The activator protein-1 (AP-1) is a pivotal transcription factor that is responsible for the regulation of the transcription of various genes in response to inflammatory cytokines, stress inducers, pathogens, and oncogenic stimuli (1). As such, AP-1 governs a variety of biological processes, including inflammation, proliferation, apoptosis, differentiation, survival, cell migration, and transformation (2). AP-1 contains Fos, Jun, and the activating transcription factor (ATF) protein dimers, all of which feature the evolutionarily-conserved basic region-leucine zipper (B-ZIP) DNA binding domain (1, 3). The composition of the dimers thereby also determines the sequence elements to which the AP-1 transcription factors bind to, and thus, may regulate specificity and subsequently execute certain distinct biological functions (4).
Jun, originally referred to as c-Jun, was first identified as a viral oncoprotein, and is one of the most characterized AP-1 subunits (5). There is long-standing evidence that c-Jun appears to be a major component of the AP-1 transcription factor complex, and that the identity of the c-Jun dimer partners determines the ability of the entire complex to transactivate AP-1-dependent transcription (6). It is well known that the AP-1 protein is a major target of and is primarily controlled by the mitogen-activated protein kinases (MAPKs), which both increase the abundance of AP-1 components and then directly stimulate their activity (6). Subfamilies of the MAPKs consist of extracellular signal-regulated protein kinases (ERK1 and ERK2), c-Jun N-terminal kinases (JNK1, JNK2, and JNK3), and p38s (p38α, p38β, p38γ, and p38δ), which are sequentially activated by MAPK kinases (MAPKKs) and their upstream MAPKK kinases (MAPKKKs) (7). During inflammatory responses, MAPK signaling is activated by phosphorylation, followed by the promotion of c-Jun transcriptional activity onto inflammatory cytokine genes. Growing evidence suggests that the c-Jun protein can also be regulated by other mechanisms, such as mRNA turnover and protein stability, thereby involving not only phosphorylation, but also cross-talking with other types of post-translational modifications, such as ubiquitination and sumoylation (2, 8).
c-Jun phosphorylation on its N-terminus is stabilizing, although the underlying mechanisms for c-Jun ubiquitination and degradation still remain largely undetermined, though recent findings have highlighted new players involved in the regulation of c-Jun degradation. For example, MEKK1 has been identified as mediating the ubiquitination of c-Jun in response to osmotic stress (8). Other ubiquitin ligases have also been reported to induce the ubiquitination and degradation of c-Jun, such as COP1, Icth, and Fbw7 (9). Each player utilizes a similar, but different mechanism, depending on the c-Jun phosphorylation status. The overall regulation of c-Jun degradation is complex, as the phosphorylation status of c-Jun itself is a distinct process dictated by MAPKs depending on the cellular context; hence, the specific nature of the regulatory mechanism remains elusive.
Therefore, we deliver insights on a new player in c-Jun degradation regulation within the specific context of inflammatory responses. In efforts to elucidate the mechanism of c-Jun degradation, we utilize a phosphatase inhibitor, sodium orthovanadate (SO, Fig. 1A), and MAPK selective inhibitors within lipopolysaccharide (LPS)-stimulated murine macrophages.
The inhibitory activity of SO was analyzed by determining the production level of PGE2 amongst LPS-treated RAW264.7 cells in order to observe whether SO exhibited anti-inflammatory activity. As shown in Fig. 1B, C, SO strongly diminished the release of PGE2 at up to 95% at 400 μM, without altering cell viability. Indomethacin (Indo) also strongly downregulated PGE2 production at 2.5, 5, and 10 μM. Simultaneously, the mRNA levels of COX-2 were measured by semiquantitative and real-time PCR to determine whether the inhibition of PGE2 release by SO occurred at the gene expression level. As indicated by Fig. 1E, SO showed a remarkable ability to inhibit the mRNA expression of COX-2 at 200 and 400 μM.
In order to obtain a better understanding of the regulation of COX-2 expression by SO, we observed the level of c-Jun, as this transcription factor is a major subunit of AP-1, which plays a critical role in controlling COX-2 expression in RAW264.7 cells under LPS-stimulated conditions (10). An increased level of c-Jun was observed 45, 60, and 75 minutes after LPS treatment, while pre-treatment with SO showed a decrease in c-Jun levels, as caused by nuclear fractionation, immunofluorescence, and reporter gene assay (Fig. 2A). A decrease of c-Jun was observed in both the nuclear fraction and the cytosolic fraction (Fig. 2A, Left panel). Similar to these results, the nuclear levels of c-Jun were also remarkably decreased within the SO/LPS-treated group, when compared to LPS-stimulated cells (Fig. 2A, Middle panel). The luciferase activity increased by AP-1 was also dose-dependently inhibited through SO exposure (Fig. 2A, Right panel). Treatment with a transcription inhibitor, actinomycin D, revealed that the decreasing pattern of c-Jun protein abundance through SO-treatment could still be observed (Fig. 2B). Additionally, c-Jun mRNA expression appeared to be similar after SO pre-treatment among LPS-stimulated RAW264.7 cells, both with and without Actino D treatment (Fig. 2C), indicating that SO did not inhibit the transcriptional process of c-Jun. Immunoblotting analysis of these proteins was then carried out to examine whether SO could also affect the phosphorylation level of MAPKs and upstream enzymes in order to control c-Jun activation. As depicted by Fig. 2D, SO increased the levels of p-p38, p-ERK, and p-JNK at 45 minutes after LPS treatment.
Under LPS stimulation, MAPKs such as p38, ERK, and JNK, are phosphorylated and subsequently induce the activation of c-Jun through phosphorylation. Previous studies have indicated that the phosphorylation of c-Jun plays a role in governing the stability of c-Jun protein levels. To confirm which upstream enzymes dominate the regulation of this process, we utilized MAPK-spe-cific inhibitors among SO/LPS-treated RAW264.7 cells. The co-treatment with U0126 (U0) or SP600125 (SP), and the selective inhibitors of ERK and JNK, respectively, were still able to further promote SO-mediated c-Jun degradation (Fig. 2E, F). Contrary to expectations, co-treatment with SB203580 (SB), a selective inhibitor of p38, showed no further decreasing of c-Jun protein levels following SO treatment among LPS-stimulated RAW264.7 cells (Fig. 2G, Left panel). Interestingly, this phenomenon also occurred following the usage of another phosphatase inhibitor, NSC95397 (NSC: 2,3-bis-[(2-hydroxyethyl)thio]-1,4-naphthoquinone), and further decreases in c-Jun levels were not shown after co-treatment with SB (Fig. 2G, Right panel). Moreover, the phosphorylation pattern of p38 and the decreased level of c-Jun were also detected through immunoblotting analysis. The phosphorylation level of c-Jun at Ser73 also exhibited a similar level, as the c-Jun total formed under all conditions after treatment with U0, SP, and SB, indicating that the direct correlation between the phosphorylation of c-Jun and the c-Jun total form level. These results thereby suggest that p38 plays a dominant role in regulating c-Jun degradation amongst LPS-stimulated macrophages. The activation of p38 by SO was also examined amongst LPS-treated RAW264.7 cells. As shown by Fig. 2H, interestingly, SO strongly enhanced the level of p-p38 from 45 to 90 minutes during LPS exposure, whereas LPS alone upregulated the c-Jun levels at these time markers.
To confirm the dependency of c-Jun degradation on p38, we performed knockdown and overexpression experiments. Initially, we performed a knockdown of p38 with siRNA, and confirmed the knockdown efficiency (up to a 70% decrease) through real-time PCR (Fig. 3A, Left panel). The knockdown of p38 indicated that no change was observed amongst c-Jun protein levels in LPS-stimulated RAW264.7 cells (Fig. 3A, Middle panel). Treatment with p38 siRNA with an increasing concentration gave clear indication of the recovery of c-Jun protein levels fol-lowing SO-mediated c-Jun degradation among RAW264.7 cells (Fig. 3A, Right panel). These data exhibited consistent results when compared to the immunoblotting results using a p38-selective inhibitor. Moreover, the overexpression of p38 was sufficient to induce c-Jun degradation among RAW264.7 cells (Fig. 3B). These results indicated that p38 positively regulates c-Jun degradation. As the ectopic expression of p38 results in a decrease of RAW264.7 cell c-Jun protein levels, while both knockdown and p38-selective inhibition showed c-Jun recovery, we hypothesized that the phosphorylation of p38 is a driving force for c-Jun degradation among RAW264.7 cells. Additionally, the co-transfection of p38 and c-Jun among HEK293T cells following SO treatment revealed that SO inhibited the binding between p38 and c-Jun through immunoprecipitation assay (Fig. 3C).
To further analyze to what extent the phosphorylation of p38 regulates c-Jun degradation, we extended the line of inquiry from our previous hypothesis, to suggest that the phosphorylation of p38 plays an important role in c-Jun degradation. The co-transfection of p38 and adaptor protein TRIF showed a strong increase in the phosphorylation of p38 while, as expected, a decreased level of c-Jun was observed (Fig. 3E). Additionally, we treated RAW264.7 cells with anisomycin, a well-characterized p38 activator and protein synthesis inhibitor. The aniso-mycin treatment increased the phosphorylation of p38, and also induced c-Jun degradation among RAW264.7 cells (Fig. 3F).
Ubiquitination-dependent c-Jun degradation has been widely analyzed (11), but whether SO-induced c-Jun degradation is ubiquitin-proteasome dependent still remains unknown. In this study, we utilized MG132, a known proteasome inhibitor, in order to identify the ubiquitin-proteasome dependencies within SO-mediated c-Jun degradation among LPS-stimulated RAW264.7 cells. Pre-treatment with MG132 led to a clear recovery of c-Jun protein levels following SO-mediated c-Jun degradation among LPS-stimulated RAW264.7 cells (Fig. 4A), which indicated that SO-mediated c-Jun degradation is a ubiquitin-proteasome-dependent event. We confirmed this regulation by inducing the overexpression of p38 and TRIF among HEK293T cells with the addition of MG132. As indicated by Fig. 4B, c-Jun protein levels were consistently recovered within the MG132-treated group, and using a similar design, we performed c-Jun immunoprecipitation and immunoblotting analysis with the usage of ubiquitin antibodies. Interestingly, increased band intensity was detected in the blot with a molecular weight around 43–48 kDa, within which c-Jun was present (Fig. 4C).
In order to expand the regulatory mechanisms of MAPK signaling with respect to c-Jun degradation, we used SO, a tyrosine phosphatase inhibitor, which has been reported to induce MAPK activation (12). LPS treatment increased both c-Jun mRNA and protein levels (Fig. 2A, C), and intriguingly, the treatment with SO reduced the c-Jun protein level, but not its mRNA level, among LPS-induced murine macrophages (Fig. 2A, C). This decrease in c-Jun protein levels could be due to a decrease in transcription or post-translational modification through proteasome degradation. According to our results, SO was found to regulate c-Jun degradation, but not at the transcriptional level. This supports the plausible implication that tyrosine phosphorylation induced the MAPK signaling during the regulation of c-Jun degradation. In addition, we observed that only the selective p38 inhibitor was able to recover the c-Jun protein levels following SO-mediated depletion, whereas JNK and ERK inhi-bitors failed to do so (Fig. 3). Interestingly, NSC95397, an ir-reversible Cdc25 dual specificity phosphatase inhibitor, was shown to also induce a decrease in c-Jun protein levels, and further decreases were not observed after treatment with p38-selective inhibitor (Fig. 2G, Right panel). The results also showed that SO strongly induced the phosphorylation of p38, while c-Jun levels were also decreased by this compound among LPS-treated RAW264.7 cells (Fig. 2H). Interestingly, the levels of p-p38 (seen between 0 to 30 min) and c-Jun (seen between 45 to 120 min) showed opposite patterns in LPS-treated RAW264.7 cells (data not shown). These results further demonstrated that p38 is a major MAPK within the regulation of c-Jun degradation under SO-treated conditions.
The knockdown and overexpression of p38 confirmed the positive regulation by p38 on c-Jun degradation. Similar to JNK, p38 has been demonstrated to directly phosphorylate c-Jun at Ser-63 and Ser-73, and thus modulate the transcriptional activity of AP-1 (13). Based on our data, the phosphorylation of p38 appears to play an important and sufficient role in inducing c-Jun ubiquitination (Fig. 4). However, the dependencies of c-Jun degradation, with respect to the particular site of c-Jun phosphorylation, still remain elusive. GSK3 phosphorylates c-Jun at Ser243 and Thr239, which are required for Fbw7-mediated c-Jun degradation (14). C-terminal Src kinase has been shown to bind with, and directly phosphorylate, c-Jun at Y26 and Y170, promoting c-Jun degradation and reduced stability (15). Of particular note, our findings indicate that there is a major involvement of p38 in c-Jun degradation during inflammatory response conditions. However, whether p38 can directly phosphorylate c-Jun at similar phosphorylation sites, and subsequently mediate c-Jun degradation, is a question that must be addressed in future studies. In addition, because phosphorylation of p38 is governed by both upstream kinases and particular phosphatases, it is also important to identify the exact molecular player, if it happens that p38 is not the direct regulator of this mechanism. Our results shed light on a plausible, novel p38/c-Jun molecular axis which governs c-Jun degradation during the inflammatory response.
Conclusively, we found that phosphatase inhibition governing MAPK signaling mediates c-Jun degradation among LPS-stimulated RAW264.7 cells. Additionally, further analysis indicated that the dominant role of p38 in the positive regulation of c-Jun degradation in an inflammatory response model, as summarized in Fig. 4D. The phosphorylation of p38 promotes c-Jun degradation through the induction of c-Jun protein ubiquitination. These results highlighted the novel regulatory role of p38 within the inflammatory response through the regulation of the protein level of c-Jun/AP-1 transcription factors.
Sodium orthovanadate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphospecific or total-protein antibodies were pur-chased from Cell Signaling Technology (Beverly, MA, USA).
RAW264.7 cells (ATCC number TIB-71) and HEK293T cells (ATCC number CRL-1573) were cultured in RPMI 1640 and DMEM media, respectively.
The inhibitory effect of SO on the production of PGE2 was determined by analyzing the level of PGE2 using EIA kits, as previously described (16).
The viability of SO-treated RAW264.7 cells was checked by a conventional MTT assay, as previously reported (17).
Preparation of whole-cell lysates was done as described previously (18). Nuclear fractionation was done according to a previous report (18). Immunoblotting analysis of both nuclear fractionation and whole-cell lysates was performed as indicated previously (18).
After washing the cells, Confocal microscopic analysis was carried out according to previous reports (19).
See Supplementary data section.
Immunoblotting analyses were then performed using these lysates prepared with immunoprecipitation buffer previously reported (21).
See Supplementary data section.
See Supplementary data section.
This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF), (Grant number: 2017R1A6A1A03015642) and by Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (Grant No.: 2020R1A6C101A191), Korea.
The authors have no conflicting interests.