Tomoko Hashikawa

Fibroblast growth factor-2 regulates the synthesis of hyaluronan by human periodontal ligament cells.

Basic fibroblast growth factor (FGF‐2) can enhance biological potentials of periodontal ligament cells and its topical application induces considerable periodontal tissue regeneration in vivo. In this study, we examined the effect of FGF‐2 on the production of hyaluronan (HA), an extracellular matrix playing important roles in homeostasis and inflammatory/wound healing responses, by human periodontal ligament (HPDL) cells. An inhibition binding‐protein assay revealed that FGF‐2 significantly increased HA production by HPDL cells in a dose dependent manner. Analysis by HPLC revealed that in conditioned medium of FGF‐2‐treated HPDL cells HA had a higher molecular mass, compared to that of untreated HPDL cells. RT‐PCR analysis revealed the enhancement of mRNA expression of hyaluronan synthase (HAS) 1 and HAS 2, both of which contribute to the production of HA with a high molecular mass, but not HAS 3 in the FGF‐2‐treated HPDL cells. In contrast, three isoforms of hyaluronidase (HYAL) transcript were unchanged in the FGF‐2‐treated HPDL cells. These results provide new evidence for the possible involvement of FGF‐2 in the regulation of HA production and its appreciable roles in not only homeostasis but also regeneration of periodontal tissues.

Fibroblast growth factor-2 regulates the cell function of human dental pulp cells.

INTRODUCTION Homeostasis and tissue repair of dentin-pulp complex are attributed to dental pulp tissue and several growth factors. Dental pulp cells play a pivotal role in homeostasis of dentin-pulp complex and tissue responses after tooth injury. Among these cytokines, fibroblast growth factor (FGF)-2 has multifunctional biologic activity and is known as a signaling molecule that induces tissue regeneration. In this study, we examined the effects of FGF-2 on growth, migration, and differentiation of human dental pulp cells (HDPC). METHODS HDPC were isolated from healthy dental pulp. Cellular response was investigated by [(3)H]-thymidine incorporation into DNA. Cytodifferentiation was examined by alkaline phosphatase (ALPase) assay and cytochemical staining of calcium by using alizarin red. Migratory activity was determined by counting the cells migrating into cleared area that had introduced with silicon block. RESULTS FGF-2 activated HDPC growth and migration but suppressed ALPase activity and calcified nodule formation. Interestingly, HDPC, which had been pretreated with FGF-2, showed increased ALPase activity and calcified nodule formation when subsequently cultured without FGF-2. These results suggest that FGF-2 potentiates cell growth and accumulation of HDPC that notably did not disturb cytodifferentiation of the cells later. Thus, FGF-2 is a favorable candidate for pulp capping agent. CONCLUSIONS These results provide new evidence for the possible involvement of FGF-2 not only in homeostasis but also in regeneration of dentin-pulp complex.

Fibroblast growth factor-2 stimulates directed migration of periodontal ligament cells via PI3K/AKT signaling and CD44/hyaluronan interaction.

Fibroblast growth factor‐2 (FGF‐2) regulates a variety of functions of the periodontal ligament (PDL) cell, which is a key player during tissue regeneration following periodontal tissue breakdown by periodontal disease. In this study, we investigated the effects of FGF‐2 on the cell migration and related signaling pathways of MPDL22, a mouse PDL cell clone. FGF‐2 activated the migration of MPDL22 cells and phosphorylation of phosphatidylinositol 3‐kinase (PI3K) and akt. The P13K inhibitors, Wortmannin and LY294002, suppressed both cell migration and akt activation in MPDL22, suggesting that the PI3K/akt pathway is involved in FGF‐2‐stimulated migration of MPDL22 cells. Moreover, in response to FGF‐2, MPDL22 showed increased CD44 expression, avidity to hyaluronan (HA) partly via CD44, HA production and mRNA expression of HA synthase (Has)‐1, 2, and 3. However, the distribution of HA molecular mass produced by MPDL22 was not altered by FGF‐2 stimulation. Treatment of transwell membrane with HA facilitated the migration of MPDL22 cells and an anti‐CD44 neutralizing antibody inhibited it. Interestingly, the expression of CD44 was colocalized with HA on the migrating cells when stimulated with FGF‐2. Furthermore, an anti‐CD44 antibody and small interfering RNA for CD44 significantly decreased the FGF‐2‐induced migration of MPDL22 cells. Taken together, PI3K/akt and CD44/HA signaling pathways are responsible for FGF‐2‐mediated cell motility of PDL cells, suggesting that FGF‐2 accelerates periodontal regeneration by regulating the cellular functions including migration, proliferation and modulation of extracellular matrix production. J. Cell. Physiol. 226: 809–821, 2011.

Fibroblast growth factor-2 regulates expression of osteopontin in periodontal ligament cells

Osteopontin is a protein found in the bone‐related matrix and plays multiple regulatory roles in mineralizing and non‐mineralizing tissue. In osteogenic cell‐lines, the expression of osteopontin increases with the progression of differentiation, but both the expression and function of osteopontin vary with the cell type and its activation state. In this study, we examined the expression of osteopontin by clones established from mouse periodontal ligament, in response to inorganic phosphate and fibroblast growth factor (FGF)‐2, which can induce periodontal tissue regeneration. The involvement of inorganic phosphate in the expression of osteopontin during the course of cell differentiation of a clone MPDL22 was confirmed by addition of foscarnet, an inorganic phosphate transport inhibitor. Although FGF‐2 decreased the mRNA expression of almost every bone‐related protein in MPDL22, FGF‐2 upregulated the expression of osteopontin in MPDL22 at both mRNA and protein levels. Interestingly, FGF‐2 enhanced the concentration of osteopontin in the culture supernatant of MPDL22, whereas inorganic phosphate did not. The FGF‐2‐induced osteopontin in the culture supernatant seems to be involved in cell survival activity. An immunohistochemical study showed that the FGF‐2‐induced osteopontin was mainly present in perinuclear matrices while the inorganic phosphate‐induced osteopontin was associated with extracellular matrices in addition to perinuclear matrices. The present results indicated that FGF‐2 induces unique expression of osteopontin, which may play a role different from the other bone‐related proteins during the process of periodontal tissue regeneration by FGF‐2. J. Cell. Physiol. 216: 640–650, 2008,

Activation of Adenosine-receptor-enhanced iNOS mRNA Expression by Gingival Epithelial Cells

A series of reports has revealed that adenosine has a plethora of biological actions toward a large variety of cells. In this study, we investigated the influence of adenosine receptor activation on iNOS mRNA expression in human gingival epithelial cells (HGEC) and SV-40-transformed HGEC. HGEC expressed adenosine receptor subtypes A1, A2a, and A2b, but not A3 mRNA. Ligation of adenosine receptors by a receptor agonist, 2-chloroadenosine (2CADO), enhanced iNOS mRNA expression by both HGEC and transformed HGEC. In addition, the adenosine receptor agonist enhanced the production of NO2 -/NO3 -, NO-derived stable end-products. An enhanced expression of iNOS mRNA and NO2 -/NO3 - was also observed when SV40-transformed HGEC were stimulated with CPA or CGS21680, A1- or A2a-selective adenosine receptor agonists, respectively. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses by HGEC in periodontal tissues.

Adiponectin regulates functions of gingival fibroblasts and periodontal ligament cells

BACKGROUND AND OBJECTIVE Adiponectin is a cytokine constitutively produced by adipocytes and exhibits multiple biological functions by targeting various cell types. However, the effects of adiponectin on primary gingival fibroblasts and periodontal ligament cells are still unexplored. Therefore, we investigated the effects of adiponectin on gingival fibroblasts and periodontal ligament cells. MATERIAL AND METHODS The expression of adiponectin receptors (AdipoR1 and AdipoR2) on human gingival fibroblasts (HGFs), mouse gingival fibroblasts (MGFs) and human periodontal ligament (HPDL) cells was examined using RT-PCR and western blotting. HGFs and MGFs were stimulated with interleukin (IL)-1β in the presence or absence of adiponectin, and the expression of IL-6 and IL-8 at both mRNA and protein levels was measured by real-time PCR and ELISA, respectively. Furthermore, small interfering RNAs (siRNAs) in MGFs were used to knock down the expression of mouse AdipoR1 and AdipoR2. The effects of adiponectin on the expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) genes were evaluated by real-time PCR. Mineralized nodule formation of adiponectin-treated HPDL cells was revealed by Alizarin Red staining. RESULTS AdipoR1 and AdipoR2 were expressed constitutively in HGFs, MGFs and HPDL cells. Adiponectin decreased the expression of IL-6 and IL-8 in IL-1β-stimulated HGFs and MGFs. AdipoR1 siRNA in MGFs revealed that the effect of adiponectin on reduction of IL-6 expression was potentially mediated via AdipoR1. Adiponectin-treated HPDL cells promoted the expression of ALP and Runx2 mRNAs and up-regulated ALP activity. Furthermore, adiponectin enhanced mineralized nodule formation of HPDL cells. CONCLUSION Our observations demonstrate that adiponectin exerts anti-inflammatory effects on HGFs and MGFs, and promotes the activities of osteoblastogenesis of HPDL cells. We conclude that adiponectin has potent beneficial functions to maintain the homeostasis of periodontal health, improve periodontal lesions, and contribute to wound healing and tissue regeneration.

Involvement of CD73 (ecto-5′-nucleotidase) in Adenosine Generation by Human Gingival Fibroblasts

Adenosine has various biological effects on human gingival fibroblasts (HGF) and epithelial cells closely associated with inflammation, such as cytokine production and cell adhesion. However, the mechanism of adenosine formation in periodontal tissues is not yet defined. In this study, we examined the involvement of CD73 (ecto-5′-nucleotidase) in adenosine generation by HGF. CD73 was detected on in vitro-maintained HGF by immunocytochemistry and flow cytometric analysis. Adenosine production was observed following the addition of 5′-AMP, the substrate of CD73-associated ecto-5′-nucleotidase. Moreover, the addition of 5′-AMP to cultured HGF resulted in the elevation of cyclic adenosine monophosphate (cAMP). The 5′-AMP-induced increase in intracellular cAMP level was inhibited markedly by xanthine amine congener, an adenosine receptor antagonist, and partially by α,β-methylene adenosine 5′-diphosphate, an ecto-5′-nucleotidase inhibitor. These results suggest that CD73 on HGF is a critical enzyme responsible for the generation of adenosine, an immunomodulator that activates adenosine receptors.

Activation of Adenosine Receptor on Gingival Fibroblasts

CD73 (ecto-5′-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5′-AMP via CD73 and the ability of 5′-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.

Induction of CD13 on T-lymphocytes by Adhesive Interaction with Gingival Fibroblasts

Lymphocytes in peripheral blood do not express CD13 (aminopeptidase N), a membrane alanyl metallopeptidase. However, it has been demonstrated that locally infiltrated lymphocytes in chronic inflammatory sites can be CD13-positive, and possible involvement of stromal cell adherence in the induction of CD13 has been suggested. In this study, we examined whether T-lymphocyte/gingival-fibroblast interaction can activate T-lymphocytes to express CD13. CD13 expression was induced on PMA-activated T-lymphocytes only when they adhered directly to human gingival fibroblasts (HGF) at 2 hrs after the co-culture began, while an increase in the enzyme activity of CD13 was also confirmed in activated T-lymphocytes that had been co-cultured with HGF. Furthermore, CD13-positive T-lymphocytes were detected in inflamed gingival tissues in vivo. Analysis of these results indicates that direct interaction with HGF is essential for the induction of CD13 expression on T-lymphocytes that was also observed in periodontitis lesions.