Young-Mi Jeon

Mechanical force induces type I collagen expression in human periodontal ligament fibroblasts through activation of ERK/JNK and AP‐1

Type I collagen (COL I) is the predominant collagen in the extracellular matrix of periodontal ligament (PDL), and its expression in PDL fibroblasts (PLF) is sensitive to mechanical force. However, the mechanism by which PLF induces COL I to respond to mechanical force is unclear. This study examined the nature of human PLF in mediating COL I expression in response to centrifugal force. Signal transduction pathways in the early stages of mechanotransduction involved in the force‐driven regulation of COL I expression were also investigated. Centrifugal force up‐regulated COL I without cytotoxicity and activated extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 kinase. ERK and JNK inhibitor blocked the expression of COL I but p38 kinase inhibitor had no effect. Centrifugal force activated activator protein‐1 (AP‐1) through dimerization between c‐Fos and c‐Jun transcription factors. ERK and JNK inhibitors also inhibited AP‐1‐DNA binding, c‐Fos nuclear translocation, and c‐Jun phosphorylation that were increased in the force‐exposed PLF. Further, transfecting the cells with c‐Jun antisense oligonucleotides almost completely abolished the force‐induced increase of c‐Jun phosphorylation and COL I induction. Our findings suggest that mechanical signals are transmitted into the nucleus by ERK/JNK signaling pathways and then stimulate COL I expression through AP‐1 activation in force‐exposed human PLF. J. Cell. Biochem. 106: 1060–1067, 2009.

Caspase-independent death of human osteosarcoma cells by flavonoids is driven by p53-mediated mitochondrial stress and nuclear translocation of AIF and endonuclease G

Flavonoids have antioxidant and antitumor promoting effects. Rhus verniciflua Stokes (RVS) is a flavonoid-rich herbal medicine that has long been used in Korea as both a food additive and antitumor agent. It was previous reported that a purified flavonoid fraction prepared from RVS, herein named RCMF (the RVS chloroform-methanol fraction), inhibited the proliferation and induced apoptosis in human osteosarcoma (HOS) cells. This study examined the mechanisms involved in the RCMF-mediated apoptosis in HOS cells. RCMF was shown to be capable of inducing apoptosis in HOS cells by inducing p53 in the cells resulting in the decrease in Bcl-2 level, activation of Bax, and cytoplasmic release of cytochrome c, which led to the translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) into the nucleus. However, the RCMF-induced apoptosis was suppressed by transfecting the cells with antisense p53 oligonucleotides but not by treating them with a MAPK or caspase inhibitor. This suppression occurred through the regulation of Bcl-2 members as well as by preventing the nuclear translocation of the mitochondrial apoptogenic factors. Overall, it appears that p53-mediated mitochondrial stress and the nuclear translocation of AIF and EndoG are mainly required for the apoptosis induced by RCMF.

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways.

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G(2)/M phase. The addition of NaF induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways.

Quercetin inhibits α-MSH-stimulated melanogenesis in B16F10 melanoma cells.

Quercetin is known to inhibit tyrosinase activity and melanin production in melanocytes. However, several reports suggest that quercetin has different and opposite effects on melanogenesis. This study examined the precise effects of quercetin on melanogenesis using cell‐free assay systems and melanocytes. Quercetin inhibited the monophenolase and diphenolase activities of tyrosinase, and melanin synthesis in cell‐free assay systems. Quercetin induced mild stimulation of the tyrosinase activity and dihydroxyphenylalaminechrome tautomerase (TRP‐2) expression but only at low concentrations (<20 μm) in B16F10 melanoma cells. In contrast, the addition of 50 μm quercetin to the cells led to a significant decrease in the activity and synthesis of tyrosinase, as well as a decrease in the expression of tyrosinase‐related protein‐1 and TRP‐2 proteins, regardless of the presence or absence of α‐melanocyte stimulating hormone (α‐MSH). Quercetin also reduced the intracellular cAMP and the phosphorylated protein kinase A levels in α‐MSH‐stimulated B16F10 cells. Moreover, quercetin (20 μm) diminished the expression and activity of tyrosinase, and melanin content in cultured normal human epidermal melanocytes. These effects were not related to its cytotoxic action. Although the in vivo effects of quercetin are still unclear, these results suggest that quercetin could play important roles in controlling melanogenesis. Copyright

Mechanical force inhibits osteoclastogenic potential of human periodontal ligament fibroblasts through OPG production and ERK-mediated signaling.

Periodontal ligament and gingival fibroblasts play important roles in bone remodeling. Periodontal ligament fibroblasts stimulate bone remodeling while gingival fibroblasts protect abnormal bone resorption. However, few studies had examined the differences in stimulation of osteoclast formation between the two fibroblast populations. The precise effect of mechanical forces on osteoclastogenesis of these populations is also unknown. This study revealed that more osteoclast‐like cells were induced in the co‐cultures of bone marrow cells with periodontal ligament than gingival fibroblasts, and this was considerably increased when anti‐osteoprotegerin (OPG) antibody was added to the co‐cultures. mRNA levels of receptor activator of nuclear factor‐kappaB ligand (RANKL) were increased in both populations when they were cultured with dexamethasone and vitamin D3. Centrifugal forces inhibited osteoclastogenesis of both populations, and this was likely related to the force‐induced OPG up‐regulation. Inhibition of extracellular signal‐regulated kinase (ERK) signaling by a pharmacological inhibitor (10 µM PD98059) or by siERK transfection suppressed the force‐induced OPG up‐regulation along with the augmentation of osteoclast‐like cells that were decreased by the force. These results suggest that periodontal ligament fibroblasts are naturally better at osteoclast induction than gingival fibroblasts, and that centrifugal force inhibited osteoclastogenesis of the periodontal fibroblasts through OPG production and ERK activation. J. Cell. Biochem. 106: 1010–1019, 2009.

Mechanical force augments the anti-osteoclastogenic potential of human gingival fibroblasts in vitro.

BACKGROUND AND OBJECTIVE The cellular response of human gingival fibroblasts to a mechanical force is considered to be primarily anti-osteoclastic because they produce relatively high levels of osteoprotegerin. However, there is little information available on the effects of compression force on the production of osteoprotegerin and osteoclastic differentiation by these cells. In this study, we examined how mechanical force affects the nature of human gingival fibroblasts to produce osteoprotegerin and inhibit osteoclastogenesis. MATERIAL AND METHODS Human gingival fibroblasts were exposed to mechanical force by centrifugation for 90 min at a magnitude of approximately 50 g/cm(2). The levels of osteoprotegerin, receptor activator of nuclear factor-kappaB ligand (RANKL), interleukin-1beta and tumor necrosis factor-alpha were measured at various time-points after applying the force. The effect of the centrifugal force on the formation of osteoclast-like cells was also determined using a co-culture system of human gingival fibroblasts and bone marrow cells. RESULTS Centrifugal force stimulated the expression of osteoprotegerin, RANKL, interleukin-1beta and tumor necrosis factor-alpha by the cells, and produced a relatively high osteoprotegerin to RANKL ratio at the protein level. Both interleukin-1beta and tumor necrosis factor-alpha accelerated the force-induced production of osteoprotegerin, which was inhibited significantly by the addition of anti-(interleukin-1beta) immunoglobulin Ig isotype; IgG (rabbit polyclonal). However, the addition of anti-(tumor necrosis factor-alpha) immunoglobulin Ig isotype; IgG1 (mouse monoclonal) had no effect. Centrifugal force also had an inhibitory effect on osteoclast formation. CONCLUSION Application of centrifugal force to human gingival fibroblasts accelerates osteoprotegerin production by these cells, which stimulates the potential of human gingival fibroblasts to suppress osteoclastogenesis. Overall, human gingival fibroblasts might have natural defensive mechanisms to inhibit bone resorption induced by a mechanical stress.

Periodontal Fibroblasts Modulate Proliferation and Osteogenic Differentiation of Embryonic Stem Cells Through Production of Fibroblast Growth Factors

BACKGROUND Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs). METHODS PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured. RESULTS In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin. CONCLUSION PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.

Fibroblast growth factor-7 facilitates osteogenic differentiation of embryonic stem cells through the activation of ERK/Runx2 signaling

Fibroblast growth factor-7 (FGF7) is known to regulate proliferation and differentiation of cells; however, little information is available on how FGF7 affects the differentiation of embryonic stem cells (ESCs). We examined the effects of FGF7 on proliferation and osteogenic differentiation of mouse ESCs. Exogenous FGF7 addition did not change the proliferation rate of mouse ESCs. In contrast, the addition of FGF7 facilitated the dexamethasone, ascorbic acid, and β-glycerophosphate (DAG)-induced increases in bone-like nodule formation and calcium accumulation. FGF7 also augmented mRNA expression of runt-related transcription factor-2 (Runx2), osterix, bone sialoprotein (BSP), and osteocalcin (OC) in the presence of DAG. FGF7-mediated increases in the mineralization and bone-specific gene expression were almost completely attenuated by pretreating with anti-FGF7 antibody. FGF7 treatment accelerated the DAG-induced activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the cells. A pharmacological inhibitor specific to ERK, but not to JNK or p38 kinase, dramatically suppressed FGF7-mediated mineralization and accumulation of collagen and OC in the presence of DAG. This suppression was accompanied by the reduction in Runx2, osterix, BSP, and OC mRNA levels, which were increased by FGF7 in the presence of DAG. Collectively, our results suggest that FGF7 stimulates osteogenic differentiation, but not proliferation, in ESCs, by activating ERK/Runx2 signaling.

A phenolic acid phenethyl urea compound inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines in cell culture

We previously used the Curtius rearrangement to synthesize various phenolic acid phenethyl urea compounds from phenolic acids and demonstrated their beneficial anti-oxidant and anti-cancer effects. Here, we investigated the effects of one of these synthetic compounds, (E)-1-(3,4-dihydroxystyryl)-3-(4-hydroxyphenethyl)urea (DSHP-U), on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and cytokine secretion in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. DSHP-U suppressed LPS-induced NO production and iNOS expression at a concentration of 50 microM and inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. Inhibitors of phosphorylated (p)-ERK and p-p38, but not of p-JNK, reduced LPS-stimulated NO production. DSHP-U also prevented the nuclear translocation of the Rel A (p65) subunit and DNA-NF-kappaB binding by suppressing IkappaBalpha phosphorylation and by the degradation of IkappaBalpha in LPS-stimulated cells. Furthermore, DSHP-U decreased the production of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 in LPS-treated macrophages. However, the LPS-stimulated expression of LPS receptors, such as Toll-like receptor 4, myeloid differentiation factor-2, and CD14, was unchanged after DSHP-U treatment at significantly high levels. Our data suggest that DSHP-U blocks NO and inflammatory cytokine production in LPS-stimulated macrophages and that these effects are mainly mediated through the inhibition of the ERK/p38- and NF-kappaB signaling pathways.

Plasma-arc generated light inhibits proliferation and induces apoptosis of human gingival fibroblasts in a dose-dependent manner.

OBJECTIVE This study examined the effects of blue light exposure on the proliferation and cytotoxicity of human gingival fibroblasts (HGF). Cellular mechanism by which blue light causes cytotoxic effects was also investigated. METHODS HGF were exposed to the plasma-arc generated blue light with various energy densities ranging from 2 to 48J/cm(2). After light exposure of the cells, they were processed for analyzing tritium incorporation, succinate dehydrogenase (SDH) activity, trypan blue exclusion, and DNA fragmentation. In addition, possible mechanism of the light-mediated cytotoxicity was investigated through flow cytometric and Western blot analyses. RESULTS Blue light exposure significantly inhibited proliferation and SDH activity of HGF in a dose-dependent manner; exposure more than 12J/cm(2) had a toxic effect on the cells. The blue light-induced cytotoxicity of the cells resulted from apoptosis, as proven by the migration of many cells to the sub-G(1) phase of cell cycle and the appearance of DNA ladders. Additional experiments revealed that blue light induces apoptosis of HGF through mitochondrial stress and poly (ADP ribose) polymerase cleavage. SIGNIFICANCE This study suggests that plasma-arc generated blue light exerts some harm to cells, particularly damaging effect to DNA, and thus a long curing time more than recommended can cause biological damage on the oral tissue.