RESUMO
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.
Assuntos
Movimento Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Ligamento Periodontal/citologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/enzimologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Osteopontina/metabolismo , Ligamento Periodontal/citologia , Animais , Apoptose , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Camundongos , Osteopontina/genética , Ligamento Periodontal/metabolismo , Fosfatos/química , Fosfatos/metabolismoRESUMO
Heparan sulfate (HS) proteoglycan is a widely distributed biological molecule that mediates a variety of physiological responses in development, cell growth, cell migration, and wound healing. We examined the effects of basic fibroblast growth factor-2 (FGF-2), which is known to modulate extracellular matrix (ECM) production of various cell types, on the production of HS proteoglycan by human periodontal ligament (HPDL) cells. We also examined the effects of FGF-2 on the expression of syndecans, a major family of membrane-bound HS proteoglycans. Treatment of HPDL cells with FGF-2 for 72 h resulted in a pronounced increase in the level of HS in the culture supernatant in a dose-dependent manner. However, reverse transcription-polymerase chain reaction data (RT-PCR) revealed that FGF-2 marginally reduced the gene expression of syndecan-1, -2, and -4, and did not alter the level of syndecan-3 mRNA. Furthermore, FGF-2 did not have an effect on the mRNA expression of enzymes associated with HS biosynthesis. Interestingly, FACS analysis revealed that the syndecan family displayed diverse alterations in response to FGF-2. FGF-2 barely altered the expression of syndecan-1, but decreased the expression of syndecan-2 and -4 on HPDL cells. Moreover, dot blot analysis showed that FGF-2 did not alter the level of syndecan-1 and -2, but enhanced the level of syndecan-4 in culture supernatants of FGF-2-stimulated HPDL cells. These results suggest that the FGF-2-activated increase in the level of HS in conditioned medium may be a result of shedding of syndecan-4 from the HPDL cell surface. Taken together, FGF-2 may differentially regulate the expression of HS proteoglycans in a HS-proteoglycan-subtype-dependent manner. The diversity of the expression patterns of HS proteoglycans may be associated with the FGF-2-induced biological functions of HPDL cells.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Heparitina Sulfato/metabolismo , Ligamento Periodontal/citologia , Linhagem Celular , Separação Celular , Meios de Cultura/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Imuno-Histoquímica/métodos , Modelos Biológicos , RNA Mensageiro/metabolismo , Fatores de Tempo , CicatrizaçãoRESUMO
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.
Assuntos
Adenosina Desaminase/fisiologia , Receptores Purinérgicos P1/metabolismo , Linfócitos T/enzimologia , Adenosina/antagonistas & inibidores , Adenosina/farmacologia , Linhagem Celular , Células Cultivadas , AMP Cíclico/metabolismo , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Humanos , Células Jurkat , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Timo/metabolismo , TransfecçãoRESUMO
Hyaluronan (HA), is a high molecular mass extracellular matrix constituting connective tissue and plays a critical role in not only homeostasis but also inflammatory and wound-healing responses. In this study, we investigated the effect of fibroblast growth factor (FGF)-2 on the production of HA by human dental pulp cells (HDPC). An inhibition binding-protein assay showed that FGF-2 increased HA production by HDPC. In addition, expression of mRNA of hyaluronan synthase (HAS) 1 and HAS 2, both of which are related to the production of high molecular mass of HA, but not HAS 3, was enhanced in FGF-2-stimulated HDPC. These results provide new evidence for the involvement of FGF-2 in the regulation of HA production by HDPC possibly through HAS 1 and HAS 2.
Assuntos
Polpa Dentária/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Ácido Hialurônico/análise , Células Cultivadas , Polpa Dentária/citologia , Glucuronosiltransferase/análise , Glucuronosiltransferase/efeitos dos fármacos , Humanos , Hialuronan Sintases , Isoenzimas/análise , Isoenzimas/efeitos dos fármacos , RNA Mensageiro/análiseRESUMO
Adipose tissue is an attractive source for somatic stem cell therapy. Currently, human adipose tissue-derived stromal cells/mesenchymal stem cells (hADSCs/MSCs) are cultured with fetal bovine serum (FBS). Recently, however, not only human embryonic stem cell lines cultured on mouse feeder cells but also bone marrow-derived human MSCs cultured with FBS were reported to express N-glycolylneuraminic acid (Neu5Gc) xenoantigen. Human serum contains high titers of natural preformed antibodies against Neu5Gc. We studied the presence of Neu5Gc on hADSCs/MSCs cultured with FBS and human immune response mediated by Neu5Gc. Our data indicated that hADSCs/MSCs cultured with FBS expressed Neu5Gc and that human natural preformed antibodies could bind to hADSCs/MSCs. However, hADSCs/MSCs express complement regulatory proteins such as CD46, CD55, and CD59 and are largely resistant to complement-mediated cytotoxicity. hADSCs/MSCs cultured with FBS could be injured by antibody-dependent cell-mediated cytotoxicity mechanism. Further, human monocyte-derived macrophages could phagocytose hADSCs/MSCs cultured with FBS and this phagocytic activity was increased in the presence of human serum. Culturing hADSCs/MSCs with heat-inactivated human serum for a week could markedly reduce Neu5Gc on hADSCs/MSCs and prevent immune responses mediated by Neu5Gc, such as binding of human natural preformed antibodies, antibody-dependent cell-mediated cytotoxicity, and phagocytosis. Adipogenic and osteogenic differentiation potentials of hADSCs/MSCs cultured with heat-inactivated human serum were not less than that of those cultured with FBS. For stem cell therapies based on hADSCs/MSCs, hADSCs/MSCs that presented Neu5Gc on their cell surfaces after exposure to FBS should be cleaned up to be rescued from xenogeneic rejection.
Assuntos
Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Antígenos Heterófilos/metabolismo , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Ácidos Neuramínicos/imunologia , Ácidos Neuramínicos/metabolismo , Adipogenia , Tecido Adiposo/citologia , Animais , Anticorpos Heterófilos/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Bovinos , Diferenciação Celular , Células Cultivadas , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Macrófagos/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Osteogênese , Fagocitose , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/metabolismo , Engenharia TecidualRESUMO
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.
Assuntos
Polpa Dentária/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fosfatase Alcalina/análise , Antraquinonas , Calcificação Fisiológica/efeitos dos fármacos , Cálcio/análise , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corantes , DNA/biossíntese , Polpa Dentária/citologia , Homeostase/efeitos dos fármacos , Humanos , Compostos Radiofarmacêuticos , Regeneração/efeitos dos fármacos , Timidina/metabolismo , TrítioRESUMO
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.