RANKL Upregulates TF Transcription in Macrophage Cells

TF is induced in macrophages, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs) of atherosclerotic lesions and triggers thrombosis after plaque rupture.² Therefore, we investigated whether RANKL induces TF expression in those cell types. RANKL increased TF mRNA expression and its procoagulant activity specifically in mouse macrophage RAW 264.7 cells (Online Figure I and Figure 1A through 1C), and also in human monocytic leukemia THP-1 cells (Figure 1D; Online Figure II, A and B). Moreover, TF antigen was increased on the surface of THP-1 cells in response to RANKL treatment (Online Figure II, C and D). The possibility of endotoxin contamination in our RANKL protein was excluded by the LAL assay and experiment with commercially available RANKL protein (Online Figure III). Taken together, our findings demonstrate that RANKL is a novel inducer of TF expression.

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Figure 1.

RANKL increases TF mRNA expression and activity. A and B, TF mRNA levels in RAW 264.7 cells after exposure to RANKL (3 μg/mL) for the indicated times (A) or with indicated doses of RANKL for 2 hours (B), as assessed by semiquantitative RT-PCR analysis. C and D, Chromogenic assay of TF activity after RANKL (3 μg/mL) treatment for 2 hours in RAW 264.7 cells (C) and THP-1 cells (D). E, The schematics of mouse TF promoter constructs. F, Luciferase activity was determined in RAW 264.7 cells transiently transfected with deletion mutants and treated with RANKL (3 μg/mL) for 2 hours. *P<0.05; **P<0.01 (RANKL-treated vs untreated cells).

Along with TF increase in RANKL-treated macrophages, we also detected very faint bands representing alternatively spliced form of TF (asTF) in human and murine cells (Online Figure II, A). The asTF was initially reported as a soluble TF, but its procoagulant activity is still unclear.^12,13 RANKL-induced asTF might exert a biological effect in thrombosis. However, because the elucidation of the function of asTF is beyond this study, we focused on the effects of RANKL on full-length TF.

The RANKL-induced increase in TF mRNA was abolished by pretreatment with actinomycin D, suggesting that RANKL increased TF mRNA by increasing transcription rather than by stabilizing preexisting mRNA (Online Figure IV). To identify the key transcription factors involved in RANKL-induced TF transcription, we evaluated the luciferase activity of the deletion mutants depicted in Figure 1E. Deletion of a putative nuclear factor (NF)-κB binding site decreased luciferase activity only slightly (Figure 1F). For a more elaborate discussion regarding involvement of NF-κB in RANKL induced TF expression, see the expanded discussion section in the Online Supplemental Material. Interestingly, deletion of the AP-1 site and/or Egr-1 site resulted in a marked decrease in luciferase activity (Figure 1F). It implies that AP-1 and Egr-1 is of importance in RANKL-induced TF transcription.

MAPK Pathways Control RANKL-Mediated TF Expression and Activity

Activated c-Jun NH₂-terminal kinase (JNK) and extracellular signal-related kinase (ERK)1/2 enhance the activity of c-Jun and Egr-1, respectively. We found that RANKL stimulated both JNK and ERK1/2 phosphorylation (Figure 2A; Online Figure II, E) and their targets, phospho–c-Jun and Egr-1 (Figure 2B) in macrophage cells. Furthermore, RANKL increased the DNA-binding activity of AP-1 and Egr-1, which was completely abolished by an excess of unlabeled oligonucleotides (Figure 2C and 2D). These results support a role for AP-1 and Egr-1 in RANKL-induced TF transcription.

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Figure 2.

RANKL activates MAPK and increases phospho-c-Jun and Egr-1 in RAW 264.7 cells. Western blot analysis showed phosphorylated JNK and ERK1/2 (A) and phosphorylated c-Jun and Egr-1 (B) in the nuclear fraction after RANKL (3 μg/mL) treatment. Nuclear extracts from RANKL-stimulated cells were incubated with ³²P-labeled AP-1 (C) and Egr-1 (D) probes and analyzed by electrophoretic mobility-shift assay. In competition experiments, unlabeled oligonucleotides were added to the RANKL-treated cells before the addition of labeled probes.

To confirm whether JNK and ERK1/2 pathways are required for RANKL-induced TF expression and activity, we cotreated RAW 264.7 cells with RANKL and chemical inhibitors. The JNK inhibitor SP600125 reduced RANKL-induced TF mRNA levels (Figure 3A) and TF activity (Figure 3C). The MEK1 inhibitor U0126 produced similar results (Figure 3B and 3D). These findings were confirmed via inhibition of phospho-c-Jun and Egr-1 in the nuclear fraction (Figure 3E and 3F). Taken together, these results suggest that phospho-c-Jun and Egr-1 via JNK and ERK1/2 signaling respectively are involved in mediating the RANKL-induced TF expression in RAW 264.7 cells.

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Figure 3.

RANKL-induced TF expression is attenuated by MAPK inhibitors and knockdown of c-Jun and Egr-1. A through F, After 1-hour pretreatment with inhibitors, RAW 264.7 cells were incubated with RANKL (3 μg/mL) for 2 hours. TF mRNA expression was determined by RT-PCR (A and B). TF activity was determined using a chromogenic assay (C and D). **P<0.01. Nuclear extracts were analyzed by Western blot (E and F). G and H, RAW 264.7 cells were infected with c-Jun small hairpin RNA lentivirus, followed by Egr-1 siRNA transfection. The cells were then treated for 2 hours with RANKL (3 μg/mL), and knockdown was confirmed by Western blot (G). TF mRNA levels were determined by semiquantitative RT-PCR (H). *P<0.05. SC indicates nontargeting scrambled siRNA.

Knockdown of c-Jun and Egr-1 Attenuates RANKL-Induced TF Transcription

To confirm the results of chemical inhibitor experiments, we determined the effects of c-Jun and Egr-1 by using siRNA against Egr-1 and/or lentivirus-based small hairpin (sh)-c-Jun. Knockdown of either Egr-1 or c-Jun attenuated RANKL-induced TF expression and that of both genes demonstrated an additive effect (Figure 3G and 3H). These results suggest that a multiprotein complex containing c-Jun and Egr-1 may play a key role in RANKL-induced TF expression in RAW 264.7 cells.

RANKL Increases TF Activity by JNK/AP-1 and ERK1/2/Egr-1 Signaling in Mouse Peritoneal Macrophages

Finally, we determined the effect of RANKL on TF using the ex vivo system of mouse peritoneal macrophages. As expected, exposure of mouse peritoneal macrophages to RANKL increased TF mRNA expression (Figure 4A) and its activity (Figure 4B). In addition, RANKL induced phosphorylation of JNK and ERK1/2 (Figure 4C), the same signaling pathways involved in TF expression in RAW 264.7 cells. Also, RANKL-induced TF expression was attenuated by SP600125 and U0126 (Figure 4D and 4E), suggesting the roles of JNK and ERK signaling in RANKL-induced TF expression in peritoneal macrophages.

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Figure 4.

RANKL induction of TF is mediated by AP-1 and Egr-1 in peritoneal macrophages. After 2 hours with RANKL treatment in peritoneal macrophages, TF mRNA levels were assessed by semiquantitative RT-PCR (A), and TF activity was assessed with a chromogenic assay (B). *P<0.05; **P<0.01. C, Western blot analysis revealed phosphorylation of JNK and ERK1/2 in peritoneal macrophages after RANKL treatment (3 μg/mL). D and E, After 1-hour pretreatment with inhibitors, peritoneal macrophages were incubated with RANKL (3 μg/mL) for 2 hours, and TF mRNA expression was determined by semiquantitative RT-PCR. **P<0.01.

In atherosclerotic plaques, TF is detected in macrophages and foam-cells as well as a lipid-rich core.⁴ The level of TF is higher in plaques from symptomatic than asymptomatic patients and plaque thrombogenicity is directly correlated to their TF content.^3,4 Given that RANKL is expressed by mast cells and T cells in activated plaques and its cognate receptor RANK is present in macrophages,⁶ ligation of RANKL and its receptor on macrophage could contribute to upregulation of TF in advanced lesions. Our data show that RANKL can induce membrane-bound TF and enhance procoagulant activity in macrophages. Notably, strong immunostaining of RANKL was detected in vulnerable plaques as well as in thrombus material from the site of ruptured plaques.^7,11 Thus, it is speculated that RANKL could participate in 2 ways at the later stage of atherosclerosis: by atherosclerotic plaque calcification^7,8,14 and subsequently by thrombus formation through strong induction of TF from macrophages.

We show that RANKL induces TF expression mainly via JNK/ERK1/2-mediated AP-1/Egr-1 signaling in macrophages. Also, our data indicate a potential role of NF-κB in TF expression by RANKL. Interestingly, it has been shown that sphingomyelin synthase 2 deficiency in macrophages diminished NF-κB and MAPK activation and reduced atherosclerosis in mice,¹⁵ and Egr-1 in the procoagulant response was higher in apoE^−/− compared to Egr-1^−/−/apoE^−/− mice.¹⁶ This supports the pathway of RANKL-mediated TF expression in macrophages and further raises the likeliness of RANKL involvement in atherothrombosis. Given that NFAT (nuclear factor of activated T cells), which is responsible for the transcription of TF, is activated by RANKL,¹⁷ we suggest that NFAT may also play a role in RANKL-mediated activation of TF. Considering our findings together with the previously identified role of RANKL as an endothelial activator^9,10 and an inducer of VSMCs calcification via the bone morphogenetic protein 4 pathway,^8,14 RANKL appears to be multiply involved in vascular pathophysiology. Although current data are limited to conclude physiological and clinical relevance, our findings support the notion that RANKL could be a causable risk factor for cardiovascular diseases including atherosclerosis.