ABSTRACT
Cell migration towards a chemotactic stimulus relies on the re-arrangement of the cytoskeleton, which is triggered by activation of small G proteins RhoA, Rac1 and Cdc42, and leads to formation of lamellopodia and actin polymerisation amongst other effects. Here we show that Rac1 is important for CXCR4 induced chemotaxis but not for CCR1/CCR5 induced chemotaxis. For CXCL12-induced migration via CXCR4, breast cancer MCF-7 cells are reliant on Rac1, similarly to THP-1 monocytes and Jurkat T-cells. For CCL3-induced migration via CCR1 and/or CCR5, Rac1 signalling does not regulate cell migration in either suspension or adherent cells. We have confirmed the involvement of Rac1 with the use of a specific Rac1 blocking peptide. We also used a Rac1 inhibitor EHT 1864 and a Rac1-GEF inhibitor NSC23766 to probe the importance of Rac1 in chemotaxis. Both inhibitors did not block CCL3-induced chemotaxis, but they were able to block CXCL12-induced chemotaxis. This confirms that Rac1 activation is not essential for CCL3-induced migration, however NSC23766 might have secondary effects on CXCR4. This small molecule exhibits agonistic features in internalisation and cAMP assays, whereas it acts as an antagonist for CXCR4 in migration and calcium release assays. Our findings strongly suggest that Rac1 activation is not necessary for CCL3 signalling, and reveal that NSC23766 could be a novel CXCR4 receptor ligand.
Subject(s)
Chemokine CXCL12/pharmacology , Chemotaxis/drug effects , Cytoskeleton/drug effects , DNA-Binding Proteins/genetics , Transcription Factors/genetics , rac1 GTP-Binding Protein/genetics , Amino Acid Sequence , Aminoquinolines/pharmacology , Chemokine CCL3/pharmacology , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Jurkat Cells , MCF-7 Cells , Peptides/chemical synthesis , Peptides/pharmacology , Pyrimidines/pharmacology , Pyrones/pharmacology , Quinolines/pharmacology , Receptors, CCR1/genetics , Receptors, CCR1/metabolism , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Signal Transduction , THP-1 Cells , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/antagonists & inhibitors , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
Chemotaxis or directed cell migration is mediated by signalling events initiated by binding of chemokines to their cognate receptors and the activation of a complex signalling cascade. The molecular signalling pathways involved in cell migration are important to understand cancer cell metastasis. Therefore, we investigated the molecular mechanisms of CXCL12 induced cell migration and the importance of different signalling cascades that become activated by CXCR4 in leukemic cells versus breast cancer cells. We identified Src kinase as being essential for cell migration in both cancer types, with strong involvement of the Raf/MEK/ERK1/2 pathway. We did not detect any involvement of Ras or JAK2/STAT3 in CXCL12 induced migration in Jurkat cells. Preventing PKC activation with inhibitors does not affect migration in Jurkat cells at all, unlike in the adherent breast cancer cell line MCF-7 cells. However, in both cell lines, knock down of PKCα prevents migration towards CXCL12, whereas the expression of PKCζ is less crucial for migration. PI3K activation is essential in both cell types, however LY294002 usage in MCF-7 cells does not block migration significantly. These results highlight the importance of verifying specific signalling pathways in different cell settings and with different approaches.
Subject(s)
Breast Neoplasms/metabolism , Chemokine CXCL12/pharmacology , Chemotaxis/drug effects , Leukemia/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Chromones/pharmacology , Female , Humans , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Jurkat Cells , Leukemia/genetics , Leukemia/pathology , MCF-7 Cells , Morpholines/pharmacology , Protein Kinase C-epsilon/genetics , Protein Kinase C-epsilon/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolismABSTRACT
The HIV viral entry co-receptors CCR5 and CXCR4 function physiologically as typical chemokine receptors. Activation leads to cytosolic signal transduction that results in a variety of cellular responses such as cytoskeletal rearrangement and chemotaxis (CTX). Our aim was to investigate the signalling pathways involved in CC and CXC receptor-mediated cell migration. Inhibition of dynamin I and II GTPase with dynasore completely inhibited CCL3-stimulated CTX in THP-1 cells, whereas the dynasore analogue Dyngo-4a, which is a more potent inhibitor, showed reduced ability to inhibit CC chemokine-induced CTX. In contrast, dynasore was not able to block cell migration via CXCR4. The same activation/inhibition pattern was verified in activated T lymphocytes for different CC and CXC chemokines. Cell migration induced by CC and CXC receptors does not rely on active internalization processes driven by dynamin because the blockade of internalization does not affect migration, but it might rely on dynamin interaction with the cytoskeleton. We identify here a functional difference in how CC and CXC receptor migration is controlled, suggesting that specific signalling networks are being employed for different receptor classes and potentially specific therapeutic targets to prevent receptor migration can be identified.
