CCR7/CCL21 Migration on Fibronectin Is Mediated by Phospholipase Cγ1 and ERK1/2 in Primary T Lymphocytes

Abstract

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 ␤1 integrins, which is a primary mechanism of T lymphocyte migration; however, the signaling pathways involved are unclear. We report that rapid (within 2 min) and transient phosphorylation of ERK1/2 is required for T cell migration on fibronectin in response to CCL21. Conversely, prevention of ERK1/2 phosphorylation by inhibition of its kinase, MAPK/MEK, prevented T lymphocyte migration. Previous studies have suggested that phospholipase C␥1 (PLC␥1) can mediate phosphorylation of ERK1/2, which is required for ␤1 integrin activation. Paradoxically, we found that inhibition of PLC␥1 phosphorylation by the general PLC inhibitor U73122 was associated with a delayed and reduced phosphorylation of ERK1/2 and reduced migration of T lymphocytes on fibronectin. To further characterize the relationship between ERK1/2 and PLC␥1, we reduced PLC␥1 levels by 85% using shRNA and observed a reduced phosphorylation of ERK1/2 and a significant loss of CCR7-mediated migration of T lymphocytes on fibronectin. In addition, we found that inhibition of ERK1/2 phosphorylation by U0126 resulted in a decreased phosphorylation of PLC␥1, suggesting a feedback loop between ERK1/2 and PLC␥1. Overall, these results suggest that the CCR7 signaling pathway leading to T lymphocyte migration on fibronectin is a ␤1 integrin-dependent pathway involving transient ERK1/2 phosphorylation, which is modulated by PLC␥1. G protein-coupled receptors (GPCRs)2 are responsive to many types of stimuli, such as hormones, neurotransmitters, and chemoattractants. Ligand activation of a GPCR leads to a conformational change in the receptor, resulting in an exchange of GDP for GTP in the G␣ subunit and dissociation of G␣ from the G␤␥ subunits. The subunits then initiate downstream signaling events to regulate the activation state of adenylyl cyclase or phospholipase C (PLC) (1). CCR7 (C-C chemokine receptor 7), a G protein-coupled receptor, is expressed on naive T lymphocytes and is required for migration into and within lymph nodes. CCR7 binds to two ligands, CCL19 and CCL21. CCL21 is expressed in high endothelial venules, the entry route into lymph nodes (2). However, it is currently unknown how T lymphocytes signal through CCR7 to mediate migration via ␤1 integrins in response to CCL21.Integrins, which are key mediators of lymphocyte adhesion and migration, are heterodimeric adhesion proteins consisting of an ␣ and a ␤ subunit, for which they are named. ␤1 integrins, which are expressed by T lymphocytes, bind to the vascular cell adhesion molecule, collagen, and fibronectin (3). Fibronectin is a major component of the lymph node and is highly expressed in the cortex in the presence of fibroblastic reticular cells, which express CCL21 (4, 5). This network provides an environment that allows CCR7-expressing T lymphocytes to migrate throughout the lymph node.PLC␤ is a...

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