Dopamine and Mesenchymal Stem Cells Figure 5. Dopamine (DA) through its D2 receptors inhibits VEGF induced migration of BM-MSCs by regulating phosphorylation of VEGFR-2 and Akt and actin polymerization. (A and B) Effect of DA on VEGF induced phosphorylation of VEGFR-2 and Akt in murine MSCs. Lane 1: Cells stimulated with VEGF (10 ng/ml). Lane 2: Cells pretreated with 1 mM DA before being exposed to VEGF (10 ng/ml). Lane 3: Cells treated with 100 mM eticlopride followed by DA and VEGF. Addition of DA significantly inhibited VEGF induced phosphorylation of both VEGFR-2 receptors and its downstream target Akt when compared with VEGF treated control. Pre-treatment with eticlopride, abrogated DA induced dephosphorylation of both VEGFR-2 and Akt. However, expression of total VEGFR-2 and total Akt remained unchanged. Results are representative of six separate experiments each yielding similar results. (C) In actin polymerization assay, VEGF induced MSCs showed significant polymerization of actin cytoskeleton leading to formation of F-actin. However, no such alterations were observed in case of Akt silenced MSCs which showed strikingly lower migratory activity against VEGF than normal MSCs. (D) Effect of DA on VEGF induced actin polymerization in MSCs. Treatment with 1 mM DA had significant inhibitory effect on actin polymerization dynamics in MSCs when compared to VEGF treated control. However, pre-treatment with specific DA D2 receptor antagonist eticlopride (100 mM) abrogated the DA induced changes in the actin polymerization dynamics of the VEGF induced MSCs. Original magnifications, 61000. doi:10.1371/journal.pone.0031682.g005 indicates that the physiological concentration of DA (1 mM) [40] present in synaptic clefts can inhibit the incorporation of exo- genously transplanted MSCs into wound bed. Several recent reports have indicated that VEGF is the prime growth factor that regulates wound angiogenesis and migration of MSCs both in vitro and in vivo [14,30–47], therefore in the present investigation while exploring the effect of DA on MSC mobilization, VEGF was used in vitro to induce chemotaxis. Furthermore, phosphorylation of Akt induced by different growth factors has been shown to be one of the major signaling pathways regulating migration of MSCs [41–44]. In the present investigation, DA-mediated inhibition of MSCs migration was observed due to inhibition of Akt phosphorylation in murine MSCs. However, this effect of DA was abrogated after treatment with specific DA D2 receptor antagonist, thereby indicating that the action of DA was mediated through its DA D2 receptors. Since PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31682