Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4

Abstract
Cell migration in response to a chemotactic stimulus depends on cytoskeletal reorganization, which is initiated by the activation of small G proteins such as RhoA, Rac1, and Cdc42. This process facilitates lamellipodia formation and actin polymerization, among other cellular changes. In this study, we demonstrate that Rac1 plays a crucial role in CXCR4-mediated chemotaxis but is not required for CCR1/CCR5-driven chemotaxis. Specifically, CXCL12-induced migration via CXCR4 in breast cancer MCF-7 cells, as well as in THP-1 monocytes and Jurkat T-cells, is Rac1-dependent. In contrast, Rac1 signaling does not influence CCL3-induced migration via CCR1 and/or CCR5 in either adherent or suspension cells.

To validate Rac1 involvement, we employed a specific Rac1-blocking peptide along with the Rac1 inhibitor EHT 1864 and the Rac1-GEF inhibitor NSC23766. While both inhibitors effectively blocked CXCL12-induced chemotaxis, they had no impact on CCL3-induced migration, confirming that Rac1 activation is not essential for this pathway. Interestingly, NSC23766 may exert additional effects on CXCR4, displaying agonistic properties in internalization and cAMP assays but acting as an antagonist in migration and calcium release assays. Our findings highlight that Rac1 activation is dispensable for CCL3 signaling and suggest that NSC23766 may function as a novel ligand for the CXCR4 receptor.