Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.     

Possible involvement of extracellular signal-regulated kinases 1/2 in mitogenic response of periodontal ligament cells to enamel matrix derivative

The efficacy of enamel matrix derivative (EMD) as an adjunct to periodontal regenerative therapy has been demonstrated in recent clinical studies, however, little is known about its molecular mechanism (s). We examined the mitogenic response of cultured periodontal ligament (PDL) cells to EMD and characterized associated changes in proliferation-related intracellular signaling molecules, including mitogen-activated protein kinases (MAPK) and Akt kinases/protein kinase B (Akt/PKB) kinases. The DNA synthesis of PDL cells increased following treatment with EMD at concentrations higher than 1 microg ml(-1). This mitogenic response to EMD was associated with the selective activation of extracellular signal-regulated kinase (ERK) 1/2. No other MAPKs, or Akt/PKB kinases, responded to EMD stimulation. The EMD induction of DNA synthesis and activation of ERK 1/2 were diminished by pretreatment with suramin, an inhibitor of receptor tyrosine kinases (RTK). The signaling pathway induced by EMD from RTK to ERK 1/2 was similar to that activated by epidermal growth factor (EGF), although the specific binding of 125I-EGF to PDL cells was not affected by pretreatment or concomitant treatment with EMD. These findings suggest that EMD elicits its mitogenic signal through an EMD-specific RTK towards ERK 1/2.     

Fluid Shear Stress Stimulates Osteogenic Differentiation of Human Periodontal Ligament Cells via the Extracellular Signal‐Regulated Kinase 1/2 and p38 Mitogen‐Activated Protein Kinase Signaling Pathways

Background: Fluid shear stress (FSS) is a major type of mechanical stress that is loaded on human periodontal ligament cells (hPDLCs) during mastication and orthodontic tooth movement. This study aims to clarify the effect of FSS on the osteogenic differentiation of hPDLCs and to further verify the involvement of mitogen‐activated protein kinase (MAPK) signaling in this process. Methods: After isolation and characterization, hPDLCs were subjected to 2‐hour FSS at 12 dynes/cm², and cell viability, osteogenic gene mRNA expression, alkaline phosphatase (ALP) activity, secretion of Type I collagen (COL‐I), and calcium deposition were assayed. The levels of phosphorylated p38 and phosphorylated extracellular signal‐regulated kinase 1/2 (ERK1/2) in response to FSS were detected by Western blot, and the involvement of ERK1/2 and p38 MAPK signaling pathways in hPDLC osteogenesis under FSS was investigated using the specific MAPK inhibitors U0126 (2Z,3Z)‐2,3‐bis[amino(2‐aminophenylthio)methylene]succinonitrile,ethanol) and SB203580 (4‐[4‐(4‐fluorophenyl)‐2‐(4‐[methylsulfinyl]phenyl)‐1H‐imidazol‐5‐yl]pyridine). Results: The application of FSS on hPDLCs induced an early morphologic change and rearrangement of filamentous actin. ALP activity, messenger RNA (mRNA) levels of osteogenic genes, COL‐I, and osteoid nodules were significantly increased by FSS. Moreover, ERK1/2 and p38 were activated in different ways after FSS exposure. U0126 and SB203580 completely blocked the FSS‐induced increases in ALP activity and osteogenic gene mRNA expression and osteoid nodules formation. Conclusions: FSS is an effective approach for stimulating osteogenic differentiation of hPDLCs. The ERK1/2 and p38 MAPK signaling pathways are involved in this cellular process.     

Induction of osteoclast differentiation by NOC-18, a long-acting nitric oxide donor

It is well established that osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites. However, the mechanism of osteoclast differentiation/activation in orthodontic tooth movement remains unclear. Nitric oxide (NO) has been reported to play a crucial role in bone remodeling induced by mechanical loading, and several reports showed that inhibition of NO synthases prevented orthodontic tooth movement in animal models. Here, we investigated whether osteoclast differentiation can be induced by NO in vitro. Osteoclast differentiation was slightly induced by NOC-18, a long-acting NO donor, and RANKL-induced osteoclast differentiation was further augmented by NOC-18. Short-acting NO donors including NOC-15, NOC-9 and sodium nitroprusside (SNP) did not significantly induce osteoclast differentiation. Since it has been reported that mitogen-activated protein kinase (MAPK) signaling plays a crucial role in RANKL-induced osteoclast differentiation and RANKL-induced osteoclast differentiation was enhanced by inhibition of extracellular signal-regulated kinase (ERK) whereas it was suppressed by inhibition of p38 MAPK, involvement of MAPK signaling in stimulation of osteoclast differentiation by NOC-18 was investigated. Western blot analysis demonstrated that pretreatment with NOC-18 inhibited RANKL-induced phosphorylation of ERK whereas it augmented RANKL-induced phosphorylation of p38 MAPK. Furthermore, mouse M-CSF-dependent bone marrow macrophages were induced to differentiate into osteoclasts by NOC-18. These results suggest that NOC-18 enhanced osteoclast differentiation via MAPK signaling in accordance with RANKL-induced osteoclast differentiation.     

The C‐terminus of the amelogenin peptide influences the proliferation of human bone marrow mesenchymal stem cells

1 Background

Amelogenins are a family of enamel matrix proteins that are important for formation of enamel. Amelogenins may induce division of mesenchymal stem cells (MSCs), among others. Recently, the C‐terminus of the amelogenin peptide (AMG‐CP) has been shown to enhance the proliferation of cementoblast lineage cells. The role of the amelogenin peptide on the proliferation of human MSCs and related alterations in the intracellular signaling pathway were studied.

2 Methods

MSCs were exposed to AMG‐CP in vitro. The MTS and 5‐bromo‐2′‐deoxyuridine (BrdU) assays were used to determine proliferation. Expression of the amelogenin receptor, lysosomal‐associated membrane protein 1 (LAMP1), was examined in MSCs with western blotting. Binding of AMG‐CP to LAMP1 was inhibited with anti‐LAMP1 antibody. Components of the mitogen‐activated protein kinase (MAPK)–extracellular signal‐regulated kinase (ERK) pathway were studied with western blotting and enzyme‐linked immunosorbent assay, and U0126, an MAPK inhibitor, was used to inhibit ERK activity.

3 Results

MSC proliferation was significantly increased in the presence of AMG‐CP and significantly inhibited by anti‐LAMP1 antibody or U0126. Increased phosphorylated ERK1/2 was observed in the presence of AMG‐CP, and decreased phosphorylated ERK1/2 was seen in the presence of anti‐LAMP1 antibody or U0126.

4 Conclusion

A C‐terminal amelogenin variant increased the proliferation of MSCs via an interaction with LAMP1 and the MAPK–ERK signaling pathway, indicating the possibility of using MSCs for tissue regeneration in the craniofacial region.     

Cot/Tpl2 is essential for RANKL induction by lipid A in osteoblasts

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-kappa B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-alpha by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-kappa B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.     

Signaling pathways regulating IL-1alpha-induced COX-2 expression

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.     

Effects of C‐Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells

Background: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. Methods: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full‐length amelogenin (rh174); 2) amelogenin cleavage product lacking the C‐terminal (rh163); 3) amelogenin cleavage product lacking the N‐terminal (rh128); and 4) the C‐terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast‐like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐ tetrazolium assay and cell proliferation enzyme‐linked immunosorbent assay. Mitogen‐activated protein kinase (MAPK)–extracellular signal‐regulated kinase (ERK) (MAPK–ERK) pathway was examined by Western blot analysis. Results: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). Conclusion: C‐terminal amelogenin cleavage product increased proliferation of HCEM through MAPK–ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.     

Proliferation of dental pulp fibroblasts in response to thrombin involves mitogen-activated protein kinase signalling

AIM: To examine the involvement of mitogen-activated protein kinases (MAPK) signalling on thrombin-stimulated human dental pulp fibroblasts (DPF). METHODOLOGY: Dental pulp fibroblasts were isolated from dental pulp connective tissue of third molars and expanded in vitro. Expression of thrombin receptors was analysed by RT-PCR, and cell proliferation was measured by 3[H]-thymidine incorporation assay. Phosphorylation levels of MAPK were determined by Western blot analysis, and alkaline phosphatase activity was measured to serve as a marker for odontogenic differentiation. Statistical analysis was performed by Student's t-test. RESULTS: Dental pulp fibroblasts express the thrombin receptors protease-activated receptor-1 (PAR-1), PAR-3 and PAR-4. Measurement of 3[H]-thymidine incorporation revealed a dose-dependent increase of DNA synthesis in response to thrombin treatment. The thrombin-induced mitogenic activity was decreased by the extracellular signal-regulated protein kinase (ERK) signalling inhibitor PD98059 (P < 0.05), and by SB203580 (P < 0.05), a p38 MAPK inhibitor. Western blot analysis demonstrated increased phosphorylation of ERK in DPF following stimulation with thrombin, while p38 MAPK and c-Jun NH2-terminal kinase (JNK) were not activated. Alkaline phosphatase activity of DPF remained unchanged upon incubation with thrombin. CONCLUSIONS: These results suggest that signalling via MAPK mediates the mitogenic activity of thrombin on DPF and may thus play a role during the early stages of pulp repair.     

Aloin Inhibits Interleukin (IL)‐1β−Stimulated IL‐8 Production in KB Cells

Background: Interleukin (IL)‐1β, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL‐8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL‐1β in IL‐8 production and determines if aloin inhibits IL‐1β?stimulated IL‐8 production in epithelial cells. Methods: Saliva was collected from volunteers to determine IL‐1β and IL‐8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL‐1β levels. KB cells were stimulated with IL‐1β or saliva with or without IL‐1 receptor agonist or specific mitogen‐activated protein kinase (MAPK) inhibitors. IL‐8 production was measured by enzyme‐linked immunosorbent assay (ELISA). MAPK protein expression involved in IL‐1β?induced IL‐8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL‐1β?induced IL‐8 production was examined by ELISA and Western blot analysis. Results: Saliva with high IL‐1β strongly stimulated IL‐8 production in KB cells, and IL‐1 receptor agonist significantly inhibited IL‐8 production. Low IL‐1β–containing saliva did not increase IL‐8 production. IL‐1β treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor‐kappa B. IL‐1β?induced IL‐8 production was decreased by p38 and extracellular signal‐regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL‐1β?induced IL‐8 production in a dose‐dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva‐induced IL‐8 production. Conclusions: Results indicated that IL‐1β in saliva stimulates epithelial cells to produce IL‐8 and that aloin effectively inhibits salivary IL‐1β–induced IL‐8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.     

The potential role of transient receptor potential type A1 as a mechanoreceptor in human periodontal ligament cells

Transient receptor potential type A1 (TRPA1) is reported to be a Ca²⁺‐permeable channel and is activated by cold temperatures and mechanical stimuli in the hair cells and in dorsal root ganglion. Using a DNA microarray, we found that TRPA1 was significantly up‐regulated in human periodontal ligament (hPDL) cells 2 d after intermittent mechanical stimulation (iMS) loading compared with unloaded cells. Although hPDL cells are known to respond to mechanical stimulation induced by occlusal force, little is known about the expression and functional role of TRPA1 in these cells. Therefore, we investigated the effects of iMS on TRPA1 expression and its signaling pathway in hPDL cells. Intermittent mechanical stimulation loading up‐regulated TRPA1 expression in hPDL cells in a time‐dependent manner, but had no effect on other mechanoreceptors. Furthermore, iMS significantly increased the phosphorylation of mitogen‐activated protein kinases (MAPKs), especially extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38, and the expression of C‐C chemokine ligand 2 (CCL2). Transient receptor potential type A1 agonists also increased MAPK phosphorylation and the intracellular Ca²⁺ concentration. By contrast, inhibition or silencing of TRPA1 partially suppressed iMS‐induced MAPK phosphorylation. In summary, iMS during occlusion activates TRPA1 and MAPK signaling in periodontal ligament tissues, suggesting that TRPA1 regulates the mechanosensitivity of occlusal force via activation of MAPKs in hPDL cells.     

MAPK—ERK在牙本质基质蛋白1信号通路中的作用

目的 探讨在人骨髓间充质干细胞（hMSC）、小鼠前成骨细胞（MC3T3）和成牙本质细胞（MDPC-23）中,牙本质基质蛋白1（DMP1）是否通过激活丝裂原活化蛋白激酶（MAPK）-细胞外调节蛋白激酶（ERK）信号通路发挥作用.方法 Western blot 检测不同时间点rDMP1C 和rDMP1F 蛋白处理hMSC、MC3T3-E1 和MDPC-23 后,对MAPK-ERK 的激活情况;并检测使用MAPK 抑制剂后,ERK 的激活是否被抑制;细胞免疫荧光技术观察MAPK 抑制剂抑制激活的ERK 从胞浆向胞核的转位情况.Western blot 检测siRNA 沉默Ras 基因后,对rDMP1 引起MAPK-ERK 信号通路激活的影响.结果 三种细胞中,rDMP1F 和rDMP1C 在5 min ～ 3 h 均可以激活MAPK-ERK 通路,而总ERK 在各时间点均无显著变化;rDMP1F 激活信号通路的持续时间均明显长于rDMP1C;MAPK 抑制剂处理组的p-ERK 条带与rDMP1F/C 处理组的p-ERK 条带差别明显.hMSC 中,rDMP1F 激活MAPK 信号通路持续时间要长于其在MC3T3 和MDPC-23 中的作用.细胞免疫荧光检测发现,MAPK 抑制剂组可以阻断ERK 向细胞核内的转位.MC3T3 和MDPC-23 在siRNA 沉默Ras 基因后,ERK 的磷酸化水平与rDMP1F 单独处理组相比显著降低.结论 rDMP1C 和rDMP1F 都通过Ras 激活MAPK-ERK信号通路,而rDMP1F 比rDMP1C 具有更强的信号功能.     

Suppression of lipopolysaccharide-induced cytokine production of gingival fibroblasts by a soybean, Kunitz trypsin inhibitor

BACKGROUND: Human bikunin, a Kunitz-type trypsin inhibitor, inhibits inflammation by down-regulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in tumor cells and inflammatory cells. OBJECTIVES: We analyzed the effect of a soybean-derived Kunitz trypsin inhibitor (KTI) on TNF-alpha production in human gingival fibroblasts stimulated by lipopolysaccharide (LPS), an inflammatory inducer. MATERIAL AND METHODS: Mitogen-activated protein kinase (MAPK) activation and cytokine levels were monitored using western blot and a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: Here, we show (i) a soybean KTI abrogates LPS-induced up-regulation of TNF-alpha mRNA and protein expression in a dose-dependent manner in gingival fibroblasts, (ii) KTI also blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins, (iii) inhibition by KTI of TNF-alpha induction correlates with the suppressive capacity of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 signaling pathways, implicating repressed ERK1/2 and p38 signalings in the inhibition, and (iv) pretreatment of cells with KTI blocked LPS-induced nuclear factor kappaB (NFkappaB) activation. CONCLUSION: Our results indicate that KTI inhibits LPS-induced up-regulation of cytokine expression possibly through suppression of ERK1/2 and p38 kinase-mediated NFkappaB activation. These findings may identify anti-inflammatory properties of KTI at the level of gingival fibroblasts and may be relevant to the use of KTI in modulating inflammation, including periodontal disease.     

平阳霉素引发细胞凋亡与MAPK信号转导通路的关联性

目的 探讨平阳霉素引发细胞凋亡与分裂原激活的蛋白激酶家族（mitogen-activated protein kinase,MAPK)信号转导通路的关联性。方法 采用Western印迹法（western blot)检测分裂原激活蛋白激酶的表达。结果 平阳霉素作用KB细胞，随作用时间延长或药物浓度增加，磷酸化细胞外信号调节激酶的表达越不明显；而p-38MAPK蛋白激酶的表达无明显变化。结论 平阳霉素引发细胞凋亡与分裂原激活的蛋白激酶家族信号转导通路有关。     

Hinokitiol increases the angiogenic potential of dental pulp cells through ERK and p38MAPK activation and hypoxia-inducible factor-1α (HIF-1α) upregulation

Hinokitiol, a natural iron-chelating agent, is known to have diverse biological and pharmacological activities in various cell types. However, the effect of hinokitiol on dental pulp cells has not yet been reported. In this study, hinokitiol increases hypoxia-inducible factor-1α (HIF-1α) protein levels and vascular endothelial growth factor (VEGF) secretion in human dental pulp cells. The extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways are involved in hinokitiol-induced HIF-1α protein expression in dental pulp cells. Conditioned media from hinokitiol-treated pulp cells enhances angiogenesis in vitro and in vivo. Overall, these results show that hinokitiol promotes ERK and p38MAPK activation and HIF-1α-induced VEGF production, thus increasing the angiogenic potential of dental pulp cells.     

MEK inhibition enhances bleomycin A5-induced apoptosis in an oral cancer cell line: signaling mechanisms and therapeutic opportunities

BACKGROUND: Bleomycin A5 is an anti-neoplastic glycoprotein antibiotic used for the treatment of various cancers. Previous work has shown that bleomycin A5 exerts its apoptotic effects on tumor cells. This was to study the signal transduction pathways that might exert the apoptotic effects of bleomycin A5 on tumor cells, as well as to examine the possibility of lower dosing of such drug in combinative treatment with other compounds in vitro. METHODS: Bleomycin A5 was applied on a human oral epidermoid carcinoma cell line, human oral epidermoid carcinoma (KB), and the apoptotic activity was determined by the presence of DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. The signal transduction pathway was measured through Western blotting and in vitro kinase assay. RESULTS: The apoptotic effect was associated with the sustained activation of c-Jun N-terminal kinases (JNK) and the inhibition of extracellular signal-regulated kinases1 (ERK1) and -2 activities, suggesting that JNK plays a positive role in the death process. ERK1 and -2 might exert a protection pathway from cell death. Here, it was determined that a combination treatment of bleomycin A5 and the MAP kinase-ERK kinase (MEK) inhibitor, PD98059, could lead to enhanced apoptosis. The activities of ERK1 and -2 are required for cell survival signaling using stable cell clones expressing MEK1. Upon bleomycin A5 treatment, cells expressing MEK1 exhibited significant delays in the onset of apoptosis, where the presence of MEK1 inhibitor enhanced cell death. Moreover, the increased activity of ERK1 and -2 coincided with cell survival. The survival signals exerted by MEK1 most likely result from the activation of ERK1 and -2. CONCLUSIONS: The apoptosis enhancement through such combinative treatment in vitro has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anti-cancer treatments.