducer Snail, but upregulated epithelial CK9 expression, indicating that BMP-2 prefers to induce MET rather than EMT. Moreover, TGF-ß1 dampened BMP-2-induced epithelial gene expression by inhibiting Smad1/5/9 expression and phosphorylation. Functional analysis revealed that TGF-ß1 and BMP-2 significantly enhanced HSC-4 cell migration and proliferation, respectively. Collectively, these data suggest that TGF-ß positively regulates hOSCC invasion in the primary tumor, whereas BMP-2 facilitates cancer cell colonization at secondary metastatic sites. Thus, the invasive and metastatic characteristics of hOSCC appear to be reciprocally regulated by BMP and TGF-ß.

Zoledronic acid suppresses transforming growth factor-ß-induced fibrogenesis by human gingival fibroblasts.

Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-ß (TGF­ß)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF­ß-induced promotion of cell viability, ii) the TGF­ß-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF­ß-induced migratory activity of hGFs and iv) the expression level of TGF­ß type I receptor on the surfaces of hGFs, as well as the TGF­ß-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF­ß-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad­dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level.

Transforming growth factor-ß1 induces invasion ability of HSC-4 human oral squamous cell carcinoma cells through the Slug/Wnt-5b/MMP-10 signalling axis.

Molecular mechanism underlying the invasion of oral cancer cells remains to be clarified. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) induces the expression of mesenchymal markers in human oral squamous cell carcinoma HSC-4 cells. Intriguingly, the expression of the epithelial-mesenchymal transition-related transcription factor Slug was also significantly upregulated upon TGF-ß1 stimulation. However, the mechanism by which Slug transduces the TGF-ß1-induced signal to enhance the invasiveness of HSC-4 cells is poorly understood. Proteomic analysis revealed that the expression of matrix metalloproteinase (MMP)-10 was upregulated in TGF-ß1-stimulated cells. Additionally, a Boyden chamber assay revealed that the TGF-ß1-induced increase in invasiveness of HSC-4 cells was significantly inhibited by MMP-10 small interfering RNA (siRNA). Intriguingly, Slug siRNA suppressed TGF-ß1-induced expression of MMP-10. These results suggest that TGF-ß1 induces invasion in HSC-4 cells through the upregulation of MMP-10 expression in a Slug-dependent manner. On the other hand, Slug siRNA suppressed TGF-ß1-induced Wnt-5b expression. Wnt-5b significantly induced MMP-10 expression, whereas Wnt-5b siRNA suppressed the TGF-ß1-induced increase in invasiveness, suggesting that TGF-ß1-induced expression of MMP-10 and the resulting upregulation of invasiveness are mediated by Wnt-5b. Overall, these results suggest that TGF-ß1 stimulates HSC-4 cell invasion through the Slug/Wnt-5b/MMP-10 signalling axis.

Transforming growth factor-ß1 induces epithelial-mesenchymal transition and integrin α3ß1-mediated cell migration of HSC-4 human squamous cell carcinoma cells through Slug.

We investigated whether transforming growth factor (TGF)-ß1 promoted epithelial-mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-ß target genes expression were most clearly upregulated following TGF-ß1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-ß1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-ß1 stimulation. Interestingly, E-cadherin and ß-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-ß1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-ß1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-ß1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3ß1-targeted proteins were upregulated in TGF-ß1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and ß1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-ß1-induced cell migration. These results suggest that the EMT and integrin α3ß1/FAK pathway-mediated migration of TGF-ß1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.

Mechanism of Fas-mediated cell death and its enhancement by TNF-alpha in human salivary gland adenocarcinoma cell line HSG.

Fas-mediated cell death in a human salivary gland adenocarcinoma cell line (HSG) was induced by treatment of the cells with agonistic anti-Fas antibody (CH-11), and this cell death was enhanced by pretreatment with tumor necrosis factor alpha (TNF-alpha). The mode of cell death was apoptosis, because it was accompanied by caspase activation and the cleavage of poly(ADP-ribose) polymerase. The TNF-alpha treatment of the cells increased the expression of Fas, which was accompanied by the activation of nuclear factor kappaB (NFkappaB). These results suggest that the enhancement of the apoptosis caused by TNF-alpha resulted from increased sensitivity of the HSG cells to CH-11-mediated apoptosis due to induction of Fas protein by TNF-alpha via the activation of NFkappaB. In order to elucidate the apoptosis signaling pathway, we examined the effect of various caspase inhibitors on the apoptosis induced by CH-11. Fas-mediated apoptosis of HSG cells was slightly inhibited by the caspase-9 inhibitor although it was mainly inhibited by that for caspase-8. Based on this finding, we consider CH-11-induced apoptosis in HSG cells to be mainly mediated by the type I death signaling pathway that is caused by a caspase cascade initiated by the activation of caspase-8 at the death-inducing signaling complex (DISC).