Murine Cementoblast Cell Line Research Articles

Effect of irisin on the expression of osteoclast-related genes in cementoblasts.

Cementoblasts can communicate with osteoclasts by synthesis and secretion of cytokines, such as RANKL, OPG, and M-CSF. Previously, we reported that irisin promotes the differentiation of cementoblasts, while the effect of irisin on cementoblast-mediated osteoclastogenesis remains inconclusive. This study aimed to explore the effect of irisin on the expression of osteoclastogenesis-related cytokines in cementoblasts. An immortalized murine cementoblast cell line OCCM-30 was used. Immunofluorescence and Western Blot were performed to identify the expression of irisin receptor integrin alphaV and the activation of its downstream signals in OCCM-30 cells. Cells were treated with irisin (100 ng/ml) for various time lengths ranging from 0 to 72 hours, and then qRT-PCR was used to detect the expression of osteoclastogenesis-related genes, including RANKL, IL-6, M-CSF, OPG, Wnt5A, Sema3A. Cells were also incubated with irisin in a series of concentrations (0-200 ng/ml) for 24 hours, and then qRT-PCR and ELISA were performed to examine the above osteoclastogenesis-related cytokines. Irisin receptor integrin alphaV was expressed in OCCM-30 cells and its downstream signaling pathways were markedly activated by irisin. Both qRT-PCR and ELISA results revealed that RANKL and IL-6 were up-regulated by irisin while M-CSF, OPG, Wnt5A, Sema3A remained unaffected. OCCM-30 cells were responsive to the stimulation of irisin. The expression of RANKL and IL-6 was significantly enhanced by irisin, suggesting a possible promotive effect on cementoblast-mediated osteoclastogenesis.

Bmal1 promotes cementoblast differentiation and cementum mineralization via Wnt/β-catenin signaling

Remodeling of the cementum plays a crucial role in periodontal regenerative therapy, while the precise mechanism of cementogenesis has yet been adequately understood. Recent studies have indicated the connection between osteogenic differentiation and Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (Bmal1). Besides, Wnt/β-catenin signaling is proven to be an essential regulator in cementogenesis. In this study, we found a robust expression of Bmal1 in cementoblasts in the mandibular first molar of mice by immunohistochemical staining. To further explore the role of Bmal1 in cementogenesis, we examined the expression pattern of Bmal1 in OCCM-30, an immortalized murine cementoblast cell line by qRT-PCR and western blot. Our data demonstrated the upregulation of Bmal1 at both mRNA and protein levels during differentiation. Additionally, stable knockdown of Bmal1 in OCCM-30 cells resulted in downregulation of osteogenic markers such as alkaline phosphatase (Alp), osteopontin (Opn), and osteocalcin (Ocn), and reduced formation of mineralized nodules. Moreover, qRT-PCR and western blot results exhibited that the expression of β-catenin was attenuated by Bmal1 deficiency. We also found that the mRNA levels of Tcf1 and Lef1, the target transcription factors of β-catenin, were reduced by Bmal1 deficiency. In conclusion, this study preliminarily confirms that Bmal1 promotes cementoblast differentiation and cementum mineralization via Wnt/β-catenin signaling, which contributes to a potential strategy in periodontal regenerative therapy.

Effects of long noncoding RNA H19 on cementoblast differentiation, mineralisation, and proliferation

Objective Cementum which is a layer of thin and bone-like mineralised tissue covering tooth root surface is deposited and mineralised by cementoblasts. Recent studies suggested long noncoding RNA H19 (H19) promotes osteoblast differentiation and matrix mineralisation, however, the effect of H19 on cementoblasts remains unknown. This study aimed to clarify the regulatory effects of H19 on cementoblast differentiation, mineralisation, and proliferation. Material and methods An immortalised murine cementoblast cell line OCCM-30 was used in this study. H19 expression was examined by real-time quantitative polymerase chain reaction (RT-qPCR) during OCCM-30 cell differentiation. OCCM-30 cells were transfected with lentivirus or siRNA to up-regulate or down-regulate H19, then the levels of runt-related transcription factor 2 (Runx2), osterix (Sp7), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), osteocalcin (Bglap) were tested by RT-qPCR or western blot. Alizarin red staining, ALP activity assay and MTS assay were performed to determine the mineralisation and proliferation ability of OCCM-30 cells. Results H19 was dramatically increased during OCCM-30 cell differentiation. Overexpression of H19 increased the levels of Runx2, Sp7, Alpl, Ibsp, and Bglap and enhanced ALP activity and the formation of mineral nodules. While down-regulation of H19 suppressed the above cementoblast differentiation genes and inhibited ALP activity and mineral nodule formation. However, the proliferation of OCCM-30 cells was not affected. Conclusions H19 promotes the differentiation and mineralisation of cementoblasts without affecting cell proliferation.

Gene expression and phosphorylation of ERK and AKT are regulated depending on mechanical force and cell confluence in murine cementoblasts

Cementoblasts, located on the tooth root surface covered with cementum, are considered to have tooth protecting abilities. They prevent tissue damage and secure teeth anchorage inside the periodontal ligament during mechanical stress. However, the involvement of cementoblasts in mechanical compression induced periodontal remodeling needs to be identified and better understood.Here, we investigated the effect of static compressive stimulation, simulating the compression side of orthodontic force and cell confluence on a murine cementoblast cell line (OC/CM). The influence of cell confluence in cementoblast cells was analyzed by MTS assay and immunostaining. Furthermore, mRNA and protein expression were investigated by real-time RT-PCR and western blotting at different confluence grades and after mechanical stimulation.We observed that cementoblast cell proliferation increases with increasing confluence grades, while cell viability decreases in parallel. Gene expression of remodeling markers is regulated by compressive force. In addition, cementoblast confluence plays a crucial role in this regulation. Confluent cementoblasts show a significantly higher basal expression of Bsp, Osterix, Alpl, Vegfa, Mmp9, Tlr2 and Tlr4 compared to sub-confluent cells. After compressive force of 48 h at 60% confluence, an upregulation of Bsp, Osterix, Alpl, Vegf and Mmp9 is observed. In contrast, at high confluence, all analyzed genes were downregulated through mechanical stress. We also proved a regulation of ERK, phospho-ERK and phospho-AKT dependent on compressive force. In summary, our findings provide evidence that cementoblast physiology and metabolism is highly regulated in a cell confluence-dependent manner and by mechanical stimulation.

Chemerin/ChemR23 regulates cementoblast function and tooth resorption in mice via inflammatory factors.

Periodontitis and orthodontic treatment can lead to inflammatory root resorption (IRR) through an unclear mechanism. Chemerin, a novel chemoattractant protein, is closely associated with inflammation, affects osteoblast and osteoclast differentiation, and may play a role in IRR. We aimed to explore possible roles of the chemerin/ChemR23 interaction in cementoblast function and IRR and reveal a new IRR therapeutic target. Cementoblast function-related gene and protein expression in the immortalized murine cementoblast cell line OCCM-30 after treatment with chemerin and siChemR23 was examined by qRT-PCR and Western blotting. The roles of the MAPK and PI3K-Akt signaling pathways were studied using specific inhibitors. Cementoblast cytokine production under different treatment conditions was measured by enzyme-linked immunosorbent assay and qRT-PCR. Additionally, we modeled IRR in wild-type and chemerin-overexpressing mice and injected transgenic mice with anti-ChemR23 antibody to block ChemR23. We then calculated the root resorption volume and examined periodontal tissue cathepsin K, Runx2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression. Chemerin suppressed cementoblast differentiation and mineralization and exerted a proinflammatory effect on cementoblasts. These effects were partially reversed by siChemR23 and reversed to different extents by p38, Erk1/2 and PI3K-Akt pathway inhibition, suggesting p38, Erk1/2 and PI3K-Akt pathways as signaling pathways downstream of chemerin/ChemR23. In vivo, chemerin overexpression worsened IRR. Moreover, chemerin expression was positively correlated with TNF-α, IL-6, and cathepsin K expression and negatively correlated with Runx2 expression. ChemR23 downregulation reversed these effects. Chemerin/ChemR23 induced TNF-α and IL-6 expression dependent on Erk1/2, p38 MAPK, and PI3K-Akt signaling pathway activation, thereby regulating cementoblast function and affecting IRR.

Regulatory effects of bone morphogenetic protein-4 on tumour necrosis factor-α-suppressed Runx2 and osteoprotegerin expression in cementoblasts.

Root resorption is a common phenomenon presented in periodontitis and orthodontic treatment, both of which are accompanied by an elevated TNF-α expression level in the periodontal tissues. Previously, we proved that TNF-α showed an inhibitory effect on cementoblast differentiation, mineralization and proliferation. However, the effect of TNF-α on Runx2 and osteoprotegerin (OPG) expression remains undetermined. This study aimed to identify the influence of TNF-α on Runx2 and OPG expression in cementoblasts and to test whether BMP-2,-4,-6,-7 would affect TNF-α-regulated Runx2 and OPG. An immortalized murine cementoblast cell line OCCM-30 was used in this study. The expression of Runx2 and OPG were examined by qRT-PCR after stimulating cells with TNF-α. The role of signalling pathways, including MAPK, PI3K-Akt and NF-κB, were studied with the use of specific inhibitors. Cells were treated with TNF-α in combination with BMP-2,-4,-6 or -7, then the expression of Runx2 and OPG, the activity of MAPK and NF-κB pathways, and the proliferation ability were evaluated by qRT-PCR, Western blot and MTS assay respectively. TNF-α inhibited Runx2 and OPG mRNAs in OCCM-30 cells, and the inhibitory effects were further aggravated by blocking p38 MAPK or NF-κB pathway. TNF-α-inhibited Runx2 and OPG were up-regulated by BMP-4. The p38 MAPK and Erk1/2 pathways were further activated by the combined treatment of BMP-4 and TNF-α compared with TNF-α alone. Finally, the TNF-α-suppressed proliferation was not obviously affected by BMP-2,-4,-6 or -7. TNF-α inhibited Runx2 and OPG in cementoblasts, and the p38 MAPK and NF-κB pathways acted in a negative-feedback way to attenuate the inhibitory effects. TNF-α-inhibited Runx2 and OPG could be effectively up-regulated by BMP-4; however, further investigations are needed to fully elaborate the underlying mechanisms.

Screening the Expression Changes in MicroRNAs and Their Target Genes in Mature Cementoblasts Stimulated with Cyclic Tensile Stress.

Cementum is a thin layer of cementoblast-produced mineralized tissue covering the root surfaces of teeth. Mechanical forces, which are produced during masticatory activity, play a paramount role in stimulating cementoblastogenesis, which thereby facilitates the maintenance, remodeling and integrity of cementum. However, hitherto, the extent to which a post-transcriptional modulation mechanism is involved in this process has rarely been reported. In this study, a mature murine cementoblast cell line OCCM-30 cells (immortalized osteocalcin positive cementoblasts) was cultured and subjected to cyclic tensile stress (0.5 Hz, 2000 µstrain). We showed that the cyclic tensile stress could not only rearrange the cell alignment, but also influence the proliferation in an S-shaped manner. Furthermore, cyclic tensile stress could significantly promote cementoblastogenesis-related genes, proteins and mineralized nodules. From the miRNA array analyses, we found that 60 and 103 miRNAs were significantly altered 6 and 18 h after the stimulation using cyclic tensile stress, respectively. Based on a literature review and bioinformatics analyses, we found that miR-146b-5p and its target gene Smad4 play an important role in this procedure. The upregulation of miR-146b-5p and downregulation of Smad4 induced by the tensile stress were further confirmed by qRT-PCR. The direct binding of miR-146b-5p to the three prime untranslated region (3′ UTR) of Smad4 was established using a dual-luciferase reporter assay. Taken together, these results suggest an important involvement of miR-146b-5p and its target gene Smad4 in the cementoblastogenesis of mature cementoblasts.

TNF-α stimulates MMP-3 production via PGE2 signalling through the NF-kB and p38 MAPK pathway in a murine cementoblast cell line

BackgroundCementoblasts are considered to play an important role in the homeostasis of periodontal tissues under both physiologic and pathologic conditions. Matrix metalloproteinases (MMPs) is the key family of enzymes participating in extracellular matrix remodelling. In the present study, the effects and regulatory mechanisms of tumour necrosis factor (TNF)-α on the expression of MMPs and their inhibitors (tissue inhibitor of metalloproteinases; TIMPs) were investigated. Materials and methodsOCCM-30, an immortalised murine cementoblast cell line, was stimulated with TNF-α at 1 and 10ng/ml for 24h. The expression of Mmp-2, Mmp-3, Mmp-13, Mmp-14, Timp-1, and Timp-2 as well as PGE2 was determined. Inhibitors of MAPKs, PI3K/Akt, NF-kB and Cox-2 were employed to reveal possible TNF-α induced regulatory signalling pathway(s). The mRNA and protein expression were analysed by (semi)quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. ResultsTNF-α dose-dependently stimulated MMP-3 expression by cementoblasts. This was found for mRNA as well as protein expression. No significant differences were found in the mRNA expression of Mmp-2, Mmp-13, Mmp-14, Timp-1, and Timp-2 upon TNF-α stimulation. The level of PGE2, however, was significantly increased along with MMP-3. Treatment with a selective Cox-2 inhibitor resulted in partial suppression of TNF-α-induced Mmp-3 mRNA expression. Addition of PGE2 enhanced Mmp-3 mRNA in a dose dependent manner, suggesting an inductive effect of TNF-α partly via PGE2. The up-regulation of Mmp-3 by TNF-α was completely suppressed by a combination of NF-kB and p38 MAPK inhibitors, while partial suppression was found with each inhibitor. The effect of PGE2 on Mmp-3 expression was abolished by treating cells with an NF-kB inhibitor; a p38 MAPK inhibitor had only a small effect. ConclusionsThe present study indicates that cementoblasts respond to TNF-α by increasing MMP-3 production partially via PGE2 and signalling through the NF-kB and p38 MAPK pathway. MMP-3 may participate in periodontal tissue degradation/remodelling.

The effect of N-acetyl- l-cysteine and ascorbic acid on visible-light-irradiated camphorquinone/ N, N-dimethyl- p-toluidine-induced oxidative stress in two immortalized cell lines

Recent studies have revealed that visible-light (VL)-irradiated camphorquinone (CQ), in the presence of a tertiary amine (e.g., N, N-dimethyl- p-toluidine, DMT), generates initiating radicals that may indiscriminately react with molecular oxygen forming reactive oxygen species (ROS). In this study, the ability of the antioxidants N-acetyl- l-cysteine (NAC) and ascorbic acid (AA) to reduce intracellular oxidative stress induced by VL-irradiated CQ/DMT or VL-irradiated hydrogen peroxide (H 2O 2) was assessed in an immortalized Murine cementoblast cell line (OCCM.30) and an immortalized Murine fibroblast cell line, 3T3-Swiss albino (3T3). Intracellular oxidative stress was measured with the membrane permeable dye, 2′,7′-dichlorodihydrofluorescein diacetate (H 2DCF-DA). VL-irradiated CQ/DMT and VL-irradiated H 2O 2 each produced significantly ( p < 0.001 ) elevated intracellular oxidative levels in both cell types compared to intracellular ROS levels in VL-irradiated untreated cells. OCCM.30 cementoblasts were found to be almost twice as sensitive to VL-irradiated CQ/DMT and VL-irradiated H 2O 2 treatment compared to 3T3 fibroblasts. Furthermore, 10 m m NAC and 10 m m AA each eliminated oxidative stress induced by VL-irradiated CQ/DMT and VL-irradiated H 2O 2 in both cell types. Our results suggest that NAC and AA may effectively reduce or eliminate oxidative stress in cells exposed to VL-irradiated CQ/DMT following polymerization.