A Role for the Rho-p160 Rho Coiled-Coil Kinase Axis in the Chemokine Stromal Cell-Derived Factor-1α-Induced Lymphocyte Actomyosin and Microtubular Organization and Chemotaxis

Abstract

The possible involvement of the Rho-p160ROCK (Rho coiled-coil kinase) pathway in the signaling induced by the chemokine Stromal cell-derived factor (SDF)-1α has been studied in human PBL. SDF-1α induced activation of RhoA, but not that of Rac. RhoA activation was followed by p160ROCK activation mediated by RhoA, which led to myosin light chain (MLC) phosphorylation, which was dependent on RhoA and p160ROCK activities. The kinetics of MLC activation was similar to that of RhoA and p160ROCK. The role of this cascade in overall cell morphology and functional responses to the chemokine was examined employing different chemical inhibitors. Inhibition of either RhoA or p160ROCK did not block SDF-1α-induced short-term actin polymerization, but induced the formation of long spikes arising from the cell body, which were found to be microtubule based. This morphological change was associated with an increase in microtubule instability, which argues for an active microtubule polymerization in the formation of these spikes. Inhibition of the Rho-p160ROCK-MLC kinase signaling cascade at different steps blocked lymphocyte migration and the chemotaxis induced by SDF-1α. Our results indicate that the Rho-p160ROCK axis plays a pivotal role in the control of the cell shape as a step before lymphocyte migration toward a chemotactic gradient.

The RhoA Effector mDia Is Induced During T Cell Activation and Regulates Actin Polymerization and Cell Migration in T Lymphocytes

“…Downstream effectors of RhoA involved in actin reorganization and cell migration include the kinase p160ROCK and mDia, but the roles of these effectors in lymphoid motility have been assessed only in the former (20,21). Here, it is demonstrated that mDia was present in very small amounts in freshly isolated PBL, but was induced both in vivo and in vitro during T cell activation.…”

“…In fact, exposure of lymphoid cells to chemokines induces a robust, but transient, increase in the cellular F-actin pool (42). Chemokines such as SDF-1␣ activate Cdc42 (43) and RhoA (20), and a detailed kinetics study of these two GTPases revealed that Cdc42 activation occurs earlier than RhoA. Furthermore, inhibition of RhoA by the ADP-ribosyltransferase C3 did not impair the increase in F-actin elicited by chemokines (20).…”

“…Human PBL and polymorphonuclear leukocytes (PMN) were obtained as previously described (20,25). T lymphoblasts were obtained from human PBL by treatment with 1 g/ml PHA-L (Sigma-Aldrich, St. Louis, MO) for 48 h and were maintained with 50 U/ml IL-2.…”

“…Interaction of chemokines such as stromal cell-derived factor 1␣ (SDF-1␣) with its receptor CXCR4 activates RhoA and its effector p160ROCK, leading to myosin L chain phosphorylation and actomyosin contraction (20), as well as to tail retraction of lymphocytes (20) and monocytes (21), which are absolutely required for chemokine-induced lymphoid migration (20). The contribution of mDia to cytoskeletal reorganization has been assessed in adherent cells such as fibroblasts (22,23) and epithelial cells (24), but no information is available on mDia expression or function in hemopoietic cells.…”

Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis

“…Some of them (e.g., CCR5 and CXCR4) participate in HIV infection of CD4 ϩ T lymphocytes as coreceptors (Berger et al, 1999). Ligand binding to the chemokine receptors triggers various signaling cascades, including activation of G proteins, phosphotidylinositol 3-kinase, Janus kinase/signal transducers and activators of transcription proteins, the Rho-p160 ROCK axis, and the MAPK pathway (Wu et al, 1993;Ganju et al, 1998;Mellado et al, 1998;Vicente-Manzanares et al, 1999;Vicente-Manzanares et al, 2002). Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane.…”