CCR7/CCL21 migration on fibronectin is mediated by PLCγ1 and ERK1/2 in primary T lymphocytes

Abstract C-C Chemokine receptor 7 (CCR7) binds to its cognate ligand, CCL21, to mediate the migration of circulating naive T lymphocytes to the lymph nodes. T lymphocytes can bind to fibronectin, a constituent of lymph nodes, via their β1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) is required for T cell migration on fibronectin in response to CCL21 with a rapid (within 2 minutes) and transient phosphorylation of ERK1/2. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MEK, prevented T lymphocyte migration. Previous studies have suggested that Phospholipase C gamma 1 (PLCγ1) can mediate phosphorylation of ERK 1/2, which is required for β1 integrin activation. Paradoxically, we found that inhibition of PLCγ1 phosphorylation by the general PLC inhibitor, U73122, was associated with an enhanced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLCγ1, we reduced PLCγ1 levels by 85% using shRNA and observed a sustained phosphorylation of ERK1/2 and a significant reduction in CCR7 mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK 1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLCγ1 suggesting a feedback loop between ERK 1/2 and PLCγ1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a β1 integrin dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLCγ1.