Extracellular adenosine 5'-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear. METHODS AND RESULTS: Reverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5'-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C-C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression. CONCLUSION: ADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.

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.

Effects of tongue cleaning on bacterial flora in tongue coating and dental plaque: a crossover study.

BACKGROUND: The effects of tongue cleaning on reconstruction of bacterial flora in dental plaque and tongue coating itself are obscure. We assessed changes in the amounts of total bacteria as well as Fusobacterium nucleatum in tongue coating and dental plaque specimens obtained with and without tongue cleaning. METHODS: We conducted a randomized examiner-blind crossover study using 30 volunteers (average 23.7 ± 3.2 years old) without periodontitis. After dividing randomly into 2 groups, 1 group was instructed to clean the tongue, while the other did not. On days 1 (baseline), 3, and 10, tongue coating and dental plaque samples were collected after recording tongue coating score (Winkel tongue coating index: WTCI). After a washout period of 3 weeks, the same examinations were performed with the subjects allocated to the alternate group. Genomic DNA was purified from the samples and applied to SYBR® Green-based real-time PCR to quantify the amounts of total bacteria and F. nucleatum. RESULTS: After 3 days, the WTCI score recovered to baseline, though the amount of total bacteria in tongue coating was significantly lower as compared to the baseline. In plaque samples, the bacterial amounts on day 3 and 10 were significantly lower than the baseline with and without tongue cleaning. Principal component analysis showed that variations of bacterial amounts in the tongue coating and dental plaque samples were independent from each other. Furthermore, we found a strong association between amounts of total bacteria and F. nucleatum in specimens both. CONCLUSIONS: Tongue cleaning reduced the amount of bacteria in tongue coating. However, the cleaning had no obvious contribution to inhibit dental plaque formation. Furthermore, recovery of the total bacterial amount induced an increase in F. nucleatum in both tongue coating and dental plaque. Thus, it is recommended that tongue cleaning and tooth brushing should both be performed for promoting oral health.

Connective tissue growth factor enhances TGF-ß1-induced osteogenic differentiation via activation of p38 MAPK in mesenchymal stem cells.

OBJECTIVES: Cellular differentiation is based on the effects of various growth factors. Transforming growth factor (TGF)-ß1 plays a pivotal role in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the influence of connective tissue growth factor (CTGF), known to function synergistically with TGF-ß1, on osteogenic differentiation in MSCs. METHODS: UE7T-13 cells were treated with TGF-ß1 and/or CTGF. Subsequently, protein levels of intracellular signaling pathway molecules were determined through western blot analysis. The mRNA expression levels of osteogenic differentiation markers were investigated using reverse transcription-quantitative polymerase chain reaction. Bone matrix mineralization was evaluated through alizarin red staining. RESULTS: Co-treatment with TGF-ß1 and CTGF resulted in the suppression of TGF-ß1-induced phosphorylation of extracellular signal-regulated kinase 1/2, an intracellular signaling pathway molecule in MSCs, while significantly enhancing the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In MSCs, co-treatment with CTGF and TGF-ß1 led to increased expression levels of alkaline phosphatase and type I collagen, markers of osteogenic differentiation induced by TGF-ß1. Osteopontin expression was observed only after TGF-ß1 and CTGF co-treatment. Notably, bone sialoprotein and osteocalcin were significantly upregulated by treatment with CTGF alone. Furthermore, CTGF enhanced the TGF-ß1-induced mineralization in MSCs, with complete suppression observed after treatment with a p38 MAPK inhibitor. CONCLUSIONS: CTGF enhances TGF-ß1-induced osteogenic differentiation and subsequent mineralization in MSCs by predominantly activating the p38 MAPK-dependent pathway.

EGF positively regulates the proliferation and migration, and negatively regulates the myofibroblast differentiation of periodontal ligament-derived endothelial progenitor cells through MEK/ERK- and JNK-dependent signals.

BACKGROUND/AIMS: Remodeling of fibrous and vascular tissues in the periodontal ligament (PDL) around the tooth root was observed during tooth movement by orthodontic force application. We previously demonstrated that a single cell-derived culture (SCDC) of primarily cultured PDL fibroblasts, called SCDC2, has an endothelial progenitor cell (EPC)-like character and can form endothelial cell (EC) marker-positive blood vessel-like structures. However, the types of molecular mechanisms that control the in vivo kinetic properties and the differentiation of the PDL-derived EPC-like cells into myofibroblasts (MFs), which are known to expand fibrous tissues, require clarification. METHODS: Using specific mitogen activated protein kinase (MAPK) inhibitors, we examined how epidermal growth factor (EGF)-mediated MAPK signals affected the proliferation, migration, and MF differentiation of these cells. RESULTS: EGF induced SCDC2 cell proliferation in MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)- and c-Jun N-terminal kinase (JNK)-dependent manners. In addition, EGF suppressed the expression of MF differentiation markers in these cells in a MEK/ERK-dependent manner, and, moreover, stimulated the cell migration in a MEK/ERK-dependent manner. CONCLUSION: EGF regulates fibrous tissue remodeling in PDLs through MEK/ERK- and JNK-mediated signals by affecting the proliferation, migration, and MF differentiation of the PDL-derived EPC-like cells.

Expression of Wilms' tumor 1 (WT1) in oral squamous cell carcinoma.

BACKGROUND: The product of the Wilms' tumor gene, WT1 protein, is a tumor antigen for various kinds of cancer, and WT1 peptide-based cancer immunotherapy is widely anticipated as a new possibility for cancer treatment. The aim of this study was to investigate the expression of WT1 from quantitative and morphological perspectives in oral squamous cell carcinoma (OSCC), the most widespread malignant neoplasm of the oral cavity. METHODS: Six OSCC cell lines and tissue sections from 29 OSCC patients were analyzed. To detect WT1 expression, reverse transcription-polymerase chain reaction analysis (RT-PCR), real-time PCR, Western blots, and immunofluorescence flow cytometry for WT1 were performed on the cell lines, and immunohistochemistry and fluorescence in situ hybridization (FISH) were performed on the tissue sections. RESULTS: WT1 mRNA was found overexpressed in one of the six OSCC cell lines, with expression levels higher than that seen in human leukemia cell line (K562). Immunohistochemical analysis of tissue sections showed overexpression of WT1 protein in two patients, concentrated mainly in the cytoplasm of the outer one to three cell layers of the cancer nests. This was consistent with the expression of WT1 mRNA observed by FISH. Meanwhile, WT1 was not detected on normal oral epithelium. WT1 protein was detected on actively proliferating cancer nests and even on elongated epithelial ridge where new droplet-cancer-nests were being formed and starting infiltration toward subepithelial layer. CONCLUSIONS: The results suggest that WT1 plays an important role in the pathogenesis of some types of OSCC, particularly in proliferation of the cancer cells.

Hydrogen peroxide-induced oxidative stress promotes expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in fibroblast-like synoviocytes derived from mouse temporomandibular joint.

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJ-OA) is a multifactorial disease caused by inflammation and oxidative stress. It has been hypothesized that mechanical stress-induced injury of TMJ tissues induces the generation of reactive oxygen species (ROS), such as hydroxyl radical (OH∙), in the synovial fluid (SF). In general, the overproduction of ROS contributes to synovial inflammation and dysfunction of the subchondral bone in OA. However, the mechanism by which ROS-injured synoviocytes recruit inflammatory cells to TMJ-OA lesions remains unclear. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of chemoattractant molecules. The phosphorylation levels of intracellular signaling molecules were evaluated using western blot analysis. RESULTS: Hydrogen peroxide (H2O2) treatment significantly promoted mRNA expression of neutrophil chemoattractant CXCL15/Lungkine in a dose-dependent manner (100-500 µM) in fibroblast-like synoviocytes (FLSs) derived from mouse TMJ. H2O2 (500 µM) significantly upregulated the phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 in FLSs. Intriguingly, the mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor U0126 (10 µM) nullified H2O2-induced increase in CXCL15/Lungkine mRNA expression. Additionally, H2O2 (500 µM) administration significantly upregulated OH∙ production in FLSs, as assessed by live-cell permeant fluorescent probe targeted against OH∙ under fluorescence microscopy. Furthermore, the ROS inhibitor N-acetyl-l-cysteine (5 mM) partially but significantly reversed H2O2-mediated phosphorylation of ERK1/2. CONCLUSIONS: H2O2-induced oxidative stress promoted the expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in mouse TMJ-derived FLSs, suggesting that FLSs recruit neutrophils to TMJ-OA lesions through the production of CXCL15/Lungkine and exacerbate the local inflammatory response.

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity.

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Spätzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

High-cell density-induced VCAM1 expression inhibits the migratory ability of mesenchymal stem cells.

MSCs (mesenchymal stem cells) migrate into damaged tissue and then proliferate and differentiate into various cell lineages to regenerate bone, cartilage, fat and muscle. Cell-cell adhesion of MSCs is essential for the MSC-dependent tissue regeneration after their homing into a damaged tissue. However, it remains to be elucidated what kinds of adhesion molecules play important roles in the cell-cell communication between MSCs. In order to identify adhesion molecules that facilitate mutual contact between MSCs, a comprehensive analysis of mRNA expression in adhesion molecules was performed by comparing profiles of expression status of adhesion molecules in MSCs at low- and high-cell density. We found that the expression level of VCAM1 (vascular cell adhesion molecule-1)/CD106 was clearly up-regulated in the human bone marrow-derived MSCs-UE7T-13 cells - under a condition of high cell density. Intriguingly, the migratory ability of the cells was clearly accelerated by a knockdown of VCAM1. Furthermore, the migratory ability of UE7T-13 cells was decreased by the over expression of exogenous VCAM1. In addition, the high cell density-induced expression of VCAM1 was clearly suppressed by NF-κB (nuclear factor-κB) signalling-related protein kinase inhibitors such as an IKK-2 (IκB kinase-2) inhibitor VI. In conclusion, the high cell density-induced VCAM1 expression through the NF-κB pathway inhibits the migratory ability of human bone marrow-derived MSCs.

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.

Two novel mechanisms for maintenance of stemness in mesenchymal stem cells: SCRG1/BST1 axis and cell-cell adhesion through N-cadherin.

Mesenchymal stem cells (MSCs) retain the ability to self-renew and differentiate into mesenchymal cells. Therefore, human MSCs are suitable candidates for use in regenerative medicine and cell therapies. Upon activation by tissue damage, MSCs contribute to tissue repair through a multitude of processes such as self-renewal, migration, and differentiation. However, loss of self-renewal and multi-lineage differentiation potential occurs at a high rate during cell doubling. Effective MSC therapies require the establishment of new techniques that preserve MSC multipotency after lengthy cell expansions. Here, two novel mechanisms are described for maintenance of stemness in MSCs via scrapie responsive gene 1 (SCRG1)/bone marrow stromal cell antigen-1 (BST1) ligand-receptor combination and cell-cell adhesion through N-cadherin. These two mechanisms findings provide a valuable tool for regenerative medicine and cell therapeutic methods that require the ex vivo expansion of human MSCs while maintaining native stem cell potential.

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.

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.

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.

Enhancement of Anti-Inflammatory and Osteogenic Abilities of Mesenchymal Stem Cells via Cell-to-Cell Adhesion to Periodontal Ligament-Derived Fibroblasts.

Mesenchymal stem cells (MSCs) are involved in anti-inflammatory events and tissue repair; these functions are activated by their migration or homing to inflammatory tissues in response to various chemokines. However, the mechanism by which MSCs interact with other cell types in inflammatory tissue remains unclear. We investigated the role of periodontal ligament fibroblasts (PDL-Fs) in regulating the anti-inflammatory and osteogenic abilities of bone marrow-derived- (BM-) MSCs. The expression of monocyte chemotactic protein- (MCP-)1 was significantly enhanced by stimulation of PDL-Fs with inflammatory cytokines. MCP-1 induced the migratory ability of BM-MSCs but not PDL-Fs. Expression levels of anti-inflammatory and inflammatory cytokines were increased and decreased, respectively, by direct-contact coculture between MSCs and PDL-Fs. In addition, the direct-contact coculture enhanced the expression of MSC markers that play important roles in the self-renewal and maintenance of multipotency of MSCs, which in turn induced the osteogenic ability of the cells. These results suggest that MCP-1 induces the migration and homing of BM-MSCs into the PDL inflammatory tissue. The subsequent adherence of MSCs to PDL-Fs plays an immunomodulatory role to terminate inflammation during wound healing and upregulates the expression stem cell markers to enhance the stemness of MSCs, thereby facilitating bone formation in damaged PDL tissue.

ROCK/actin/MRTF signaling promotes the fibrogenic phenotype of fibroblast-like synoviocytes derived from the temporomandibular joint.

Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast­like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho­associated coiled­coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α­smooth muscle actin (α­SMA) and type I collagen; and ii) an inhibitor against the actin­polymerizing agent ROCK, Y­27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor­regulated transcription, CCG­100602, clearly suppressed the mRNA levels of α­SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor­1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ­OA and may aid in identifying drug targets for treating this condition at the molecular level.

SCRG1 suppresses LPS-induced CCL22 production through ERK1/2 activation in mouse macrophage Raw264.7 cells.

Recently, we identified the scrapie responsive gene 1 (SCRG1) secreted from mesenchymal stem cells (MSCs) and its receptor bone marrow stromal cell antigen 1 (BST1) as positive regulators of stem cell qualities such as self­renewal, migration abilities, and osteogenic differentiation potential. Here, we examined the effect of the paracrine activity of SCRG1 in macrophages. The mouse macrophage­like cell line Raw264.7 expressed BST1/ß1 or BST1/ß2 integrin as possible SCRG1 receptors. Unexpectedly, recombinant SCRG1 did not enhance cell proliferation, migration, or adhesion in these macrophages. However, further examination of the effect of SCRG1 in Raw264.7 cells did reveal a potent anti­inflammatory effect whereby SCRG1 suppressed LPS­induced CCL22 production. SCRG1 also induced the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2) in these cells and, moreover, a mitogen­activated protein kinase (MAPK)/ERK kinase inhibitor U0126 significantly suppressed the effect of SCRG1 on LPS­induced chemokine CCL22 production. Taken together, these data indicate that SCRG1 signals through the MAPK pathway and suppresses the LPS signaling pathway. CCL22 is generally known to be chemotactic for monocytes, dendritic cells, natural killer cells and chronically activated T lymphocytes, suggesting that MSC­derived SCRG1 may block infiltration of these cells. A mechanism is proposed by which MSCs play their immunosuppressive role through suppressing chemokine expression in monocyte/macrophage lineage cells.

VEGF-C and TGF-ß reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-ß (TGF-ß)-responsive SG­2 cells, bone morphogenetic protein (BMP)-responsive SG­3 cells, and TGF-ß/BMP-non-responsive SG­5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-ß-responsive SG­2 cells. Among the genes that were highly expressed in the SG­2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG­2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-ß significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG­2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-ß reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

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.