Extracellular ATP is one of the most common signalling molecules that induce an increase in the intracellular Ca2+ concentration ([Ca2+]i) via P2 receptors, namely ligand-gated ion channels P2X and/or G protein-coupled P2Y receptors. ATP is shown to evoke robust Ca2+ signalling in mesenchymal stem cells (MSCs) and regulate MSC proliferation, migration and differentiation. However, there are noticeable discrepancies in the mechanisms underlying ATP-induced Ca2+ signalling and regulation of MSC functions. Furthermore, the Ca2+-dependent downstream signalling pathways in ATP-induced regulation of MSC functions are poorly understood. MSCs have been documented to release ATP in response to mechanical stimulation, but the mechanism mechanosensing and mediating ATP release remains elusive. The studies presented in this thesis, using human dental pulp-derived MSCs (hDP-MSCs), aimed to investigate ATP-induced purinergic Ca2+ signalling and Ca2+-dependent signalling pathways in regulating cell migration and adipogenic differentiation and examine the hypothesis that the mechanically activated Ca2+-permeable Piezo1 channel regulates hDP-MSC migration via ATP release and activation of the P2 receptors.
The study presented in chapter 3, first of all, showed that exposure to ATP increased cell migration, using wound healing and trans-well migration assays. Exposure to BzATP and NF546, a P2Y11 selective agonist, also enhanced cell migration. ATP-induced cell migration was inhibited by PPADS, a P2 generic antagonist. It was also reduced by KN-93, a CaMKII inhibitor, chelerythrine chloride, a PKC inhibitor, and PF431396, a PYK2 inhibitor, and furthermore, by U0126, an inhibitor MEK/ERK, and SB202190, a p38 MAPK inhibitor. Collectively, these results provide evidence to confirm the recent findings that extracellular ATP stimulates hDP-MSC migration and identify CaMKII, PKC, PYK2 and MAPKs as downstream signalling pathways in transducing ATP-induced Ca2+ signalling to cell migration.
The study in chapter 4 focused on the Piezo1 channel in hDP-MSCs. Piezo1 mRNA and protein expression were detected using RT-PCR and immunostaining, respectively. Exposure to Yoda1, a chemical activator of the Piezo1 channel, elevated the [Ca2+]i via Ca2+ influx. Yoda1-induced Ca2+ response was inhibited by ruthenium red and GsMTx4, two structurally different Piezo1 inhibitors, or Piezo1-specific siRNA, supporting the functional expression of the Piezo1 channel. Exposure to Yoda1 stimulated cell migration, which was inhibited by Piezo1-specific siRNA. Yoda1-induced cell migration was also prevented by apyrase, an ATP-scavenger as well as PPADS, suggesting that Piezo1 channel activation stimulates cell migration via ATP release and activation of the P2 receptors. Consistently, Yoda1-induced cell migration was inhibited by KN-93, chelerythrine chloride, PF431396, and U0126, indicating engagement of CaMKII, PKC and PYK2 and MEK/ERK as Ca2+-dependent downstream signalling mechanisms.
The study presented in chapter 5 started with examining the molecular mechanisms participating in ATP-induced Ca2+ signalling. ATP-induced increase in the [Ca2+]i in extracellular Ca2+-containing solution was inhibited by PPADS. ATP and BzATP induced increases in the [Ca2+]i in extracellular Ca2+-free as well as Ca2+-containing solutions. ADP and NF546 also evoked an increase in the [Ca2+]i. These results support a significant role for the P2X7, P2Y1 and P2Y11 receptors in ATP-induced Ca2+ signalling. The study, next, examined the expression of these receptors and also the P2Y2 receptor during adipogenic differentiation. There was an increase in the P2Y1 and a decrease in the P2Y2 mRNA expression, but no alteration in the expression of the P2Y11 and P2X7 after hDP-MSC adipogenesis. Exposure to ATP during adipogenesis resulted in a significant increase in adipogenic differentiation, examined using oil red O staining and, furthermore, up-regulation of the P2Y11 expression with no effect on the other receptors. Moreover, ATP-induced adipogenic differentiation was reduced by KN-93 and PF431396, suggesting the involvement of CaMKII and PYK2 in ATP-induced upregulation of adipogenesis.
In summary, the studies presented in this thesis gain a better understanding of ATP-induced Ca2+ downstream signalling pathways in the regulation of MSC migration and adipogenic differentiation, and also provide evidence to support an important role for the Piezo1 channel in regulating MSC migration via ATP release and subsequent activation of P2 receptors. Such information should be useful for the development of better use of MSCs in tissue engineering and cell-based therapies.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)