1D) (migration index, NECA + HGF: 1.45 ± 0.13; 8-PST + NECA + HGF: 2.67 ± 0.3; P < 0.05). Adenosine signals through four receptor subtypes: A1, A2a, A2b, and A3. Adenosine subtype–specific antagonists were used to determine the role of receptor subtypes (DPCPX [10 nM], A1; ZM241385 [1 μM], A2a;

MRS-1706 [10 nM], A2b; and MRS-1523 (5 μM], A3). The ability of adenosine to inhibit MSC chemotaxis to HGF was significantly blocked by the A2a receptor subtype antagonist but not by the A1, A2b, and A3 receptor-selective antagonists (Fig. 1D) (migration index, NECA + HGF: 1.45 ± 0.13; Zm241385 + NECA + HGF: 3.6 ± 0.3; selleck compound P < 0.05). This demonstrates that the A2a receptor subtype is responsible for the inhibition of HGF-induced MSC chemotaxis. Signaling downstream of the A2a receptor is mediated predominantly via adenylate cyclase activation, resulting in elevation in cAMP.20 We found a central role for cAMP downstream of signaling of A2aR in MSCs. The adenosine agonist NECA increased cAMP levels, and this effect was blocked by the A2a receptor antagonist (Fig. 2A). Forskolin, which induces elevations in cAMP independent of adenosine receptor activation, mimics the inhibitory effect of NECA on HGF-induced MSC chemotaxis (Fig. 2B) (migration index, HGF: 2.02 Gefitinib chemical structure ± 0.15; forskolin + HGF 1.08 ± 0.05, P < 0.05). Cyclic AMP mediates downstream

effects through activation of protein kinase A (PKA) in many cells.21 We tested the ability of the PKA inhibitor ST-HT31 Ribonucleotide reductase to reverse forskolin-inhibited chemotaxis and found that forskolin-induced inhibition of MSC migration was antagonized by the PKA inhibitor ST-HT31 (Fig. 2B) (migration index, forskolin + HGF: 1.08 ± 0.05, ST-HT31+ forskolin + HGF: 2.25

± 0.38; P < 0.05). We further found that the PKA inhibitor could block the effect of NECA on HGF-induced MSC chemotaxis (Fig. 2C) (migration index, NECA+ HGF: 1.53 ± 0.19, ST-HT31 + NECA + HGF: 4.2 ± 0.69; P < 0.05). These findings demonstrate that adenosine inhibits HGF-induced MSC chemotaxis through a cAMP/PKA-dependent pathway. To elucidate the intracellular signaling pathways responsible for adenosine and HGF interaction, we investigated pathways responsible for HGF-induced chemotaxis in MSCs. HGF signaling via c-met is known to increase cytosolic Ca++ and lead to activation of Rac1.22 To determine whether Rac1 inhibition may be a mechanism by which adenosine inhibits HGF-induced chemotaxis, we tested the ability of a Rac 1 inhibitor (NSC23766) and a Rho kinase inhibitor (Y27632) to inhibit HGF-induced chemotaxis. The Rho kinase inhibitor had no significant effect on HGF-induced migration (Fig. 3A). However, the Rac1 inhibitor significantly blocked HGF-induced MSC migration (Fig. 3A). These findings support a requirement for Rac1 in HGF-induced MSC chemotaxis (migration index, HGF: 2.0 ± 0.2; Rac1 inhibitor + HGF: 1.2 ± 1.2; P < 0.05).