Masahide Takedachi

CD73-Generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73−/− mice have normal sized lymphoid organs in the steady state, but ∼1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73+/+ and cd73−/− lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A2B receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-α. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73−/− mice is largely normalized by pretreatment with the selective A2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

CD73-generated adenosine promotes osteoblast differentiation.

CD731 is a GPI‐anchored cell surface protein with ecto‐5′‐nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73‐generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73‐deficient (cd73−/−) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild‐type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3‐E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3‐E1 cells expressed adenosine A2A receptors (A2AAR) and A2B receptors (A2BAR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3‐E1 cells over expressing CD73 were suppressed by treatment with an A2BAR antagonist but not with an A2AAR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A2BAR signaling. J. Cell. Physiol. 227: 2622–2631, 2012.

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.

Regulation of adenosine receptor engagement by ecto-adenosine deaminase

Adenosine deaminase (ADA) can localize to the cell surface through its interaction with CD26. Using CD26‐transfected cells, we demonstrate that cell surface ADA (ecto‐ADA) can regulate adenosine receptor engagement by degrading extracellular adenosine (Ado) to inosine. This ability was dependent upon CD26 expression, the extent of CD26 saturation with ecto‐ADA, and the kinetics of the cAMP response. Thus, the cAMP response was markedly decreased when CD26‐transfected cells were incubated with an exogenous source of ADA to increase ecto‐ADA expression. The ability of ecto‐ADA to inhibit the cAMP response was demonstrated by treatment with the specific ADA inhibitor 2′‐deoxycoformycin. This inhibited the ability of ecto‐ADA to degrade Ado and increased the cAMP response. Although CD26 expression on human thymocytes was low compared with that of CD26‐transfected cells, it was saturated with ecto‐ADA. When thymocytes incubated at high densities (to mimic the situation in tissues) were exposed to exogenous adenosine, the cAMP response was dramatically decreased by ecto‐ADA. We conclude that ecto‐ADA has the potential to regulate adenosine receptor‐mediated cAMP responses in vivo in tissues with CD26+ cells and sufficient cell death caused by apoptosis or inflammation to provide a source of ADA to bind to CD26.

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.

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells ☆

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.

Effects of L-ascorbic acid 2-phosphate magnesium salt on the properties of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE L-Ascorbic acid 2-phosphate magnesium salt (APM) is an L-ascorbic acid (AsA) derivative developed to improve AsA stability and display effective biochemical characteristics. This study aimed to investigate the effects of APM on the functions and properties of human gingival fibroblasts with respect to the prevention of periodontal disease in comparison with those of AsA. MATERIAL AND METHODS Human gingival fibroblasts were incubated in the presence or absence of APM or L-ascorbic acid sodium salt (AsANa). Intracellular AsA was analysed by HPLC. Collagen synthesis was measured by ELISA and real-time RT-PCR. Intracellular reactive oxygen species (ROS) induced by hydrogen peroxide (H(2)O(2)) were quantified using a fluorescence reagent, and cell damage was estimated with calcein acetoxymethyl ester. Furthermore, intracellular ROS induced by tumor necrosis factor-α (TNF-α) were quantified, and expression of TNF-α-induced interleukin-8 expression, which increases due to inflammatory reactions, was measured by ELISA and real-time RT-PCR. RESULTS APM remarkably and continuously enhanced intracellular AsA and promoted type 1 collagen synthesis and mRNA expression. Furthermore, APM decreased cell damage through the suppression of H(2)O(2)-induced intracellular ROS and inhibited interleukin-8 production through the suppression of TNF-α-induced intracellular ROS. These effects of APM were superior to those of AsANa. CONCLUSION These results suggest that APM is more effective than AsANa in terms of intake, collagen synthesis, decreasing cell damage and inhibiting interleukin-8 expression in human gingival fibroblasts. This suggests that local application of APM can help to prevent periodontal disease.