NFKB1/NR3C1-MAPK4 axis regulates the pathology of acute lung injury

Acute lung injury (ALI) is a serious disease with highly morbidity and mortality that causes serious health problems worldwide. MAPK4, a member of atypical MAPK family, has been implicated in the development of cancer. Herein, the current study aimed to investigate the possible role of MAPK4 in the pathology of ALI to identify potential candidates for ALI therapy. Murine ALI model was established in WT or MAPK4 -/- mice and the expressions of MAPK4 were measured. The survival ratio of ALI model mice was observed. Moreover, the changes of pathologic injury and infiltration of inflammatory cells, as well as the related signaling pathways, in lung tissues were analyzed. Furthermore, the possible molecular mechanism of MAPK4 expression in ALI was analyzed by massARRAY and EMSA assay. Finally, the effect of MAPK4 silencing using shRNA interference on the pathology of ALI was identified. Data showed that MAPK4 was up-regulated in lung tissues in LPS-induced murine ALI model. Importantly, MAPK4 deficiency mice exhibited prolonged survival time after LPS challenge, accompanied by alleviated inflammatory injury in lung tissues characterized with reduced production of pro-inflammatory cytokines, infiltration of immune cells and altered transduction of related signaling pathways. Besides, massARRAY results showed no aberrant change in CpG methylation levels between control and ALI mice. Bioinformatics analysis and EMSA assay showed that transcriptional factor NFKB1 and NR3C1 could negatively regulate the expression of


4
MAKP4. Finally, MAPK4-shRNA treatment could ameliorate the pathology of lung tissues and prolong the survival time of mice after LPS challenge.

Conclusions
Our data demonstrated that MAPK4, orchestrated by NFKB1 and NR3C1, could regulate the pathology of ALI, indicating that MAPK4 might be a new therapeutic target for ALI treatment.

Background
Acute lung injury (ALI) and it's more serious form acute respiratory disease syndrome (ARDS) are critical diseases characterized with diffuse inflammations in lungs, which could be triggered by various pathologies, such as sepsis and severe trauma [1,2]. Despite numerous therapeutic strategies have been used to ALI treatment [3][4][5], the worldwide incidence and mortality of ALI are still showing no sign of amelioration in the past decades [6,7]. One of the reasons why these therapeutic strategies are invalid is that the molecule mechanism of development of ALI is very complex and remains to be fully elucidated. Therefore, further investigation on the molecular mechanism of pathology of ALI is still urgent and critical for the development of novel therapeutic strategies against ALI, which ultimately benefits the clinical outcome of ALI patients.
MAPK4 (alias ERK4, p63 MAPK) is the member of atypical MAPKs and closely related to MAPK6 with 73% amino acid identity in the kinase domain [8]. Accumulating evidences have shown that MAPK4 is implicated in the development of various diseases including cancer and infection diseases [9][10][11]. To lung development and diseases, Rousseau et al. found that the loss of MAPK4 didn't affect intrauterine growth and lung maturation [12], suggesting that MAPK4 might be not involved in the development of lung. Interestingly, EI-Aarag et al. found that MAPK4 was closely related to the development of lung cancer [13]. Most recently, Wang et al. further reported that MAPK4 overexpression could promote the progression of lung cancer, indicating MAPK4 is a potential target for lung cancer therapy [14]. These researches have raised an interesting question that MAPK4 might be, as an important regulator, involved in the development of lung diseases. However, up to now, the potential role of MAPK4 in the lung related inflammatory diseases including ALI remains largely unknown.
To this aim, in present study, we evaluated the expression of MAPK4 in LPS-induced murine ALI and accessed the possible effects of MAPK4 deficiency on the pathology of ALI. We found that MAPK4 was up-regulated in lung tissues of LPS-induced murine ALI model. Importantly, MAPK4 deficiency could attenuate the inflammatory injury of ALI, accompanied by reduced infiltration of immune cells and production of proinflammatory cytokines, as well as altered transduction of related signaling pathways. Moreover, we found that transcription factor NFKB1 and NR3C1 could negatively regulate the expression of MAPK4 by binding to the core sequence of MAPK4 promoter. Importantly, MAPK4-shRNA treatment could significantly reduce the pathology of lung tissues and prolong survival time of ALI mice. Altogether, our study reveals an unknown role of MAPK4 in the pathology of ALI, indicating that MAPK4 might be a new therapeutic target for clinic therapy against ALI.

Mice
MAPK4 deficiency (MAPK4 -/-) mice breeding pair in a C57BL/6 background were purchased from The Jackson Laboratory (027666). Animals were housed under 6 specific pathogen-free conditions at Zunyi Medical University.

Cell culture
Raw264.7 cells were purchased from Conservation Genetics CAS Kunming Cell Bank (KCB200603YJ), and were cultured in high glucose DEME containing 10% fetal bovine serum at 37°C in 5% CO 2 .

Establishment of ALI Model
WT and MAPK4 -/mice (7 to 9 week-old) were challenged with i.p. injection of 10mg/kg LPS (Escherichia coli 0111:B4; Sigma) dissolved in sterile PBS as shown in our previous study [17]. Then the body weight and lung weight index (lung weight/body weight) was detected at indicated time.

Bronchoalveolar Lavage
Immediately after euthanasia, 1 ml aliquots of PBS were slowly infused in the murine lungs through the tracheostomy and then withdrawn gently. This lavage was repeated three times using the same syringe. The collected lavage fluid was stored in a 10ml tube on ice. The fluid was centrifuged at 1000rpm and 4°C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000g and 4°C for 10 min, stored at −80°C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.

Lung edema determination
Lungs from mice were excised and completely dried in the oven at 60°C 24h for calculation of lung wet/dry ratio.

Histology and immunohistochemistry
7 Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4μm-thick sections. For histology analysis, the lung sections were stained with hematoxylin and eosin (HE). For immunohistochemistry, the lung sections were deparaffinized with xylene and rehydrated in graded ethanol (100% to 70%). To eliminate endogenous peroxidase activity, the slides were treated with 3% H 2 O 2 for 30min and washed with PBS. Then, the slides were blocked with Goat serum (BOSTER, AR1009 ) for 2h at room temperature and incubated overnight at 4°C with corresponding antibodies (p-JNK: Cell Signaling Technology, 4668; p-p38 MAPK: Cell Signaling Technology, 42390). After washed with PBS, the slides were incubated with secondary antibodies and visualized with DAB kit (Solarbio, DA1010). Finally, the slides were counterstained with hematoxylin and analyzed by Olympus microscope.

RNA extraction and quantitative real time PCR
Total RNA was isolated from mice lungs using RNAiso Plus (TAKARA, 9108) according

DNA extraction and methylation analysis
Genomic DNA was extracted from 12 lungs of control and ALI model mice by using DNeasy Blood and Tissue kit (QIAGEN, 69504) according to manufacture's instructions. The quality and quantified were evaluated by gel electrophoresis and a NanoDrop spectrophotometer (Thermo). The genomic DNA from each sample was treated with sodium bisulfite using an EZ DNA methylation kit (Zymo Reasearch).
Altogether, 35 CpG sites were tested in this region. The spectra methylation ratios of each CpG site were generated by MassARRAY EpiTYPER software (Agena).

Western blot
Lung tissues were homogenized in ice-clod lysis buffer (KeyGEN BioTECH, KGP2100) according to manufacture's instructions. Equal amounts of protein were separated by 10% SDS-PAGE and protein were transferred onto polyvinyldifluoride membranes.
Membranes were incubated with 5% skim milk in PBS for 1 hour. Immunobloting was

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software. 1-way ANOVA followed by Bonferroni's post-hoc was applied for multiple comparisons and student's t-test was used when two conditions were compared. P <0.05 was considered statistically significant and two-sided tests were performed. All date are shown as a mean ± standard error of the mean (SEM). Survival was evaluated by the Kaplan-Meier method.

MAPK4 is up-regulated in the lung tissues of murine ALI model.
To investigate the possible role of MAPK4 in the development of ALI, we first detected the expression of MAPK4 in LPS-induced ALI model ( Figure 1A). As shown in figure 1B, the relative expression of MAPK4 increased obviously in the lung tissues and reached the peak at 24 hours post LPS challenge (P<0.05). Moreover, compared with that in control group, the expression level of MAPK4 protein in lung tissues in ALI group also increased significantly ( Figure 1C, P<0.05). To confirm this phenomenon, we also verified the expression of MAPK4 in lung tissues using immunohistochemistry assay and obtained similar results ( Figure 1D).
Microphages played important roles in the inflammatory process of ALI [15,16].
Then, we also detected the infiltration of F4/80 + cells in lung tissues in LPS-induced ALI model. Immunofluorescence assay data showed that the infiltration of F4/80 + cells increased obviously in the lung tissues in ALI model ( Figure 1E), which was consistent with our previous work [17]. Moreover, in line with above findings, the expression of MAKP4 protein was also elevated in the lung tissues ( Figure 1E).
Interestingly, we found that there was co-localization of F4/80 + cells and MAPK4 protein in lung tissues ( Figure 1E). Altogether, these findings indicated that MAPK4 might be involved in the pathology of ALI.

MAPK4 deficiency ameliorates the pathology of ALI.
Next, we observed the possible effects of MAPK4 deficiency on the pathology of ALI.
As shown in figure 2A, MAPK4 -/mice had a prolonged survival time compared with that in LPS-treated WT mice (P<0.05). Even though the body weight index and lung weight index did not change significantly ( Figure 2B and 2C, P>0.05), the lung edema reduced obviously in LPS-treated MAPK4 -/mice compared with that in LPStreated WT mice ( Figure 2D, P<0.05), which was a typical symptom of inflammation in lung injury [18]. Moreover, the protein level of BAL fluid also decreased markedly in LPS-treated MAPK4 -/mice ( Figure 2E, P<0.05). Furthermore, HE staining results showed that the infiltration of inflammatory cells and alveolar interstitium thickening in lungs significantly reduced in LPS-treated MAPK4 -/mice ( Figure 2F).
Hence, these data demonstrated that MAPK4 deficiency could obviously reduce the pathology of LPS-induced ALI.

Loss of MAPK4 alters the production of related cytokines in ALI.
Previous evidences have shown that pro-inflammatory cytokines, such as IL-1β and TNF-α, were involved in the pathology of lung injury [19,20]. To confirm the effects of MAPK4 deficiency on the pathology of ALI, we further detected the levels of inflammatory related cytokines in ALI mice. As shown in figure 3A-F, the mRNA levels of IL-1β and TNF-α in lung tissues in LPS-treated MAPK4 -/mice significantly decreased compared with those in LPS-treated WT mice (P<0.05). By contrast, the mRNA levels of anti-inflammatory cytokines TGF-β, IL-4 and IL-10 were markedly higher in LPS-treated MAPK4 -/mice (P<0.05). To verify these data, we also analyzed the protein levels of these cytokines in BAL fluid from LPS-treated WT or MAPK4 -/mice and obtained similar results ( Figure 3G-K, P<0.05). Thus, these results demonstrated that MAPK4 deficiency could affect the production of related inflammatory cytokines in ALI.

Loss of MAPK4 affects the immune cells composition in BAL fluid of ALI.
The infiltration and activation of related immune cells in lung tissues were involved in inflammatory response in ALI [21], therefore we assessed the composition of Collectively, these results demonstrated that MAPK4 deficiency could affect the compositions and functions of infiltrated immune cells in ALI, which could contribute to the attenuated pathology of ALI.

ALI.
It is well known that AKT, ERK1/2, JNK, p38 MAPK and NF-κb signaling pathways play important roles in the development of inflammation response [23][24][25]. To elucidate whether MAPK4 deficiency ameliorated the pathology of ALI was related to these signaling pathways, we detected the transduction of those signaling pathways in lung tissues from LPS-treated WT or MAPK -/mice, respectively. Western blot results showed that the expression levels of p-AKT, p-JNK and p-p38 MAPK significantly decreased in LPS-treated MAPK4 -/mice compared with these in LPS-treated WT mice ( Figure 5A, P<0.05). Previous study has shown that MK5 was a downstream molecular of MAPK4 [26], and we also found that the expression level of p-MK5 markedly reduced in LPS-treated MAPK4 -/mice ( Figure 5B, P<0.05). To confirm these data, we further analyzed the expressions of p-p38 MAPK and p-MK5 in lung tissues by immunohistochemistry assay and similar results were obtained ( Figure   5C). Token together, these data indicated that the protective effects of MAPK4 deficiency on the pathology of ALI was related to the altered transduction of AKT, JNK, p38 MAPK and MK5 signaling pathways.

NFKB1 and NR3C1 negatively regulate the expression of MAPK4 in ALI.
Next, we further explore the potential molecular mechanism of up-regulated expression of MAPK4 in ALI. Recent decades, DNA methylation is commonly invoked as a mechanism for transcriptional repression [27][28][29], we wondered whether DNA demethylation of CpG island in MAPK4 promoter enhanced the expression of MAPK4 in LPS-induced ALI. Expectedly, bioinformatics analysis indicated that there was a CpG island in the promoter of MAPK4 ( Figure 6A). However, sequenom massARRAY assay data showed that the CpG methylation level of MAPK4 promoter did not change significantly between control and ALI mice ( Figure 6B and 6C, P>0.05), indicating that DNA demethylation maybe not contribute to the up-regulation of MAPK4 in ALI.
To further analyze the molecular mechanism of up-regulated MAPK4 in ALI, we next sought to screen the core transcription factors of MAPK4 promoter and performed 3' deletion assay to access the core sequence of MAPK4 promoter ( Figure 7A). Results showed that luciferase activity increased significantly between 2.2kb and 1.5kb region in MAPK4 promoter, indicating that this region might be the core sequence of and MRF4 (myogenic factor 6) ( Figure 7C and 7D). Previous studies showed that Sp1, Egr-1, NFKB1, NR3C1 and PU.1 have been reported to play roles in inflammatory response [30][31][32][33][34], so we analyzed the mRNA levels of these transcription factors and found that, compared with those in control group, the mRNA levels of NFKB1, NR3C1 and Egr-1 were obviously lower in ALI group, while the mRNA levels of Sp1 and PU.1 significantly increased ( Figure 7E, P<0.05).
Because the luciferase activity results showed that the putative transcription factors could negatively regulate MAPK4 expression and accumulating evidences have shown that NFKB1 and NR3C1 were anti-inflammatory genes [31,32], so we presumed that NFKB1 and NR3C1 might be critical transcription factors in regulating MAPK4 expression in ALI. Expectedly, further analysis showed that the protein levels of NFKB1 and NR3C1 decreased obviously in lung tissues in ALI mice ( Figure 7F, P<0.05), which is consistent with our above data. Importantly, electrophoretic mobility shift assay further showed that NFKB1 and NR3C1 could directly bind to the promoter of MAPK4 ( Figure 7G and 7H), Together, these results demonstrated that NFKB1 and NR3C1 are new core transcription factors in regulating the expression of MAPK4 in ALI.

MAPK4-shRNA treatment protects mice against LPS-induced ALI.
Finally, we further explored whether MAPK4 might be a novel valuable target in ALI treatment using shRNA technique. As shown in figure 8A and B, compared with that in control group, the expression of MAPK4 decreased markedly in MAPK4-shRNA treatment group (P<0.05). Expectedly, the edema and inflammatory injury of lung tissues reduced significantly in MAPK4-shRNA treatment group ( Figure 8C and 8D).
Meanwhile, the levels of pro-inflammatory cytokines, including IL-1β and TNF-α, decreased significantly ( Figure 8E, P<0.05); conversely, the level of antiinflammatory cytokine IL-10 increased obviously in MAPK4-shRNA treatment group ( Figure 8E, P<0.05). To confirm the effect of MAPK4-shRNA in the transduction of related signaling pathways, we further analyzed the expressions of these pathways.
Date showed that the expression levels of p-AKT, p-JNK and p-p38 MAPK significantly decreased in MAPK4-shRNA treatment mice compared with these in LPS-treated WT mice ( Figure 8F), which were consistent with our above data. Of note, MAKP4-shRNA treatment could prolong survival time of ALI mice ( Figure 8G). All together, these data demonstrated that MAPK4-shRNA treatment could effectively ameliorate the pathology of ALI, indicating it might be a new valuable therapeutic target in ALI treatment.
For example, Carnesecchi et al. found that hyperoxia led to the phosphorylation of JNK and ERK, which was involved in cell death signaling and was related with oxidative stress induced acute lung injury [38]. Schnyder-Candrian et al. showed that LPS induced ARDS was mediated by p38 MAPK [39]. However, the studies focused on the roles of atypical MAPKs in inflammation related diseases is still scare. Herein, we found that the expression of MAPK4 was up-regulated in ALI.
Importantly, we verified that MAPK4 deficiency could significantly ameliorate the pathology of lung tissues, accompanied with reduced infiltration of immune cells and production of pro-inflammatory cytokines, including IL-1β and TNF-α. Therefore, our current data revealed an unknown role of MAPK4, a member of atypical MAPKs, in the pathology of ALI, which might provide a light on the role of atypical MAPKs in inflammation related diseases. Given the fact that immune cells, such as macrophages, expressed MAPK4 in ALI model, then, which types of immune cells are mainly involved in the pathology process of ALI in the condition of MAPK4 deficiency need to be further elucidated.
It is well known that there are complex networks among AKT, JNK and ERK pathways in biological process. For example, Wu et al. reported that IL-22 tethered to apolipoprotein A-I could ameliorate acute liver injury by altering EKT and AKT signaling transductions [40], suggesting the complicated connection among these pathways in liver injury. Moreover, in our previous work, we also found that miR-7 could affect the development of ALI, accompanied with altered transduction of AKT and ERK pathways [17]. In present study, we found that MAPK4 deficiency could  [14]. Therefore, these data might highlight the underlying connection among conventional MAPKs, atypical MAPKs and other signaling pathways. Finally, previous work found that MAPK4 could regulate MK5 thereby controlling the biological process [26]. Consistent with this finding, we also noticed that, in present study, the level of p-MK5 significantly decreased in the condition of MAPK4 deficiency. Interestingly, Perander et al. reported that the expression of DUSP2 could inhibit ERK3 and ERK4 mediated activation of its downstream substrate MK5 [41]. Therefore, further study on the roles of other couple molecules of MAPK4, which did not be screened in current study, is much valuable for illustration on the exact connections among MAPK4/MK5, AKT and other signaling pathways in the pathology of ALI.
Recent decades, numerous studies have revealed that the mechanisms of transcriptional regulation on gene expression were complex, including DNA 20 methylation and transcriptional factors [42][43][44][45]. In present study, bioinformatics analysis showed that there was a CpG island in the promoter of MAPK4.
Unexpectedly, we found that there was not significant change in CpG methylation level of MAPK4 promoter in ALI model, indicating that DNA demethylation of MAPK4 promoter might not be critical for the up-regulation of MAPK4 in ALI. Interestingly, we identified the core sequence region of MAPK4 promoter (-470 to +173 relative to the TSS). Of note, we verified that two important transcription factors NFKB1 and NR3C1 could directly bind to the core sequence region of MAPK4 promoter, indicating that NFKB1/NR3C1 negatively regulated the expression of MAPK4 in ALI.
Consistent with these findings, Adamzik et al. showed that genotypes of NFKB1 promoter polymorphism were associated with the severity in ARDS [46]. Moreover, Zhao et al. reported that somatostatin could alleviate the pathology of murine ALI, which was closely related with the affinity of glucocorticoid receptor [47].
Therefore, these data further verified the important role of NFKB1/NR3C1 in the pathology of ALI.
The gene silencing technique, including antisense oligonucleotides (ASOs) and short hairpin RNA (shRNA), could effectively knock down the expressions of interesting genes and was an important strategy for gene therapy against various diseases [48][49][50][51]. Our previous studies showed that ASOs against miR-21 might be a useful

Conclusions
For the first time, our study revealed an unknown role of MAPK4, which was regulated by NFKB1 and NR3C1, in the pathology of ALI. Importantly, MAPK4 silencing could obviously reduce the pathology of lung tissue in ALI model. This work might provide a novel insight for future ALI therapy.

Ethical approval and consent to participate
All animal experiments were performed according to the guidelines for the Care and

Consent for publication
All listed authors consent to the submission and all data are used with the consent of the person generating the data.

Availability of supporting data
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.