Abstract
Objectives
We have previously shown that vasopressin could inhibit the upregulation of inflammatory mediators. Expression of inflammatory mediators is tightly regulated by the upstream transcriptional pathway mitogen-activated protein kinases (MAPKs) and activated protein-1 (AP-1). In this study, we elucidated whether vasopressin could inhibit the upregulation of MAPKs/AP-1.
Methods
Murine macrophages (RAW264.7 cells) randomly received lipopolysaccharide (LPS; 100 ng/mL) or LPS plus vasopressin (1000 pg/mL) (designated as the LPS and the LPS+V groups, respectively). Control groups were run simultaneously. For MAPKs, cells were harvested at 0 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after reaction. For AP-1, cells were harvested at 60 minutes after reaction. Between-group differences in MAPKs (i.e., extracellular regulated kinase, c-Jun N-terminal kinase, and p38 MAPK) and AP-1 expressions were compared.
Results
Immunoblotting assay data revealed that extracellular regulated kinase concentrations of the LPS +V group that harvested at 45 minutes and 60 minutes, but not at 15 minutes and 30 minutes, were significantly lower than those of the LPS group (p = 0.005 and p = 0.013). C-Jun N-terminal kinase concentrations of the LPS+V group that harvested at 15 minutes, 30 minutes, 45 minutes, and 60 minutes were also significantly lower than those of the LPS group (all p < 0.001). Concentrations of p38 MAPK of the LPS+V group that harvested at 15 minutes, 30 minutes, and 45 minutes, but not at 60 minutes, were also significantly lower than those of the LPS group (all p < 0.001). In addition, immunohistochemistry assay revealed that the AP-1 fluorescence signals of the LPS+V group were weaker than those of the LPS group.
Conclusion
Vasopressin inhibits MAPKs and AP-1 in endotoxin-activated macrophages.
Keywords
activated protein-1; c-Jun N-terminal kinase; extracellular regulated kinase; p38 mitogen-activated protein kinase; RAW264.7 cells;
1. Introduction
Vasopressin is a nanopeptide that is synthesized in paraventricular and supraoptic nuclei in the hypothalamus.1 The established physiological characteristics of vasopressin include vasoconstriction, antidiuresis, and behavioral regulation.1, 2, 3 Moreover, clinical data revealed that septic shock patients tend to have low circulating levels of endogenous vasopressin.3, 4Therefore, clinical guidelines have suggested exogenous vasopressin supplement as part of the therapies against severe sepsis.5
The concept of vasopressin supplement against sepsis is supported by previous data that exogenous vasopressin replacement could decrease sepsis-induced pulmonary inflammation6 and preserve renal and mesenteric blood flow.7, 8 We have further confirmed that vasopressin could significantly inhibit the upregulation of inflammatory mediators.9 Together, these data conform the potent anti-inflammatory effects of vasopressin.
Expression of inflammatory molecules is tightly regulated by the upstream regulatory pathway mitogen-activated protein kinases (MAPKs) and activated protein-1 (AP-1).10 To date, the question of whether vasopressin can exert significant effects on inhibiting the upregulation of MAPKs/AP-1 remains unstudied. To elucidate further, we thus conducted this cellular study with the hypothesis that vasopressin can inhibit the upregulation of MAPKs [i.e., extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK] and AP-1 in endotoxin-activated murine macrophages.
2. Materials and methods
2.1. Cell cultures and cell activation protocols
This study employed RAW264.7 cells, an immortalized murine macrophage-like cell line, to facilitate the investigation. Cultured with mixtures of Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum, and 1% penicillin/streptomycin (Life Technologies), RAW264.7 cells were incubated in a humidified chamber at 37°C and maintained with a gas mixture of 95% air and 5% CO2. To activate cells, confluent RAW264.7 cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL), a gram (−) endotoxin from Escherichia coli (serotype 0127:B8; Sigma-Aldrich, St. Louis, MO, USA), according to our previous report.9
2.2. Experimental protocols
Confluent RAW264.7 cells randomly received phosphate buffered saline (PBS, Life Technologies), vasopressin (1000 pg/mL, Life Technologies), LPS (100 ng/mL, Sigma-Aldrich), or LPS plus vasopressin and designated as the PBS, the V, the LPS, and the LPS+V groups, respectively. Vasopressin was administered immediately after LPS. The dosage and timing of administration of vasopressin were also determined according to our previous report.9
2.3. Immunoblotting assay for MAPKs
Cell cultures were harvested at 0 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes after reaction with LPS or at comparable time points in groups without LPS. Cell harvesting and cell culture processes were performed as we have previously reported.11 In brief, RAW264.7 cells were washed, scraped, and centrifuged at 1500g for 5 minutes. The cell pellet was resuspended in 5 mL cell lysis buffer [10mM HEPES (pH 7.9), 1.5mM MgCl2, 10mM KCl, 0.5mM dithiothreitol, and 0.2mM phenylmethylsulfonyl fluoride] and centrifuged again at 1500g for 5 minutes. Cells were resuspended again in cell lysis buffer and allowed to swell on ice for 10 minutes followed by homogenization. Homogenates were centrifuged at 3300g for 15 minutes at 4°C. The supernatants were saved and the pellets were discarded. The protein concentration of each sample was measured using a bicinchoninic acid protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA).
Then, equal amounts of protein (65 μg) were loaded into each well of a 7.5% tris-glycine polyacrylamide gel and separated using gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking, the nitrocellulose membranes were incubated overnight at 4°C with the primary antibody solutions of ERK (1:500 dilution, polyclonal p-ERK1/2 antibody, Santa Cruz Biotechnology Inc., Dallas, TX, USA), JNK (1:500 dilution, polyclonal p-JNK1/2 antibody, Santa Cruz Biotechnology Inc.), p38 MAPK (1:200 dilution, polyclonal p-p38 MAPK antibody, Santa Cruz Biotechnology Inc.), or actin (as the internal standard; 1:500 dilution, Millipore Corporation; Burlington, MA, USA).
Horseradish peroxidase conjugated antimouse immunoglobulin-g antibody (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) was used as the secondary antibody. Bound antibody was detected with chemiluminescence (ECL plus kit, Amersham Pharmacia Biotech Inc.). The protein band densities were quantified using densitometric technology (Scion Image for Windows; Scion Corp, Frederick, MD, USA).
2.4. Immunohistochemistry assay for AP-1
Immunohistochemistry assay was performed following our previous protocol.11 Cell cultures were grown on glass coverslips and harvested at 60 minutes after reaction with LPS or at comparable time point in groups without LPS. Harvested cell cultures were fixed, permeabilized, blocked, and then incubated for 30 minutes in primary antibody solution of AP-1 (1:100 dilution, polyclonal anti-c-Jun antibody; Sigma-Aldrich) followed by washing and then incubated with fluorescent rhodamine isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) for another 30 minutes. Nuclear counterstaining was performed with diamidino-2-phenylindole (Pierce Biotechnology Inc.). Then, the cells were imaged using a confocal microscope (TCS SP5 AOBS, Leica Microsystems CMS GmbH, Mannheim, Germany).
2.5. Statistical analysis
One-way analysis of variance was used to test the differences among these groups. The Student-Newman-Keuls test was used for posthoc analysis. All data were presented as means ± standard deviations. The significance level was set as 0.05. A statistical software package (SPSS 11.5 for Windows, SPSS Science, Chicago, IL, USA) was employed for data processing and analyses.
3. Results
3.1. Vasopressin inhibits MAPKs
Immunoblotting assay data revealed that the concentrations of ERK, JNK, and p38 MAPK of these four groups that harvested at 0 minutes after reaction were low. The concentrations of ERK, JNK, and p38 MAPK of the PBS and the V groups that harvested at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after reaction were also low (data not shown). By contrast, the concentrations of ERK, JNK, and p38 MAPK of the LPS group that harvested at 15 minutes, 30 minutes, 45 minutes, and 60 minutes after reaction were significantly higher than those of the PBS group (all p < 0.001).
Moreover, the concentrations of ERK of the LPS+V group that harvested at 45 minutes and 60 minutes after reaction, but not at 15 minutes and 30 minutes, were significantly lower than those of the LPS group (p = 0.005 and p = 0.013, respectively; Figure 1). Similarly, the concentration of JNK of the LPS+V group that harvested at 15 minutes, 30 minutes, 45 minutes, and 60 minutes were also significantly lower than those of the LPS group (all p < 0.001; Figure 1). The concentrations of p38 MAPK of the LPS+V group that harvested at 15 minutes, 30 minutes, and 45 minutes, but not at 60 minutes, were also significantly lower than those of the LPS group (all p < 0.001; Figure 1).
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Figure 1. Representative gel photography and the densitometric data of protein concentrations of mitogen-activated protein kinases, including extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, in murine macrophages (RAW264.7 cells) using immunoblotting assay. Actin was used as the internal control. Data are presented as mean ± standard deviation. LPS: the lipopolysaccharide (100 ng/mL) group. LPS+V: the LPS plus vasopressin (1000 pg/mL) group. * p < 0.05 the LPS+V group versus the LPS group. ERK = extracellular regulated kinase; JNK = c-Jun N-terminal kinase; LPS = lipopolysaccharide; MAPK = mitogen-activated protein kinase.
3.2. Vasopressin inhibits AP-1
Immunohistochemistry assay data revealed that the fluorescence signals of the PBS and V groups were low (data not shown). By contrast, the fluorescence signals of the LPS group were stronger than those of the PBS group. Moreover, the fluorescence signals of the LPS+V group were weaker than those of the LPS group (Figure 2).
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Figure 2. Representative findings of the immunohistochemistry assay for activated protein-1 in RAW264.7 cells. The activated protein-1 was stained with fluorescent rhodamine isothiocyanate-conjugated antibody. Nuclei were counterstained with diamidino-2-phenylindole. The cells were imaged using a confocal microscope. LPS: the lipopolysaccharide (100 ng/mL) group. LPS+V: the LPS plus vasopressin (1000 pg/mL) group. AP-1 = activated protein-1; LPS = lipopolysaccharide.
4. Discussion
Data from this study confirm our hypothesis that vasopressin can inhibit endotoxin-induced upregualtion of MAPKs/AP-1 in activated murine macrophages. Using the same model, we have previously demonstrated that vasopressin could inhibit endotoxin-induced upregulation of inflammatory mediators9 and hence confirmed the potent anti-inflammatory effects of vasopressin. As expression of inflammatory mediators is tightly regulated by the upstream transcriptional pathway MAPKs/AP-1, data from this study thus provides clear evidence to support the concept that the mechanisms underlying the anti-inflammatory effects of vasopressin may involve its effects on inhibiting MAPKs/AP-1 and the subsequent inhibition of inflammatory mediators.
Though data from this study confirmed the effects of vasopressin on inhibiting MAPKs/AP-1 upregulation, the underlying mechanisms remain unstudied. As our data revealed that vasopressin could inhibit the upregulation of all three members of MAPKs (i.e., ERK, JNK, and p38 MAPK) as well as AP-1, these data seem to suggest that the mechanisms underlying the effects of vasopressin on inhibiting MAPKs/AP-1 should involve the regulatory pathways of MAPKs/AP-1. It is well-established that endotoxin-induced upregulation of MAPKs/AP-1 is regulated by the complex of toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2).12 For activation of the TLR4/MD2 complex, endotoxin needs to be firstly transported by the soluble lipopolysaccharide-binding protein and then be presented to the cell surface pattern recognition receptor cluster of differentiation 14 (CD14).13 CD14 then transfers LPS to the complex of TLR4/MD2, which then leads to the activation of MAPKs/AP-1 and the subsequent upregulation of inflammatory mediators.12 Judging from these data, we thus speculate that vasopressin might act through inhibiting lipopolysaccharide-binding protein, CD14, and/or the TLR4/MD2 complex to exert its effects on inhibiting MAPKs/AP-1 activation.
The mechanisms underlying the effects of vasopressin on inhibiting MAPKs/AP-1 remain to be elucidated; we thus acknowledge this as part of the study limitations. Moreover, in addition to MAPKs/AP-1, expression of inflammatory mediators is also regulated by the transcriptional pathway nuclear factor-κB.14 The question of whether vasopressin can exert similar effects on inhibiting nuclear factor-κB expression remains unanswered. We therefore also acknowledge this as part of the study limitations. Finally, this is a cellular study and therefore further data interpretation should be performed with caution.
In conclusion, vasopressin inhibits MAPKs/AP-1 activation in endotoxin-activated macrophages.
Acknowledgments
This work was supported by a grant from Taipei Tzu Chi Hospital, New Taipei City, Taiwan (TCRD-TPE-1-4-RT-1) awarded to C.J.H.
