The tissue was mechanically dissociated by trituration and filtered through a 70-m nylon mesh. a mutant form unable to be cleaved at lysine 104 (K104A-MCP1) is relatively ineffective in this assay, suggesting that the C-terminal Cbz-B3A region interferes with the MCP1-CCR2 interaction. Moreover, FL-MCP1 and K104Stop-MCP1 stimulation leads to activation of Rabbit Polyclonal to ZADH1 Rac1, a small GTPase involved in Cbz-B3A cell migration. Conversely, MCP1-stimulated microglial migration is blocked by the Rac1 inhibitor, NSC23766, demonstrating the requirement for Rac1 effector pathways in this response. Taken together, we propose a model for MCP1 localization, activation, and function based on the initial presence and then removal of its C terminus, coupled with a requisite downstream signaling pathway from CCR2 stimulation to Rac1 activation. Keywords:Cell Migration, Chemokines, Mouse, Plasmin, Protease, Activation, Microglia == Introduction == Microglia, the immune-like cells normally present in the brain, derive from the bone marrow (15), enter the central nervous system (CNS) early during development, and reside in the parenchyma in a resting state characterized by ramified morphology. In the healthy brain, they continually extend and retract their processes to sense changes in the surrounding microenvironment (6). However, when there is an injury in the CNS, microglia switch to an Cbz-B3A activated state characterized by changes in gene expression, morphology, and proliferation (616). The activated microglia then migrate to the site of injury and modify the injury outcome. The migration of microglia to the site of injury is stimulated by the local release of chemokines, a superfamily of structurally related small proteins that function as chemoattractants. A potent chemoattractant for monocytes/microglia (17), MCP1 (monocyte chemoattractant protein-1; also called CCL2), is up-regulated in many types of CNS injury, including ischemia, hemorrhage, trauma, infection, hypoxia, and peripheral nerve axotomy (1824). The MCP1 protein is highly conserved in the N terminus among different species, whereas the C terminus is much more variable. The rodent MCP1 C terminus is decorated extensively withO-linked carbohydrates (25), which bind both soluble glycosaminoglycans (GAGs) and GAGs immobilized on the cell surface. Soluble GAGs inhibit the binding of MCP1 to its high affinity receptor CCR2 (30), which is expressed by microglia, astrocytes, and microvascular endothelial cells in the brain (26,27). Cell surface GAGs, on the other hand, have been reported to concentrate MCP1 locally, promote MCP1 oligomerization, and thus facilitate the binding of MCP1 to CCR2 (2831). Whether MCP1 functions as a monomer or homodimer, however, is still under debate. Although it is believed that MCP1 exerts its function as a homodimer (25,32), it has also been suggested that MCP1 can bind to CCR2 and induce downstream signaling as a monomer (33,34). It should be noted, however, that these experiments were conducted using human MCP1, not rodent MCP1, which contains a heavily glycosylated C-terminal extension. Previous studies in our laboratory have shown that 1) the highly glycosylated C-terminal extension of mouse MCP1 is removed by plasmin, the active protease of plasminogen, and 2) the C terminus-truncated mouse MCP1 has a higher chemotactic potency than the full-length mouse MCP1 (35). Additionally, the fact that infusion of mouse MCP1 lacking the C terminus into plasminogen-deficient mice, which are more resistant to excitotoxic injury, restored microglial migration and neuronal loss, unlike the intact mouse MCP1, further suggests that the C-terminal extension negatively regulates the chemotactic ability of mouse MCP1 (35). How exactly the highly glycosylated C terminus regulates mouse MCP1 function, however, remains to be elucidated. Herein, we investigated several potential mechanisms: dimerization/oligomerization of mouse MCP1, interaction of MCP1 with CCR2, and intracellular signaling. We find Cbz-B3A that the C terminus of mouse MCP1 Cbz-B3A is required for MCP1 dimerization, attenuates the MCP1-CCR2 interaction, and suppresses MCP1-induced Rac activation and subsequent microglial migration. == EXPERIMENTAL PROCEDURES == == == == == == Cell Culture == N9 cells were originally provided by Drs. S. Barger (University of Arkansas, Fayetteville, AR) and P. Ricciardi-Castagnoli (University of Milano-Bicocca, Milan, Italy). The cells were maintained in minimum Eagle’s medium (MEM)2supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin at 37 C with 5% CO2. Primary microglia were prepared from mixed cortical cultures, as described previously, with minor modifications (9). Briefly, brains of 1-day-old pups from wild-type or CCR2/mice were collected. After removing meninges and hippocampi, the cortical tissue was digested with trypsin (0.25% in Hanks’ balanced saline solution) for 15 min at 37 C. The tissue was mechanically dissociated.