Actin cytoskeleton differentially modulates NF-κB-mediated IL-8 expression in myelomonocytic cells
Introduction
Actin cytoskeleton is involved in many aspects of cellular function, such as cell movement, muscle contraction, phagocytosis and mitosis [1], [2], [3], [4]. The dynamic assembly and spatial organization of actin filaments in response to extracellular signals are at the basis of these fundamental processes [5]. Recently, several studies established a link between actin dynamics and alteration of gene expression by demonstrating direct interactions between actin and members of signal transduction pathways. Indeed, it has been demonstrated that cytoskeleton-disrupting agents can modulate post-transcriptional and transcriptional events [6], [7], [8], [9], [10]. In another hand, many recent studies support the idea of a role for nuclear actin in the control of gene transcription. Nuclear actin is required for chromatin remodelling and for the transcription by all three RNA polymerases [11], [12], [13].
Several pro-inflammatory stimuli, such as TNFα (tumor necrosis factor α) and IL-1β (interleukin-1β) as well as some bacterial products like LPS (lipopolysaccharide) known to activate the transcription factor nuclear factor κB (NF-κB), are also able to induce actin cytoskeleton remodelling [14], [15], [16]. Moreover, many physiopathological events such as phagocytosis, pathogen invasion, cellular adhesion and chemotaxis governed by actin-based cytoskeleton are often accompanied by NF-κB activation and the expression of pro-inflammatory genes [3], [17], [18], [19]. Furthermore, we demonstrated that a strong perturbation of actin dynamics induced NF-κB activation in myelomonocytic cells [20]. For these different reasons, we decided to determine the modulating effect or the potential role of the actin cytoskeleton in the transduction pathways leading to NF-κB activation by two inducers: TNFα or LPS.
The transcription factor NF-κB regulates the expression of a large-scale of genes involved in a series of important cellular processes such as inflammatory and immune responses, cellular proliferation and differentiation as well as cell survival (for a review, see [21]). The NF-κB transcription factor binds specific DNA sequences as homo- or heterodimers composed of members of the Rel/NF-κB family [22]. The most ubiquitous complex is the heterodimer p50/p65(RelA). At the N-terminal end, the Rel proteins bear a related, but non-identical, 300-residue-long Rel homology domain (RHD), which is responsible for dimerization, nuclear translocation and specific DNA binding. In addition, some Rel proteins such as p65(RelA), contain one or two C-terminal transactivating domain (TAD).
In the resting state, NF-κB complexes are sequestered in an inactive form in the cytoplasm of the cells through its association with an inhibitory protein belonging to the inhibitory κB (IκB) family comprising notably IκBα. The classical NF-κB-activating pathway is induced by a variety of stimuli such as pro-inflammatory cytokines TNFα and IL-1β or bacterial products such as LPS [23], [24], [25], [26]. While these numerous stimuli induce the activation of NF-κB through different receptors and adaptor proteins, they all converge to a specific complex called the IKK (IκB kinase) complex. This complex is composed of two catalytic subunits, IKKα and IKKβ, a regulatory subunit, IKKγ (NEMO, NF-κB essential modulator) and different scaffold proteins [27], [28]. Upon cell stimulation, the IKK complex is activated by phosphorylation [29], [30] and then phosphorylates the IκBα protein on Ser32 and Ser36, targeting IκBα for polyubiquitination and degradation by the 26S proteasome. The released NF-κB translocates into the nucleus regulating the expression of its target genes such as those coding for cytokines, adhesion molecules and chemokines which have a crucial role in both immune and inflammatory responses [22], [29], [30], [31].
Moreover, the NF-κB functions are regulated by post-translational modifications including phosphorylation and acetylation [32], [33], [34]. Recent studies have demonstrated that p65 can be phosphorylated by various cytoplasmic and nuclear kinases at multiple sites either in the N-terminal RHD or C-terminal TAD in a stimulus- and cell-type dependent manner leading either to an increase in p65 DNA binding and/or transcriptional activity [32], [33], [34]. The best characterized phosphorylable residues are Ser276 and Ser536. The phosphorylation of these residues may change the conformation of p65 and promote its interaction with its coactivators CBP/p300 [35]. These proteins CBP/p300 are recruited to specific gene promoter by association with sequence-specific transcription factors and modulate the promoter activities by acetylation of both histone and non-histone substrates [34], [36], [37], [38], [39]. The acetylation of specific sites of histones, especially histones H3 and H4, surrounding specific genes is an important step in the chromatin remodelling which is often required to promote the access to the basal transcription machinery and can consequently favor the expression of specific genes [32].
In the present study, we showed that actin disruption by F-actin-depolymerizing compound, Cytochalasin D (CytD), up-regulated IL-8 expression in response to TNFα and LPS through transcriptional and post-transcriptional events in myelomonocytic cells. In both cases, the synergistic effect of CytD on il-8 gene transcription resulted from an increased NF-κB-mediated transcription. However, this up-regulating effect of CytD on NF-κB-mediated transcription involved different mechanisms according to the inducer. While CytD potentiated the canonical NF-κB activation pathway induced by LPS, this compound did not absolutely interfere with this pathway in the case of TNFα. Interestingly, actin disruption primed p65 phosphorylation induced by TNFα and LPS, on Ser276 and Ser536, respectively, which suggested that actin cytoskeleton could also modulate the p65 transactivating activity.
Section snippets
Chemicals
CytD was obtained from MP Biomedicals (Asse-Relegem, Belgium). LPS from Escherichia coli (serotype 0111: B4), LPS from E. coli (serotype 0111: B4) FITC conjugate and ActD were purchased from Sigma–Aldrich (Bornem, Belgium). Human recombinant TNFα was purchased from Peprotech (Tebu Bio, Boechart, Belgium).
Plasmids
The reporter construct (κB)5LUC (where LUC stands for luciferase) was obtained from Stratagene (La Jolla, CA, USA). The reporter plasmid (133-IL8)LUC contained a human IL-8 promoter fragment of
Actin disruption potentiates the expression of various inflammatory genes in TNFα- or LPS-stimulated myelomonocytic cells
To test the modulating effect or the potential role of actin cytoskeleton in TNFα- or LPS-induced NF-κB signalling, CytD, a naturally occurring actin-disrupting substance, was used. CytD inhibits actin polymerization by capping actin filaments and stimulating ATP hydrolysis on G-actin [45]. The staining of actin filaments (F-actin) in promyelocytic cells, especially HL-60 cells, showed a cortical cytoskeleton (Fig. 1A). The treatment with CytD disrupted actin cytoskeleton but did not induce a
Discussion
A growing body of evidences demonstrated that actin cytoskeleton, in addition to its well-known roles in cell morphology and motility, could modulate various signalling pathways [7], [8], [9], [10], [20]. Several classical NF-κB inducers, such as TNFα and LPS, can also lead to actin cytoskeleton reorganizations [14], [16]. For these different reasons, we decided to elucidate the role of actin cytoskeleton in the NF-κB activation pathways induced by TNFα and LPS in monocyte-like cells.
We have
Acknowledgements
We thank the platform Imaging and Flow cytometry, a CRCE/GIGA platform, and more especially we are grateful to S. Ormenese which is logistic collaborator FRS-FNRS of this platform for her technical assistance. This work was supported in part by the Interuniversity Attraction Poles (IAP)5/12 and IAP6/18 programs (Brussels, Belgium), Televie (Brussels, Belgium) and the National Fund for Scientific Research (FNRS Brussels, Belgium). GK and JH were supported by the IAP program and the Télévie, NEM
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