Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain.

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X7 ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X7 receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y2 in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y2 expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y2 sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y2 mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X7-antagonists oATP and BBG. The BzATP-evoked P2Y2 receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y2siRNA, thus supporting evidence of increased P2Y2 activity. To further investigate the mechanisms by which P2X7 receptors mediated the P2Y2 mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y2 mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X7-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X7 receptors in astrocytes enhances P2Y2 mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways

Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules

Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules

Changes in matrix extracellular phosphoglycoprotein expression before and during in vitro osteogenic differentiation of human dental papilla mesenchymal cells.

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing β-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration

La produzione di fattori neuroprotettivi mediata da recettori metabotropi delle purine e del glutammato apre nuove prospettive terapeutiche delle malattie neurovegetative

Dottorato di ricerca in farmacoterapia e farmacovigilanza. 12. ciclo. A.a 1996-99. Coordinatore Francesco Caciagli.Consiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Osteogenic Differentiation of Mesenchymal Stromal Cells: A Comparative Analysis between Human Subcutaneous Adipose Tissue and Dental Pulp

White adipose tissue is a source of mesenchymal stromal/stem cells (MSCs) that are actively studied for their possible therapeutic use in bone tissue repair/remodeling. To better appreciate the osteogenic potential of these cells, we compared some properties of MSCs from human subcutaneous adipose tissue [subcutaneous-Adipose stromal cells (S-ASCs)] and dental pulp stem cell (DPSCs) of third-impacted molars, the latter representing a well-established MSC source. Both undifferentiated cell types showed similar fibroblast-like morphology and mesenchymal marker expression. However, undifferentiated S-ASCs displayed a faster doubling time coupled to greater proliferation and colony-forming ability than DPSCs. Also, the osteogenic differentiation of S-ASCs was greater than that of DPSCs, as evaluated by the higher levels of expression of early osteogenic markers Runt-related transcription factor-2 (RUNX2) and alkaline phosphatase at days 3-14 and of extracellular matrix mineralization at days 14-21. Moreover, S-ASCs showed a better colonization of the titanium scaffold. In addition, we investigated whether S-ASC osteogenic commitment was enhanced by adenosine A1 receptor (A1R) stimulation, as previously shown for DPSCs. Although A1R expression was constant during DPSC differentiation, it increased in S-ASC at day 21 from osteogenesis induction. Accordingly, A1R stimulation by the agonist 2-chloro-N6-cyclopentyl-Adenosine, added to the cultures at each medium change, stimulated proliferation only in differentiating DPSC and enhanced the osteogenic differentiation earlier in DPSCs than in S-ASCs. These effects were counteracted by cell pretreatment with a selective A1R antagonist. Thus, our findings suggest that S-ASCs could be advantageously used in regenerative orthopedics/dentistry, and locally released or exogenously added purines may play a role in bone repair/remodeling, even though this aspect should be more thoroughly evaluated

Changes in Matrix Extracellular Phosphoglycoprotein Expression before and during in Vitro

The purpose of this study is to characterise the expression of matrix extracellular phosphoglycoprotein (MEPE) in cultured mesenchymal cells isolated from human dental papilla (PaMCs) of impacted third molars either before or during differentiation of these cells into osteo/odontoblasts. PaMCs, like mesenchymal cells deriving from human dental pulp (DPMCs), resulted positive for a number of mesenchymal markers including CD146 and STRO-1. During the first week in culture they showed a faster proliferation rate than DPMCs, coupled to an earlier down-regulation of MEPE. Also when the cells were further cultured in osteogenic medium (containing β-glycerophosphate, ascorbic acid and dexamethasone) for 40 days, MEPE down-regulation coupled to an increased expression of osteogenic markers, such as osteocalcin and alkaline phosphatase, occurred earlier in PaMCs than in DPMCs. Thus, our data, indicating that also in PaMCs MEPE expression is higher when cells proliferate, whereas it is downregulated as cells differentiated, are in favour of a role of MEPE as an early regulator of odontogenic differentiation. We also confirm the superior proliferative potential of PaMCs in comparison with DPMCs, coupled to a more rapid induction of osteogenic differentiation. Therefore, these cells represent an optimal source to be conveniently used for dental tissue engineering and tooth regeneration