Actin cytoskeleton modulates ADMA-induced NF-kappaB nuclear translocation and ICAM-1 expression in endothelial cells

Background

Asymmetric dimethylarginine (ADMA) has been described as a uremic toxin in chronic kidney disease (CKD) patients and a critical factor of cardiovascular diseases (CVD) [1,2]. On the basis of many experimental and clinical studies, it is postulated that ADMA is not only a marker but also a potent mediator of endothelial dysfunction, CVD and mortality of CKD [3–11]. However, the precise molecular mechanism underlying ADMA-induced impaired endothelial functions is not fully understood.

Recent studies demonstrated that NF-κB may play a critical role in ADMA-induced endothelial dysfunction through inflammatory cytokines such as ICAM-1 [12,13], an inducible endothelial adhesive protein that serves as a counter-receptor for β2-integrins (CD11/CD18) present on the surface of leukocytes. Interaction of ICAM-1 with β2-integrins enables polymorphonuclear leukocytes to adhere firmly and stably to the vascular endothelium, and to migrate across the endothelial barrier [14–17].

NF-κB dimers, typically a heterodimer of p50 and RelA/p65 subunits, are primarily sequestered in the cytoplasm by inhibitory protein IκBα, belonging to the IkBs family, which masks the nuclear localization signal (NLS) of RelA/p65 [18,19]. Upon stimulation, IκBα is phosphorylated by IκB kinase (IKK) complex, ubiquitinated by the ubiquitin ligase, and then degraded by the 26S proteasome [20–23]. The released NF-κB dimers undergo rapid nuclear translocation and subsequent binding to NF-κB responsive elements to activate transcription of target genes, including ICAM-1 [24–27].

Despite ADMA increases the NF-κB activity, the mechanisms underlying it are unclear. A recent study showed that the destabilization or stabilization of actin filaments inhibited NF-κB activation [28]. Hence, the present study was designed to investigate the role of actin cytoskeleton in modulating ADMA-induced NF-κB activation and ICAM-1 expression in endothelial cells.

Material and Methods

MATERIALS:

HUVEC was purchased from Sciencell Inc (Carlsbad, CA). A monoclonal antibody to ICAM-1 was obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Monoclonal antibody to p65/RelA, Texas Red Goat anti-Rabbit IgG and polyclonal antibodies to β-actin were obtained from Abcam Inc (Cambridge, MA). In addition, cytochalasin D, fluorescein isothiocyanate-phalloidin and Hoechst 33342 were obtained from Sigma Inc (Louis, MO). Jasplakinolide was purchased from Enzo Life Sciences Inc (Plymouth Meeting, PA).

CELL CULTURE:

HUVEC was cultured as described [29] in gelatin-coated flasks using endothelial cell medium (Sciencell Inc) with bullet kit additives (Sciencell Inc) and 5% fetal bovine serum (Sciencell Inc). HUVECs used in the experiments were between 3 and 6 passages.

CELL LYSIS AND IMMUNOBLOTTING:

Cells were harvested in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25 mM EDTA, pH 8.0, 1% deoxycholic acid, 1% Triton X-100, 5 mM NaF, 1 mM sodium orthovanadate) containing protease inhibitors. For immunoblotting, protein samples were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham International PLC, Cardiff, UK) electrophoretically. The membrane was preincubated in 5% (w/v) nonfat dry milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) with shaking for 2.5 h at room temperature to block the residual binding sites, followed by an overnight incubation with the indicated prime antibodies at 4°C and a second antibody at room temperature for 1 h with shaking. The membranes were processed using the enhanced chemiluminescence (ECL) kit (Millipore Co, Billerica, MA) and exposed to Kodak X-OMAT film.

IMMUNOFLUORESCENCE:

Cells grown on coverslips were fixed in 3.7% paraformaldehyde/PBS for 30 min and permeabilized with 0.1% Triton X-100 for 10 min. Then permeabilized cells were incubated in 1% bovine serum albumin/PBS for 30 min at room temperature to remove nonspecific binding of the antibody. Cells were incubated with fluorescein isothiocyanate-phalloidin (FITC-phalloidin) for 60 min at room temperature to localize F-actin filaments. RelA/p65 was detected by a rabbit monoclonal anti-RelA/p65 antibody for 60 min at 37°C and a secondary antibody conjugated to Texas Red for 60 min at room temperature. Nuclei were stained using Hoechst 33342 in PBS. The coverslips were mounted on the slide using mounting media. Images were obtained using a fluorescence microscope (Leica DM 4000B, Germany).

PREPARATION:

After treatments, cells were washed 2 times with ice-cold Tris-buffered saline and resuspended in 400 μl of bufferA (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1mMEDTA, 0.1 mMEGTA, 1mM dithiothreitol, 0.5mM phenylmethylsulfonyl fluoride). After 15 min, Nonidet P-40 was added to a final concentration of 0.6%. Samples were centrifuged to collect the supernatants containing cytosolic proteins. The pelleted nuclei were resuspended in 50 μl of buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). After 30 min at 4°C, lysates were centrifuged and supernatants were transferred to new vials containing nuclear protein.

ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA):

Ten μg of nuclear extract was incubated with 1 μg of poly(dI-dC) in a binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5mM dithiothreitol, 10% glycerol, 20-μl final volume) for 15 min at room temperature. Then end-labeled double-stranded oligonucleotides containing a NF-κB site (30,000 cpm each) were added and the reaction mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were resolved in 5% native polyacrylamide gel electrophoresis in low ionic strength buffer (0.25× Tris borate/EDTA). The oligonucleotide used for the gel shift analysis was NF-κB 5′-AGTTGAGGGGACTTTCCCAGGC-3′. The sequence motifs within the oligonucleotides are underlined.

STATISTICAL ANALYSIS:

Data are expressed as mean ±S.E. Comparisons between experimental groups were made by T test. Differences in mean values were considered significant at p<0.05.

Results

ADMA INCREASED THE FORMATION OF STRESS FIBER IN ENDOTHELIAL CELLS AND CYT D INDUCED OBVIOUS DESTABILIZATION OF THE ACTIN FILAMENTS:

HUVEC was treated with or without 100 uM ADMA for 24h and stained with FITC-phalloidin to visualize F-actin by immunofluorescence microscopy. Before the treatment, F-actin was found mostly in the cortical regions comprising the cortical actin bands (Figure 1A), while after treatment with ADMA these bands rearranged into stress fibers (Figure 1B). Pretreatment of cells with 5 μM Cyt D, the prototypic actin depolymerizing drug [30], induced obvious destabilization of the actin filaments and then prevented the formation of stress fibers induced by ADMA (Figure 1C).

STABILIZATION AND DESTABILIZATION OF THE ACTIN FILAMENTS PREVENTED ADMA-INDUCED NUCLEAR ACCUMULATION OF RELA/P65:

Immunofluorescence results showed that RelA/p65 was predominantly localized in the cytoplasm in unstimulated cells (Figure 2A). Stimulation of cells with 100 μM ADMA resulted in nuclear accumulation of RelA/p65 as indicated by the pink versus blue nuclei in the control cells (Figure 2B). Pretreatment of cells with 5 μM Cyt D for 30 min decreased RelA/p65 nuclear localization induced by ADMA (Figure 2C). Unlike Cyt D, Jas is a drug that induces actin polymerization [30], but it was found that cells pretreated with 1 μM Jas for 30 min also inhibited RelA/p65 nuclear localization induced by ADMA(Figure 2D).

STABILIZATION AND DESTABILIZATION OF ACTIN CYTOSKELETON INHIBITED ADMA-INDUCED DNA BINDING OF NF-κB:

The EMSA revealed that the control cells show a faint shift, while the cells exposed to 100 μM ADMA for 24h produced a strong shift. In subsequent experiments, it was found that 5 μM Cyt D and 1 μM Jas both reduced the DNA binding of RelA/p65 in response to ADMA challenge (Figure 3A, B). The reduction is significant in that the shift induced by Cyt D and Jas are almost equal to the control cells.

STABILIZATION AND DESTABILIZATION OF ACTIN CYTOSKELETON INHIBITED THE ICAM-1 EXPRESSION IN ENDOTHELIAL CELLS:

Western blot analysis showed that control cells expressed a low level of ICAM-1. ADMA(100 μM) challenge of HUVECs resulted in increased level of ICAM-1 protein expression; this response was significantly inhibited in cells pretreated with of Cyt D(5 μM) or Jas(1 μM) for 30 min (Figure 4).

Discussion

The results suggest that actin cytoskeleton modulated ADMA-induced NF-κB activation and the expression of ICAM-1 in endothelial cells and demonstrate that ADMA altered the actin cytoskeleton in endothelial cells. Interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevented ADMA-induced NF-κB activation and ICAM-1 expression. The effects were associated with impaired nuclear translocation of RelA/p65, thus suggesting a novel role for the actin cytoskeleton in modulating ADMA-induced nuclear translocation of RelA/p65, and thereby promoting ICAM-1 expression in endothelial cells.

It has been reported that ADMA induces NF-κB activation [13], but mechanisms underlying the activation of NF-κB are poorly understood. Previous reports demonstrated that stabilization and destabilization of the actin filaments prevented thrombin-induced NF-κB activation in endothelial cells [28]. We therefore hypothesized that cytoskeleton dynamics are essential for ADMA-induced NF-κB activation in endothelial cells. Therefore, 2 reciprocal approaches were employed to address the role of actin cytoskeleton in the mechanism of ADMA-induced NF-κB activation in endothelial cells. These included stabilization and destabilization of the actin filaments to perturb the alterations in actin cytoskeleton that occurred in response to ADMA challenge of endothelial cells. Cyt D is used to destabilize the actin filaments, while Jas could induce actin filaments stabilization [30]. ADMA increased the formation of stress fiber (Figure 1B), which is associated with the higher permeability of the endothelial barrier, and Cyt D induced obvious destabilization of the actin filaments (Figure 1C). Because Jas competitively inhibits the binding of Phalloidin to F-actin [31], we did not test the effect of Jas on the actin filaments induced by ADMA.

Nuclear translocation is an important step for NF-κB activation, thus we supposed that Cyt D may prevent the NF-κB nuclear accumulation in response to ADMA challenge. Indeed, ADMA induced NF-κB nuclear transport (Figure 2B) and Cyt D impaired ADMA-induced nuclear translocation of NF-κB (Figure 2C). Intriguingly, stabilizing the actin cytoskeleton by Jas also inhibited ADMA-induced nuclear translocation of NF-κB, thus indicating the importance of actin dynamics in modulating RelA/p65 nuclear transport following ADMA challenge of endothelial cells.

Further, the effect of Cyt D on the NF-κB DNA binding activity in response to ADMA challenge was evaluated. ADMA increased the DNA binding of NF-κB, and stabilizing and destabilizing the actin filaments both reduced the DNA binding of NF-κB in response to ADMA challenge (Figure 3). Considered together, these data raise the possibility that specific reorganization of the actin cytoskeleton as induced by ADMA in endothelial cells is essential for NF-κB activation.

ICAM-1 is involved in the recruitment of leucocytes to sites of inflammation at the endothelium and is thus involved in the pathogenesis of atherosclerosis [32]. Studies have shown that activation of NF-κB is essential for ICAM-1 expression in endothelial cells after stimulation with ADMA [12]. The effect of destabilizing or stabilizing the actin filaments on ADMA-induced ICAM-1 protein expression was then determined. Western blot analysis showed that ADMA challenge of HUVEC resulted in increased ICAM-1 protein expression and that this response was inhibited in cells pretreated with Cyt D or Jas (Figure 4). A low dose of 5 μM Cyt D or 1 μM Jas was sufficient to inhibit ICAM-1 protein expression by ADMA.

It is consistent with the confirmed reports that disturbing the dynamic of the cytoskeleton affects the activity of NF-κB in other cell sites [33–37]. In addition, actin cytoskeleton plays a regulating role in several other signaling pathways, such as the extracellular signal-regulated protein kinase (ERK) pathways [38], p38MAPK pathway [39], and the regulation of serum response factor (SRF) [40,41]. The mechanisms by which actin cytoskeleton regulates these signaling pathways have been explored, but remain to be detailed.

Conclusions

In summary, we found that ADMA increased the formation of stress fiber, and then promoted the NF-κB nuclear translocation, DNA binding to NF-κB and thereby ICAM-1 expression in endothelial cells. Disruption of ADMA-induced alterations in actin cytoskeleton, either by stabilizing or destabilizing the actin filaments, impaired the nuclear uptake of NF-κB, interfered with the DNA binding to NF-κB, and prevented the expression of ICAM-1. Thus, the specific targeting of actin cytoskeleton may be a useful strategy for preventing ADMA-activated inflammatory responses.