Mami Terakura

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 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.

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.