Neutrophil extracellular traps inhibit osteoclastogenesis.

Neutrophil extracellular traps (NETs) released by neutrophils upon inflammation or infection, act as an innate immune defense against pathogens. NETs also influence inflammatory responses and cell differentiation in host cells. Osteoclasts, which are derived from myeloid stem cells, are critical for the bone remodeling by destroying bone. In the present study, we explores the impact of NETs, induced by the inflammatory agent calcium ionophore A23187, on the differentiation and activation of osteoclasts, potentially through suppressing RANK expression. Our results collectively suggested that the inhibition of RANKL-mediated osteoclastogenesis by NETs might lead to the suppression of excessive bone resorption during inflammation.

Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Macrophage migration inhibitory factor-mediated mast cell extracellular traps induce inflammatory responses upon Fusobacterium nucleatum infection.

Mast cell extracellular traps (MCETs) released by mast cells contribute to host defense. In this study, we investigated the effects of MCETs released from mast cells after infection with a periodontal pathogen Fusobacterium nucleatum. We found that F. nucleatum induced MCET release from mast cells, and that MCETs expressed macrophage migration inhibitory factor (MIF). Notably, MIF bound to MCETs induced proinflammatory cytokine production by monocytic cells. These findings suggest that MIF expressed on MCETs, released from mast cells upon infection with F. nucleatum, promotes inflammatory responses that may be associated with the pathogenesis of periodontal disease.

Serial Cultivation of an MSC-Like Cell Line with Enzyme-Free Passaging Using a Microporous Titanium Scaffold.

In vitro studies on adherent cells require a process of passage to dissociate the cells from the culture substrate using enzymes or other chemical agents to maintain cellular activity. However, these proteolytic enzymes have a negative influence on the viability and phenotype of cells. The mesenchymal stem cell (MSC)-like cell line, C3H10T1/2, adhered, migrated, and proliferated to the same extent on newly designed microporous titanium (Ti) membrane and conventional culture dish, and spontaneous transfer to another substrate without enzymatic or chemical dissociation was achieved. The present study pierced a 10 µm-thick pure Ti sheet with 25 µm square holes at 75 µm intervals to create a dense porous structure with biomimetic topography. The pathway of machined holes allowed the cells to access both sides of the membrane frequently. In a culture with Ti membranes stacked above- and below-seeded cells, cell migration between the neighboring membranes was confirmed using the through-holes of the membrane and contact between the membranes as migration routes. Furthermore, the cells on each membrane migrated onto the conventional culture vessel. Therefore, a cell culture system with enzyme-free passaging was developed.

Pharmacological Activation of YAP/TAZ by Targeting LATS1/2 Enhances Periodontal Tissue Regeneration in a Murine Model.

Due to their multi-differentiation potential, periodontal ligament fibroblasts (PDLF) play pivotal roles in periodontal tissue regeneration in vivo. Several in vitro studies have suggested that PDLFs can transmit mechanical stress into favorable basic cellular functions. However, the application of mechanical force for periodontal regeneration therapy is not expected to exhibit an effective prognosis since mechanical forces, such as traumatic occlusion, also exacerbate periodontal tissue degeneration and loss. Herein, we established a standardized murine periodontal regeneration model and evaluated the regeneration process associated with cementum remodeling. By administering a kinase inhibitor of YAP/TAZ suppressor molecules, such as large tumor suppressor homolog 1/2 (LATS1/2), we found that the activation of YAP/TAZ, a key downstream effector of mechanical signals, accelerated periodontal tissue regeneration due to the activation of PDLF cell proliferation. Mechanistically, among six kinds of MAP4Ks previously reported as upstream kinases that suppressed YAP/TAZ transcriptional activity through LATS1/2 in various types of cells, MAP4K4 was identified as the predominant MAP4K in PDLF and contributed to cell proliferation and differentiation depending on its kinase activity. Ultimately, pharmacological activation of YAP/TAZ by inhibiting upstream inhibitory kinase in PDLFs is a valuable strategy for improving the clinical outcomes of periodontal regeneration therapies.

Introduction of tenomodulin by gene transfection vectors for rat bone tissue regeneration.

Introduction: Periodontal ligament is regenerated in association with hard tissue regeneration. Tenomodulin (Tnmd) expression has been confirmed in periodontal ligament and it reportedly inhibits angiogenesis or is involved in collagen fibril maturation. The introduction of Tnmd by gene transfection in bone tissue regeneration therapy might inhibit topical hard tissue formation and induce the formation of dense fibrous tissue. Therefore, the effect of Tnmd introduction by gene transfection technique in vitro and in vivo was investigated in this study. Methods: Osteogenesis- and chondrogenesis-related gene expression levels in osteoblastic cells (MC3T3E1) and rat bone marrow derived cells were detected using qPCR three days after gene transfection with plasmid DNA (Tnmd) using non-viral gene transfection vectors: a calcium phosphate-based gene transfection vector (CaP(Tnmd)) or a cationic polymer-based reagent (JetPEI (Tnmd)). Next, an atelocollagen scaffold with or without CaP (Tnmd) or JetPEI (Tnmd) was implanted into a rat calvaria bone defect, and the remaining bone defect volume and the tissue reaction at 28 days after surgery were evaluated. Results: Runx 2 and SP7 mRNA was reduced by JetPEI (Tnmd) in both cells, but not in CaP(Tnmd). The volume of expressed Tnmd was at 9 ng/mL in both gene transfection vector. The remaining bone defect volume of JetPEI (Tnmd) was significantly bigger than that of the other groups and CaP (EGFP), and that of CaP (Tnmd) was significantly bigger than that of CaP (EGFP). Conclusions: Tnmd introduction treatment inhibits bone formation in artificial bone defect, however, the effect of that was dependent on non-viral gene transfection vector.

Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes.

Edible mushrooms are known to exert anti-inflammatory effects. In this study, the effects of ethanol extracts from edible mushrooms, such as Hericium erinaceus, and other edible mushrooms on inflammatory responses were investigated. Experiments were conducted using the inflammatory responses of human monocytes induced by lipopolysaccharide (LPS), a bacterial component, that provokes inflammation. Notably, we demonstrated that LPS mixed with ethanol and hot water extracts derived from edible mushrooms attenuated the production of inflammatory cytokines, such as interleukin (IL)-1ß, -6, and -8, induced by LPS in human monocytic cell cultures. Moreover, we found that the ethanol extract of H. erinaceus contained ergosterol, which attenuated IL-8 production in LPS-stimulated cells. Subsequent component analysis of the ethanol extract of H. erinaceus revealed that ergosterol binds to lipid A to attenuate LPS-induced inflammation. Together, our findings suggest that ergosterol in ethanol extracts from edible mushrooms can prevent the induction of inflammation by binding to LPS.

Extracellular Vesicles Derived From Murine Cementoblasts Possess the Potential to Increase Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclastogenesis.

Cementum resorption, unlike bone resorption, is clinically known to occur only with limited pathological stimuli, such as trauma, orthodontic forces, and large apical periodontitis; however, the molecular mechanisms that control osteoclast formation on the cementum surface remain unclear. In this study, we focused on extracellular vesicles (EVs) secreted by cementoblasts and analyzed their effects on osteoclast differentiation. EVs were extracted from the conditioned medium (CM) of the mouse cementoblast cell line OCCM-30. Transmission electron microscopy (TEM) analysis confirmed the presence of EVs with a diameter of approximately 50-200 nm. The effect of the EVs on osteoclast differentiation was examined using the mouse osteoclast progenitor cell line RAW 264.7 with recombinant receptor activator of nuclear factor (NF)-κB ligand (rRANKL) stimulation. EVs enhanced the formation of tartrate-resistant acid phosphatase (TRAP) activity-positive cells upon rRANKL stimulation. EVs also enhanced the induction of osteoclast-associated gene and protein expression in this condition, as determined by real-time PCR and Western blotting, respectively. On the other hand, no enhancing effect of EVs was observed without rRANKL stimulation. A Western blot analysis revealed no expression of receptor activator of NF-κB ligand (RANKL) in EVs themselves. The effect on rRANKL-induced osteoclast differentiation was examined using the CM of cementoblasts in terms of TRAP activity-positive cell formation and osteoclast-associated gene expression. The conditioned medium partly inhibited rRANKL-induced osteoclast differentiation and almost completely suppressed its enhancing effect by EVs. These results indicate that cementoblasts secreted EVs, which enhanced RANKL-induced osteoclast differentiation, and simultaneously produced soluble factors that neutralized this enhancing effect of EVs, implicating this balance in the regulation of cementum absorption. A more detailed understanding of this crosstalk between cementoblasts and osteoclasts will contribute to the development of new therapies for pathological root resorption.

Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells.

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

DMP-1 promoter-associated antisense strand non-coding RNA, panRNA-DMP-1, physically associates with EGFR to repress EGF-induced squamous cell carcinoma migration.

Accumulating evidence suggests that specific non-coding RNAs exist in many types of malignant tissues, and are involved in cancer invasion and metastasis. However, little is known about the precise roles of non-coding RNAs in squamous cell carcinoma (SQCC) invasion and migration. Recently, the dentin matrix protein-1 (DMP-1) gene locus was identified as a transcriptionally active site in squamous cell carcinoma (SQCC) tissue and cells. However, it is unclear whether RNA associated with cell migration exist at the DMP-1 gene locus in SQCC cells. We identified a novel promoter-associated non-coding RNA in the antisense strand of DMP-1 gene locus, promoter-associated non-coding RNA (panRNA)-DMP-1, by the RACE method in SQCC cells and tissues, and characterized the functions of panRNA-DMP-1 in EGF-driven SQCC cell migration. The inhibition of endogenous panRNA-DMP-1 expression by specific siRNAs and exogenous over-expression of panRNA-DMP-1 resulted in increased and suppressed cellular migration toward EGF in SQCC cells, respectively, and nuclear expression of panRNA-DMP-1 was induced by EGF stimulation. Mechanistically, suppression of panRNA-DMP-1 expression increased EGFR nuclear localization upon EGF treatment and nuclear panRNA-DMP-1 physically interacted with EGFR, which was confirmed by RNA immunoprecipitation assay using a bacteriophage-delivered PP7 RNA labeling system. Furthermore, co-immunoprecipitation assay revealed that suppression of panRNA-DMP-1 stabilized EGFR interaction with STAT3, a known co-transcription factors of EGFR, to induce migratory properties in many cancer cells. Based on these findings, panRNA-DMP-1 is an EGFR-associating RNA that inhibits the EGF-induced migratory properties of SQCC possibly by regulating EGFR nuclear localization and EGFR binding to STAT3.

A large-scale observational study to investigate the current status of diabetic complications and their prevention in Japan (JDCP study 6): baseline dental and oral findings.

Japan Diabetes Complication and Prevention prospective (JDCP) study was conducted to examine the association between glycemic control and oral conditions in a large database of Japanese patients with diabetes. It included a total of 6099 patients with diabetes (range, 40-75 years) who had been treated as outpatients between 2007 and 2009. The mean number of present teeth at baseline was 19.8 and women with type 2 diabetes had fewer teeth than men with type 2 diabetes. Within the previous year, 17% of all patients had lost teeth. At baseline, 32% had experienced gingival swelling, 69% had brushed more than twice a day, 37% had used interdental cleaning aids, and 43% had undergone regular dental checkups. Multiple logistic regression analysis indicated that type 1 patients with HbA1c ≥ 7.0% were at higher risk of having fewer than 20 teeth (odds ratio [OR] 2.38; 95% confidence interval [CI] 1.25-4.78), and type 2 patients with HbA1c ≥ 8.0% also were at high risk of having fewer than 20 teeth (OR 1.16; 95% CI 1.00-1.34), after adjustment for nine possible confounding factors. In conclusion, patients with diabetes were found to be at high risk of tooth loss, and the poorer the glycemic control, the higher the risk of tooth loss in these patients.

Evaluation of Preosteoblast MC3T3-E1 Cells Cultured on a Microporous Titanium Membrane Fabricated Using a Precise Mechanical Punching Process.

The surface topography of Titanium (Ti) combined toughness and biocompatibility affects the attachment and migration of cells. Limited information of morphological characteristics, formed by precise machining in micron order, is currently available on the Ti that could promote osteoconduction. In the present study, a pure Ti membrane was pierced with precise 25 µm square holes at 75 µm intervals and appear burrs at the edge of aperture. We defined the surface without burrs as the "Head side" and that with burrs as the "Tail side". The effects of the machining microtopography on the proliferation and differentiation of the preosteoblasts (MC3T3-E1 cells) were investigated. The cells were more likely to migrate to, and accumulate in, the aperture of holes on the head side, but grew uniformly regardless of holes on the tail side. The topography on the both surfaces increased osteopontin gene expression levels. Osteocalcin expression levels were higher on the head side than one on the blank scaffold and tail side (p < 0.05). The osteocalcin protein expression levels were higher on the tail side than on the head side after 21 days of cultivation, and were comparable to the proportion of the calcified area (p < 0.05). These results demonstrate the capacity of a novel microporous Ti membrane fabricated using a precise mechanical punching process to promote cell proliferation and activity.

Azimuthal-rotation sample holder for molecular orientation analysis.

In this study, an azimuthal-rotation sample holder compatible with scanning transmission X-ray microscopy was developed. This holder exhibits improvement in the accuracy of rotation angles and reduces the displacement of the rotation axes during azimuthal rotation by using a crossed roller bearing. To evaluate the performance of the holder, the authors investigated the dependence of the optical density around the C K-edge absorption of π-orbital-oriented domains in natural spherical graphite on the rotational angle by using linearly horizontally and vertically polarized undulator radiation. Based on the dependence of the optical density ratio between C 1s → π* and 1s → σ* excitation on the polarization angle of the X-rays, the average two-dimensional orientation angle of the π orbital in each position in a natural spherical graphite sample was visualized.

Cyclic Stretch Force Induces Periodontal Ligament Cells to Secrete Exosomes That Suppress IL-1ß Production Through the Inhibition of the NF-κB Signaling Pathway in Macrophages.

In the oral mechanical environment, periodontal ligament cells (PDL cells) contribute to maintaining periodontal tissue homeostasis. Recent studies showed that exosomes, which are small vesicles secreted by various types of cells, play a pivotal role in cell-to-cell communication in biological processes. We examined the secretion of exosomes from PDL cells stimulated with cyclic stretch and their role in the inflammatory response of macrophages using the human macrophage cell line THP-1 and human primary monocytes/macrophages. We prepared supernatants from human PDL cells (PDL-sup) stimulated with cyclic stretch. The treatment of macrophages with PDL-sup, but not PDL-sup from unstimulated PDL cells, inhibited the production of IL-1ß in LPS/nigericin-stimulated macrophages. The pretreatment of PDL cells with GW4869, an inhibitor of exosome secretion, or siRNA for Rab27B, which controls exosome secretion, abrogated the inhibitory effects of PDL-sup. A transmission electron microscopy analysis demonstrated the existence of exosomes with diameters ranging between 30 and 100 nm in PDL-sup, suggesting that exosomes in PDL-sup contribute to this inhibition. An immunofluorescence microscopy analysis revealed that exosomes labeled with PKH67, a fluorescent dye, were incorporated by macrophages as early as 2 h after the addition of exosomes. Purified exosomes inhibited IL-1ß production in LPS/nigericin-stimulated macrophages and the nuclear translocation of NF-κB as well as NF-κB p65 DNA-binding activity in LPS-stimulated macrophages, suggesting that exosomes suppress IL-1ß production by inhibiting the NF-κB signaling pathway. Our results indicate that PDL cells in mechanical environments contribute to the maintenance of periodontal immune/inflammatory homeostasis by releasing exosomes.

Mechanical regulation of macrophage function - cyclic tensile force inhibits NLRP3 inflammasome-dependent IL-1ß secretion in murine macrophages.

Mechanical stress maintains tissue homeostasis by regulating many cellular functions including cell proliferation, differentiation, and inflammation and immune responses. In inflammatory microenvironments, macrophages in mechanosensitive tissues receive mechanical signals that regulate various cellular functions and inflammatory responses. Macrophage function is affected by several types of mechanical stress, but the mechanisms by which mechanical signals influence macrophage function in inflammation, such as the regulation of interleukin-1ß by inflammasomes, remain unclear. In this review, we describe the role of mechanical stress in macrophage and monocyte cell function.

Periodontal Regeneration by Allogeneic Transplantation of Adipose Tissue Derived Multi-Lineage Progenitor Stem Cells in vivo.

The ultimate goal of periodontal disease treatment is the reorganization of functional tissue that can regenerate lost periodontal tissue. Regeneration of periodontal tissues is clinically possible by using autogenic transplantation of MSCs. However, autologous MSC transplantation is limited depending on age, systemic disease and tissue quality, thus precluding their clinical application. Therefore, we evaluated the efficacy of allogeneic transplantation of adipose-derived multi-lineage progenitor cells (ADMPC) in a micro-mini pig periodontal defect model. ADMPC were isolated from the greater omentum of micro-mini pigs, and flow cytometry analysis confirmed that the ADMPC expressed MSC markers, including CD44 and CD73. ADMPC exhibited osteogenic, adipogenic and periodontal ligament differentiation capacities in differentiation medium. ADMPC showed high expression of the immune suppressive factors GBP4 and IL1-RA upon treatment with a cytokine cocktail containing interferon-γ, tumor necrosis factor-α and interleukin-6. Allogeneic transplantation of ADMPC in a micro-mini pig periodontal defect model showed significant bone regeneration ability based on bone-morphometric analysis. Moreover, the regeneration ability of ADMPC by allogeneic transplantation was comparable to those of autologous transplantation by histological analysis. These results indicate that ADMPC have immune-modulation capability that can induce periodontal tissue regeneration by allogeneic transplantation.

Cyclic Stretch Negatively Regulates IL-1ß Secretion Through the Inhibition of NLRP3 Inflammasome Activation by Attenuating the AMP Kinase Pathway.

Macrophages are immune cells of hematopoietic origin that play diverse roles in host defenses and tissue homeostasis. In mechanical microenvironments, macrophages receive mechanical signals that regulate various cellular functions. However, the mechanisms by which mechanical signals influence the phenotype and function of macrophages in the process of inflammation have not yet been elucidated in detail. We herein examined the effects of cyclic stretch (CS) on NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation in J774.1, a murine macrophage cell line, and mouse primary bone marrow-derived macrophages. We showed that cyclic stretch inhibited adenosine triphosphate (ATP)-stimulated interleukin (IL)-1ß secretion in lipopolysaccharide (LPS)-primed macrophages using ELISA and Western blot analyses. Cyclic stretch did not affect the degradation of the Inhibitor of κB or the nuclear translocation/transcriptional activity of nuclear factor (NF)-κB, suggesting that cyclic stretch-mediated inhibition was independent of the NF-κB signaling pathway. Consistent with these results, cyclic stretch did not affect the LPS-induced expression of inflammasome components, such as pro-IL-1ß and NLRP3, which is known to require the activation of NF-κB signaling. We showed that the cyclic stretch-mediated inhibition of IL-1ß secretion was caused by the suppression of caspase-1 activity. The addition of compound C, a specific inhibitor of adenosine monophosphate-activated protein kinase (AMPK), to LPS-primed macrophages inhibited IL-1ß secretion as well as caspase-1 activation, suggesting that AMPK signaling is involved in ATP-triggered IL-1ß secretion. Furthermore, the phosphorylation of AMPK induced by ATP in LPS-primed macrophages was significantly suppressed by cyclic stretch, indicating that cyclic stretch negatively regulates IL-1ß secretion through the inhibition of caspase-1 activity by attenuating the AMPK pathway. Our results suggest that mechanical stress functions to maintain homeostasis through the prevention of excessive inflammasome activation in macrophages in mechanical microenvironments.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells.

We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.