Intracellular signaling events at the leading edge of migrating cells

doi:10.1016/S1357-2725(00)00035-2

The International Journal of Biochemistry & Cell Biology

Volume 32, Issue 9, September 2000, Pages 931-943

https://doi.org/10.1016/S1357-2725(00)00035-2 Get rights and content

Abstract

Cell migration is an important facet of the life cycle of immune and other cell types. A complex set of events must take place at the leading edge of motile cells before these cells can migrate. Chemokines induce the motility of various cell types by activating multiple intracellular signaling pathways. These include the activation of chemokine receptors, which are coupled to the heterotrimeric G proteins. The release of Gβγ subunits from chemokine receptors results in the recruitment to the plasma membrane, with subsequent activation of various down-stream signaling molecules. Among these molecules are the pleckstrin homology domain-containing proteins and the phosphoinositide 3-kinase γ which phosphorylates phospholipids and activates members of the GTP exchange factors (GEFs). These GEFs facilitate the exchange of GTP for GDP in members of GTPases. The latter are important for reorganizing the cell cytoskeleton, and in inducing chemotaxis. Chemokines also induce the mobilization of intracellular calcium from intracellular stores. Second messengers such as inositol 1,4,5 trisphosphate, and cyclic adenosine diphosphate ribose are among those induced by chemokines. In addition, the Gβγ subunits recruit members of the G protein-coupled receptor kinases, which phosphorylate chemokine receptors, resulting in desensitization and termination of the motility signals. This review will discuss the intracellular signaling pathways induced by chemokines, particularly those activated at the leading edge of migrating cells which lead to cell polarization, cytoskeleton reorganization and motility.

Section snippets

Background

Our earlier results showed that killer lymphocytes, which kill and destroy targets such as tumor cells, are under the control of factors secreted by other cell types [1], [2]. Several of these factors have now been identified. In order for killer cells to reach their target cells, they must adhere to the substratum, extravasate and migrate toward the sites of tumor growth, or of microbial replication. This is also true for other immune cells such as T and B as well as macrophages and dendritic

Chemokines

Chemotactic cytokines (chemokines) are pro-inflammatory mediators important for the recruitment of various cell types into the inflammatory sites. Chemokines are implicated in allergic disorders, autoimmune diseases and in ischemia associated with the infiltration of leukocytes [6]. They are divided into four subfamilies; the CC chemokines (also known as ß chemokines) in which the first two cystein residues in the motif proteins are adjacent to each other. The second branch of this subfamily is

Heterotrimeric G proteins

Receptors for chemokines are proteins with seven transmembrane spanning domains (heptahelical), and are coupled to G proteins (reviewed in [15]). The proposed sites of interaction between chemokine receptors and G proteins have been recently reviewed [16]. The heterotrimeric G proteins are composed of three subunits, α, ß and γ. About 20 α subunits have been reported, which belong to four subfamilies, (1) α_s (α_s and α_olf); (2) α_q (α_q, α₁₁, α₁₄, α₁₅ and α₁₆); (3) α_i (α_i1, α_i2, α_i3, α_o1, α_o2, α_z,

Role of GTPases

At the leading edge of moving cells, cellular polarization is facilitated by the assembly of focal adhesion system, which include among other molecules, the p120 focal adhesion kinase (p120^FAK), the adaptor protein paxillin and the p130 Crk-associated substrate (p130^CAS). Also, GTPases important for the formation of filopodia and lamellipodia such as Rho A, Cdc42 and Rac are assembled at this site. GTPases exist in an inactive GDP-bound form. They exchange GDP for GTP very slowly due to the low

Receptor desensitization

Motile immune cells must cease their migration and stop at the points of contact with antigen presenting cells, or with target cells such as tumor or infected cells. GPCRs must be desensitized in order for the cells to stop moving. This occurs when the receptors are phosphorylated. Many second messengers and kinases have been described which phosphorylate these receptors. These include protein kinase C, protein kinase A and a set of G protein coupled receptor kinases (GRKs, reviewed in [52]).

Lessons from NK cell chemotaxis

Previous work suggested that chemotaxis is mediated by G_i only, and not by other G proteins such as G_q or G_s [62], [63]. In the anti-tumor and anti-microbial IL-2-activated NK cells, most chemokine receptors are coupled to more than one G protein. In some cases, the number of G proteins that bind to one chemokine receptor can be four or more [64]. It is agreed that the βγ released upon activation of a particular chemokine receptor, is responsible for initiating chemotaxis [45], [62], [63]. What

Discrimination between chemotaxis and calcium flux activities

In all the chemotaxis assays described in the literature, a bell-shaped response is observed regardless of the nature of the chemoattractants and the cell types responding. This means that intermediate concentrations of the chemoattractants are chemotactic, while very low and very high concentrations are not. However, the same high concentrations of chemokines induce calcium mobilization in these assays. This finding discriminates between the chemotaxis and the mobilization of intracellular

Concluding remarks

In order for motile cells to reach the sites of tumor growth, or the sites of microbial replication, they must respond to a set of chemoattractant peptides known as chemokines. Chemokines induce various intracellular signaling pathways inside motile cells, which include phosphoinositides 3 kinase γ and members of the GTPase family of proteins. GTPases reorganize the cell cytoskeleton, and polarize the cells toward the concentration gradients of chemokines. In addition to inducing chemotaxis,

Acknowledgements

I would like to thank my colleagues Dr John Torgils Vaage, and Knut Martin Torgersen for critically reading this manuscript. The work in the laboratory of the author is supported by grants from the Norwegian Research Council, the Norwegian Cancer Society and Anders Jahres Fond.

References (68)

M. Amano et al.
Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase)
J. Biol. Chem.
(1996)
J.A. Hedrick et al.
Lymphotactin
Clin. Immunol. Immunopathol.
(1998)
A.A. Maghazachi
Intracellular signalling pathways induced by chemokines in natural killer cells
Cell Signal
(1999)
G.J. Barritt et al.
An evaluation of strategies available for the identification of GTP-binding proteins required in intracellular signalling pathways
Cell Signal
(1997)
H.C. Lee
Cyclic ADP-ribose: a new member of a super family of signalling cyclic nucleotides
Cell Signal
(1994)
A. Galione
Cyclic ADP-ribose, the ADP-ribosyl cyclase pathway and calcium signalling
Mol. Cell. Endocrinol.
(1994)
M. Inngjerdingen et al.
Differential utilization of cyclic ADP-ribose pathway by chemokines to induce the mobilization of intracellular calcium in NK cells
Biochem. Biophys. Res. Commun.
(1999)
G. Pacheco-Rodriguez et al.
Guanine nucleotide exchange on ADP-ribosylation factors catalyzed by cytohesin-1 and its Sec7 domain
J. Biol. Chem.
(1998)
S.M. Soisson et al.
Crystal structure of the Dbl and pleckstrin homology domains from the human son of sevenless protein
Cell
(1998)
E. Mossessova et al.
Structure of the guanine nucleotide exchange factor Sec7 domain of human Arno and analysis of the interaction with ARF GTPase
Cell
(1998)

A.R. Horwitz et al.

Cell migration-movin'on

Science

(1999)

M. Nieto et al.

Roles of chemokines and receptor polarization in NK-Target cell interactions

J. Immunol.

(1998)

M.P. Keane, R.M. Strieter, Chemokine signaling in inflammation, Crit. Care Med. 28 (2000)...

K.B. Bacon et al.

Chemokines as mediators of allergic inflammation

Int. Arch. Allergy Immunol.

(1996)

P.C. Wilkinson et al.

Locomotion and chemotaxis of lymphocytes

Autoimmunity

(1997)

A.A. Maghazachi et al.

Interferon-inducible protein-10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin-sensitive and -insensitive heterotrimeric G-proteins

FASEB J.

(1997)

Cited by (55)

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity
2015, Molecular and Cellular Neuroscience
Citation Excerpt :
Also, certain results suggest a role for the L-type VGCC in the control of proliferation and migration of activated microglia. For example, CCL5, a β-chemokine involved in immune responses including migration (Maghazachi, 2000), secretion (Logan et al., 2003), and gene expression (Crabtree and Olson, 2002), specifically activates a nimodipine-sensitive calcium current in microglia (Shideman et al., 2006). The vulnerability of the CNS to excitotoxic insults and hypoxia-ischemia differs according to brain areas (Rodríguez et al., 2009b).
Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the Ca_V1.2 and Ca_V1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca²⁺ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.
Mouse monocyte chemoattractant protein 1 (MCP1) functions as a monomer
2014, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
These data suggest that MCP1 dimers lose the ability to activate Rac1 in microglia. To further investigate the effect of dimeric MCP1 in microglial migration, we examined the formation of lamellipodia, a cellular event downstream of Rac1 activation (Maghazachi, 2000; Pankov et al., 2005; Terashima et al., 2005). Exposure of primary microglia to a point source of FL-MCP1 and K104Stop-MCP1 significantly promoted the protrusion of lamellipodia (formation of cellular polarity, arrows in Fig. 5A).
Monocyte chemoattractant protein 1 (MCP1) is an important chemoattractant for microglia. Rodent MCP1 carries a heavily glycosylated C-terminus, which has been predicted to increase local MCP1 concentration, promote MCP1 dimerization/oligomerization, and facilitate receptor engagement. Previous studies have shown that MCP1 mutant lacking the glycosylated C-terminus cannot dimerize/oligomerize, but has higher chemotactic potency than the wild-type (full-length) MCP1, suggesting that rodent MCP1 may function as a monomer. Although many groups support this hypothesis, there is no direct evidence on whether rodent MCP1 dimer is functional. In this paper, using forced recombinant dimeric MCP1 proteins we show that mouse MCP1 dimer is unable to activate Rac1, promote protrusion of lamellipodia, or induce microglial migration, although it can bind to CCR2 and mediate its internalization. These results support the idea that signaling events mediated by MCP1 require the presence of the monomeric form of this chemokine.
Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration
2011, Biochemical and Biophysical Research Communications
In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.
We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.
Chronic exposure to the chemokine CCL3 enhances neuronal network activity in rat hippocampal cultures
2010, Journal of Neuroimmunology
We examined the effect of chronic CCL3 treatment on the properties of cultured rat hippocampal neurons to gain an understanding of the neuronal effects of CCL3 during neuroinflammatory disorders. Western blot assays showed that chronic exposure to CCL3 altered the level of specific neuronal and glial proteins and that CCL3 had no effect on neuronal survival. CCL3 treatment also altered intracellular Ca²⁺ dynamics and increased Ca²⁺ levels in hippocampal neurons, measured by fura-2 imaging techniques. Additionally, chronic CCL3 increased NMDA-evoked Ca²⁺ signals in the hippocampal neurons and increased NMDA receptor levels. These CCL3-induced neuroadaptive changes could play an important role in the CNS dysfunction associated with CNS disorders with a neuroinflammatory component.
The C terminus of Mouse Monocyte Chemoattractant Protein 1 (MCP1) mediates MCP1 dimerization while blocking its chemotactic potency
2010, Journal of Biological Chemistry
Citation Excerpt :
MCP1 engagement of CCR2 activates intracellular signaling cascades that differ depending on the cell type (38, 42). In microglia, one of these signaling pathways involves activation (GTP loading) of Rac, a member of the Rho family of small GTPases (38, 43). The activated Rac then promotes protrusion of lamellipodia and cell migration (38, 44, 45).
The extracellular protease plasmin cleaves mouse MCP1 (monocyte chemoattractant protein 1) at lysine 104, releasing a 50-amino acid C-terminal domain. The cleavage event increases the chemotactic activity of MCP1 and, by doing so, promotes the progression of excitotoxic injury in the central nervous system in pathological settings. The mechanism through which the cleavage event enhances MCP1-mediated chemoattraction is unknown; to investigate it, we use wild-type and mutant forms of recombinant MCP1. Full-length MCP1 (FL-MCP1) is secreted by cells as a dimer or multimer. We show that a mutant truncated at the C terminus, K104Stop-MCP1, does not dimerize, revealing that the C terminus mediates the interaction. MCP1 interacts with the monocyte/microglia receptor CCR2. The interaction is critical to the function of MCP1 because CCR2^−/− microglia do not undergo chemotaxis in response to MCP1 stimulation. We show that stimulation of microglia with FL-MCP1 or K104Stop-MCP1 triggers CCR2 internalization, whereas a mutant form unable to be cleaved at lysine 104 (K104A-MCP1) is relatively ineffective in this assay, suggesting that the C-terminal region interferes with the MCP1-CCR2 interaction. Moreover, FL-MCP1 and K104Stop-MCP1 stimulation leads to activation of Rac1, a small GTPase involved in 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.
A biophysical insight into the RANTES-glycosaminoglycan interaction
2009, Biochimica et Biophysica Acta - Proteins and Proteomics
Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES–GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine–GAG interaction. This was absent in the oligomerisation-deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES–GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with K_d values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.

View all citing articles on Scopus

View full text

ReviewIntracellular signaling events at the leading edge of migrating cells

Abstract

Section snippets

Background

Chemokines

Heterotrimeric G proteins

Role of GTPases

Receptor desensitization

Lessons from NK cell chemotaxis

Discrimination between chemotaxis and calcium flux activities

Concluding remarks

Acknowledgements

J. Biol. Chem.

Clin. Immunol. Immunopathol.

Cell Signal

Cell Signal

Cell Signal

Mol. Cell. Endocrinol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

Cell

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Trends Biol. Sci.

J. Biol. Chem.

Cell

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Detection in vivo 6 h after immunization of a mediator with possible antisuppressor activity

Eur. J. Immunol.

T cell derived factors detected in vivo 6 h after immunization enhance the immune response

Lymphokines

Cell migration-movin'on

Science

Roles of chemokines and receptor polarization in NK-Target cell interactions

J. Immunol.

Chemokines as mediators of allergic inflammation

Int. Arch. Allergy Immunol.

Locomotion and chemotaxis of lymphocytes

Autoimmunity

Interferon-inducible protein-10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin-sensitive and -insensitive heterotrimeric G-proteins

FASEB J.

Review
Intracellular signaling events at the leading edge of migrating cells