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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Expression of Wilms' tumor 1 (WT1) in ameloblastomas.

The Wilms' tumor 1 gene (WT1) was originally isolated and described as the gene responsible for Wilms' tumor. Although there is growing evidence linking the overexpression of WT1 to tumorigenesis, no reports on ameloblastoma are available at present. The aim of this study was to examine the expression of WT1 in various histological subtypes of ameloblastoma tissue specimens and in human ameloblastoma cell lines. Immunohistochemical analyses were performed on a total of 168 cases of ameloblastoma, one case of ameloblastic carcinoma, and five cases of tooth germs (control). Three immortalized human dental epithelial cell lines (HAM1, HAM2, and HAM3) derived from the same ameloblastoma patient were used for reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays. The tooth germs did not express WT1 (0%), and more than half of the ameloblastoma cases showed WT1 overexpression (54.7%). Immunoreactivity of solid-type ameloblastoma (76.1%) was more evident than that of unicystic-type ameloblastoma (40.9%). The expression level of WT1 mRNA in HAM2 was higher than that in HAM1 (moderate) and HAM3 (weak), showing the heterogeneity of tumor cells. The WT1 protein was strongly detected in HAM2 and minimally detected in HAM1 and HAM3. Our results suggest that WT1 expression influences the pathogenesis of ameloblastoma by varying its expression level in different histological types. (J Oral Sci 58, 407-413, 2016).

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.

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.

Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein-transgenic mice exhibiting a diversity in intracellular transforming growth factor-ß and bone morphogenetic protein signaling.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG­2, ­3, and ­5 from the bone marrow of green fluorescent protein (GFP)­transgenic mice. These cell lines clearly expressed mouse MSC markers Sca­1 and CD44, and SG­2 and ­5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)­ß superfamily. By contrast, SG­3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG­3 cells, which underwent osteogenesis in response to BMP, while TGF­ß receptor II was most highly expressed in SG­3 and ­5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF­ß1 in SG­2 cells but not in SG­3 or ­5 cells. Furthermore, TGF­ß1 clearly induced the expression of Smad­interacting transcription factor CCAAT/enhancer binding protein­ß in SG­2 but not in SG­3 or ­5 cells. These results demonstrated the establishment of TGF­ß­responsive SG­2 MSCs, BMP­responsive SG­3 MSCs and TGF­ß/BMP­unresponsive SG­5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF­ß superfamily affects the proliferation and differentiation status of MSCs in vivo.

Establishment of immortalized mesenchymal stem cells derived from the submandibular glands of tdTomato transgenic mice.

Transgenic mice that overexpress the red fluorescent protein tdTomato (tdTomato mice) are well suited for use in regenerative medicine studies. Cultured cells from this murine model exhibit strong red fluorescence, enabling real-time in vivo imaging through the body surface of grafted animals. Mesenchymal stem cells (MSCs) have marked potential for use in cell therapy and regenerative medicine; however, the mechanisms that regulate their dynamics in vivo are poorly understood. In the present study, an MSC line was derived from the submandibular gland fibroblasts of tdTomato mice. The fluorescent signal from this cell line was observed in organs throughout the body, as well as in salivary glands. Primary culture cells derived from the submandibular gland were immortalized with SV40 large T antigen (GManSV cells); these cells exhibited increased migratory ability, as compared with those isolated from the sublingual gland. GManSV cells were tdTomato-positive and exhibited spindle-shaped fibroblastic morphology; they also robustly expressed mouse MSC markers: Stem cell antigen-1 (Sca-1), CD44, and CD90. This cell line retained multipotent stem cell characteristics, as evidenced by its ability to differentiate into both osteogenic and adipogenic lineages. These results indicate that Sca-1+/CD44+/CD90+-GManSV cells may be useful for kinetic studies of submandibular gland-derived MSCs in the context of in vitro co-culture with other types of salivary gland-derived cells. These cells may also be used for in vivo imaging studies, in order to identify novel cell therapy and regenerative medicine for the treatment of salivary gland diseases.

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.

The homeobox gene DLX4 promotes generation of human induced pluripotent stem cells.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by defined transcription factors has been a well-established technique and will provide an invaluable resource for regenerative medicine. However, the low reprogramming efficiency of human iPSC is still a limitation for clinical application. Here we showed that the reprogramming potential of human dental pulp cells (DPCs) obtained from immature teeth is much higher than those of mature teeth DPCs. Furthermore, immature teeth DPCs can be reprogrammed by OCT3/4 and SOX2, conversely these two factors are insufficient to convert mature teeth DPCs to pluripotent states. Using a gene expression profiles between these two DPC groups, we identified a new transcript factor, distal-less homeobox 4 (DLX4), which was highly expressed in immature teeth DPCs and significantly promoted human iPSC generation in combination with OCT3/4, SOX2, and KLF4. We further show that activation of TGF-ß signaling suppresses the expression of DLX4 in DPCs and impairs the iPSC generation of DPCs. Our findings indicate that DLX4 can functionally replace c-MYC and supports efficient reprogramming of immature teeth DPCs.

Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process.

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.