In a variety of cells, the A2aR is known to be coupled with adenylate cyclase, resulting in up-regulation of cAMP and PKA activation. We extend these findings to MSCs, demonstrating an increase in cytosolic cAMP after activation by the non-hydrolyzable adenosine agonist NECA. The ability of forskolin to inhibit HGF chemotaxis demonstrates that elevations in cAMP are sufficient for such inhibition. We also show a requirement for PKA using the specific PKA peptide inhibitor ST-HT31. HGF is known to increase cytosolic Ca++, and we have previously

shown in hepatic stellate cells that signaling via the A2a receptor check details can inhibit increases in cytosolic Ca++. We therefore tested whether NECA can inhibit the HGF-induced increase in cytosolic Ca++. As can be seen from Fig. NVP-LDE225 nmr 5A, HGF induced a significant increase in cytosolic Ca++, and this was inhibited by NECA in a PKA-dependent manner. HGF has been shown to increase Rac1 activity in a Ca++-mediated manner, and we further confirm the requirement for Rac1 in HGF chemotaxis by demonstrating that a Rac1 inhibitor blocks HGF-induced chemotaxis. The inhibition of HGF-induced

cytosolic Ca++ by adenosine and the requirement for an increase in cytosolic Ca++ for Rac1 activation predict that adenosine will inhibit an HGF-mediated increase in Rac1 activity. This was found to be the case (Fig. 3B). To definitively confirm that the inhibition of HGF-induced Rac1 activation by adenosine is the mechanism of inhibition of chemotaxis, we used a well-tested plasmid (RacQL) expressing the constitutively active form of rac1. When MSCs were transfected with RacQL, NECA was unable to block HGF-induced chemotaxis. Rac1 activation is known to MCE be important in actin stress fiber formation, and to further confirm functional Rac1 inhibition

in response to NECA, we examined actin stress fibers in MSC. HGF increased the prominence of actin stress fibers, and as predicted from its ability to inhibit Rac1, NECA resulted in almost complete loss of actin stress fibers. Collectively, these studies demonstrate a novel action of adenosine on MSC via the A2a receptor, resulting in inhibition of chemotaxis through a cAMP, PKA, Rac1 pathway. This has significant implications for MSCs when they reach an area of cell death or inflammation with high levels of adenosine. The previously described model of chemotaxis of MSCs toward an increasing gradient of HGF is still valid,33, 34 but our data suggest that, on arriving at a site of cellular injury with high adenosine levels, MSC chemotaxis will be inhibited. We propose that the inhibition of chemotaxis will provide a functional stop signal and result in localization of MSC to these sites. Such a model incorporating a stop signal in addition to the known chemotaxis signals has the advantage of localizing MSC to sites of injury, where they are most needed. We propose a schema to describe the interaction between HGF and adenosine (Fig. 8).