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The Journal of Immunology, 2006, 177: 8148-8153.
Copyright © 2006 by The American Association of Immunologists, Inc.

Annexin 1 Negatively Regulates IL-6 Expression via Effects on p38 MAPK and MAPK Phosphatase-11

Yuan H. Yang*, Myew-Ling Toh*, Colin D. Clyne{dagger}, Michelle Leech*, Daniel Aeberli*, Jin Xue*, April Dacumos*, Laveena Sharma* and Eric F. Morand2,*

* Centre for Inflammatory Diseases, Department of Medicine, Monash University, and {dagger} Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Annexin 1 (Anx-1) is a mediator of the anti-inflammatory actions of glucocorticoids, but the mechanism of its anti-inflammatory effects is not known. We investigated the role of Anx-1 in the regulation of the proinflammatory cytokine, IL-6. Lung fibroblast cell lines derived from Anx-1–/– and wild-type (WT) mice were treated with dexamethasone and/or IL-1. IL-6 mRNA and protein were measured using real-time PCR and ELISA, and MAPK pathway activation was studied. Compared with WT cells, unstimulated Anx-1–/– cells exhibited dramatically increased basal IL-6 mRNA and protein expression. In concert with this result, Anx-1 deficiency was associated with increased basal phosphorylated p38, JNK, and ERK1/2 MAPKs. IL-1-inducible phosphorylated p38 was also increased in Anx-1–/– cells. The increase in IL-6 release in Anx-1–/– cells was inhibited by inhibition of p38 MAPK. Anx-1–/– cells were less sensitive to dexamethasone inhibition of IL-6 mRNA expression than WT cells, although inhibition by dexamethasone of IL-6 protein was similar. MAPK phosphatase-1 (MKP-1), a glucocorticoid-induced negative regulator of MAPK activation, was up-regulated by dexamethasone in WT cells, but this effect of dexamethasone was significantly impaired in Anx-1–/– cells. Treatment of Anx-1–/– cells with Anx-1 N-terminal peptide restored MKP-1 expression and inhibited p38 MAPK activity. These data demonstrate that Anx-1 is an endogenous inhibitory regulator of MAPK activation and IL-6 expression, and that Anx-1 is required for glucocorticoid up-regulation of MKP-1. Therapeutic manipulation of Anx-1 could provide glucocorticoid-mimicking effects in inflammatory disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Glucocorticoids are well known as potent anti-inflammatory and immunosuppressive agents. The anti-inflammatory effects of glucocorticoids are effected both by negative regulation of proinflammatory gene expression and by up-regulation of certain genes products, including annexin 1 (Anx-1).3 Evidence that Anx-1 is involved in the actions of glucocorticoids has been described in inhibition of cell proliferation (1), anti-inflammatory effects (2, 3, 4), the regulation of cell differentiation (5), and membrane trafficking (6, 7). For example, exogenous Anx-1 or its N-terminal-derived peptide mimic many inhibitory effects of glucocorticoids, including inhibition of leukocyte recruitment at inflammatory sites (8, 9, 10); inhibition of proinflammatory mediators such as phospholipase A2, cyclooxygenase-2, and inducible NO; induction of apoptosis in inflammatory cells; and induction of IL-10, an anti-inflammatory cytokine (reviewed in Ref. 11).

Recent studies using Anx-1-deficient mice have demonstrated that proinflammatory cytokine expression in carrageenin- or zymosan-induced acute inflammation, Ag-induced arthritis, and experimental endotoxemia is increased in the absence of Anx-1 (12, 13, 14). The absence of Anx-1 is also associated with reduced sensitivity to the anti-inflammatory effects of dexamethasone, indicating a major role for Anx-1 in the pathopharmacology of inflammation (12, 13).

IL-6, a member of a family of structurally related cytokines, has a range of biological activities in regulation of immune response, inflammation, hemopoiesis, and oncogenesis. It is normally tightly regulated and expressed at low levels, and increased in various conditions characterized by inflammation such as sepsis, endotoxemia, and in response to proinflammatory cytokines, including IL-1 (15, 16, 17). Overproduction of IL-6 is involved in the pathology of inflammatory diseases, including rheumatoid arthritis, Castleman’s disease, juvenile idiopathic arthritis, and Crohn’s disease (18).

The mechanism through which Anx-1 regulates inflammation is not well understood. Its broad range of effects on inflammation suggests an influence on key pathways regulating cytokine expression, but the mechanism of any effect on cytokine expression has not been investigated. We explored the effects of Anx-1 on IL-6 expression using fibroblasts derived from Anx-1–/– mice. In these studies we demonstrate a powerful tonic inhibitory effect of Anx-1 on IL-6 expression, mediated via inhibition of MAPK, and dependence on Anx-1 for glucocorticoid regulation of MAPK phosphatase-1 (MKP-1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and reagents

Lung fibroblasts were obtained from wild-type (WT) and Anx-1–/– mice on a mixed 129/SvJ x C57BL/6 background as described (12, 19). Cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in 5% CO2. Before experiments, 80% confluent fibroblasts were cultured in DMEM/0.1% BSA (Sigma-Aldrich) for 24 h. Cells were then treated with human rIL-1 (1 ng/ml) and/or dexamethasone (10–6–10–8 M) as indicated.

Actinomycin D, an inhibitor of DNA-primed RNA synthesis (Sigma-Aldrich), dexamethasone (Sigma-Aldrich), IL-1beta (Sigma-Aldrich), Abs against phospho-ERK, phospho-p38, or phospho-JNK, total p38 MAPK, and beta-actin (Cell Signaling Technology), and anti-MKP-1 Ab (Santa Cruz Biotechnology) were obtained commercially. SB203580, a p38 MAPK inhibitor, was provided by Dr. A. Badger (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). The biologically active Anx-1 N-terminal peptide 2–26 (Ac2–26) was obtained commercially (Mimotopes).

ELISA

Levels of IL-6 in culture supernatants were measured using commercially available ELISA (Quantikine M; R&D Systems). In brief, capture anti-mouse IL-6 mAb (1 µg/ml) was coated on plates overnight. Supernatants, or recombinant mouse IL-6 in a range from 8 to 1000 pg/ml as standards, were incubated for 2 h, then incubated with biotinylated Ab and subsequently streptavidin HRP (Vector Laboratories). Color was developed using 3,3',5,5'-tetramethylbenzidine liquid substrate (Sigma-Aldrich). Absorbance was measured at 450 nm. The detection limit of the assay was 31 pg/ml.

Quantitative real-time PCR analysis

Quantitative real-time PCR was performed as described (13). Briefly, total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s protocol. The 0.5–1 µg of total RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen Life Technologies) and oligo(dT)20.

PCR amplification was performed on a Rotor-Gene 3000 (Corbett Research) using SYBR Green I (Roche). For PCR, 5 µl each of the standard and sample cDNA dilutions were added to individual tubes. Amplification (40 cycles) was conducted in a total volume of 10-µl containing primer concentrations of 3 pmol and 1 µl of dNTP mix, Taq, reaction buffer, and SYBR Green I dye. The primer-specific nucleotide sequences of IL-6 (20), MKP-1 (21), and beta-actin (20) were used. Melting curve analysis was performed at the end of each PCR. Amplification efficiency was controlled by the use of an internal control (beta-actin) and external standards, which were homologous to the targets. Relative quantification of target mRNA expression was calculated and normalized to beta-actin expression. The results are presented as the fold induction of mRNA expression relative to the amount present in control samples.

Western blot analysis

Western blotting was performed as previously described (22). In brief, total cell protein was measured by BCA Protein assay kit (Pierce). The 70 µg of protein was separated on 10% SDS-PAGE and transferred to Hybond-C extra nitrocellulose membranes (Millipore). Membranes were probed with anti-MKP-1 Ab. After incubation with sheep anti-mouse HRP Ab, protein bands were detected by ECL-plus Western blotting detection system (Millipore). The membranes were then stripped and serially reprobed with Abs against phospho-ERK, phospho-p38, or phospho-JNK and total p38 or beta-actin. Densitometry ratios of MKP-1 and phospho-MAPKs were normalized to total p38 or beta-actin content and expressed as arbitrary units using Image Gauge software (version 3.46). The activity of p38 MAPK was antagonized with SB203580.

Statistical analysis

The Student’s t test was used for comparison of continuous variables. Results are expressed as mean ± SEM. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Modulation of IL-6 by Anx-1

We first investigated the effect of Anx-1 deficiency on IL-6 expression. Supernatants were collected from cultured cells, either untreated or treated with dexamethasone, IL-1 or both for 24 h. Basal release of IL-6 was ~200-fold increased in Anx-1–/– relative to WT cells (p < 0.005) (Fig. 1A). IL-1 induction of IL-6 protein was observed in WT cells, but the level of IL-6 induced by IL-1 in WT cells remained significantly lower than the basal level in Anx-1–/– cells (p < 0.005). IL-1 did not additionally increase the elevated basal IL-6 in Anx-1–/– cells. Dexamethasone (10–7 M) significantly inhibited basal and IL-1-induced IL-6 protein in both WT and Anx-1–/– cells (p < 0.005 and 0.05, respectively) (Fig. 1A).


Figure 1
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  		FIGURE 1. Anx-1 regulates IL-6 expression and dexamethasone sensitivity. A, Up-regulation of IL-6 protein in Anx-1–/– cells. Supernatant was collected from WT and Anx-1–/– fibroblasts cultured for 24 h in the presence or absence of dexamethasone 10–7 M and/or IL-1 (1 ng/ml). IL-6 was measured by ELISA (n = 3 experiments each). *, p < 0.005 Anx-1–/– cells vs WT; {dagger}, p < 0.05 IL-1/dexamethasone treatment vs IL-1; {dagger}{dagger}, p < 0.005 dexamethasone treatment vs control. B, IL-6 mRNA expression in Anx-1–/– cells. Total RNA extracted from WT and Anx-1–/– cells was reverse transcribed. cDNA samples were amplified using real-time quantitative PCR and SYBR Green detection as described (n = 4 experiments each). *, p < 0.05 Anx-1–/– vs WT cells. C and D, Dose response of dexamethasone on the inhibition of IL-6 mRNA-induced by IL-1. Cells were treated in the presence or absence of IL-1 (1 ng/ml) and/or dexamethasone at concentrations as indicated. The expression of IL-6 in cells in WT cells (C) and Anx-1–/– cells (D) is shown (n = 3 experiments each). {dagger}, p < 0.05 IL-1/dexamethasone treatment vs IL-1. All results are expressed as mean ± SEM.

 
We next examined IL-6 mRNA expression. A 3-fold increase in basal IL-6 mRNA was observed in Anx-1–/– cells in comparison to WT cells (Fig. 1B). IL-1 increased IL-6 mRNA in both WT and Anx-1–/– cells (2.2- and 1.7-fold, respectively). In contrast to the effects on IL-6 protein, dexamethasone suppressed basal and IL-1-induced IL-6 mRNA in WT cells, but not in Anx-1–/– cells.

The dose dependence of the effect of dexamethasone on WT and Anx-1–/– cells was next analyzed. All concentrations of dexamethasone tested inhibited IL-1-induced IL-6 mRNA in WT cells (Fig. 1C). In contrast, in Anx-1–/– cells, IL-1-induced IL-6 mRNA was only suppressed by 10–6 M dexamethasone, whereas 10–7 or 10–8 M dexamethasone did not suppress IL-6 mRNA (Fig. 1D). These data indicate a reduced sensitivity to dexamethasone of IL-1-induced IL-6 mRNA in the absence of Anx-1.

Modulation of MAPKs by Anx-1

The expression of IL-6 is under the regulatory control of intracellular signal transduction pathways, including those involving MAPK. We therefore investigated MAPK activation in Anx-1–/– cells. Markedly increased basal phospho-p38, phospho-JNK, and phospho-ERK1/2 was observed in Anx-1–/– cells compared with WT cells (Fig. 2A). IL-1beta-induced MAPK activity was next investigated. In addition to high basal levels, phosphorylation of p38 induced by IL-1 was markedly increased in Anx-1–/– cells in comparison with WT cells (Fig. 2B). The duration of p38 MAPK phosphorylation induced by IL-1 was also noted to be extended as late as 3 h in Anx-1–/– cells. In contrast, IL-1 did not further increase ERK and JNK activity in the absence of Anx-1 in comparison to WT cells (data not shown). These data indicated that deficiency of Anx-1 was associated with enhanced basal and IL-1-induced p38 MAPK.


Figure 2
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  		FIGURE 2. Requirement of Anx-1 for control MAPK activation. A, Up-regulation of phospho-MAPKs in Anx-1–/– cells. MAPK activation was measured in WT and Anx-1–/– cells by Western blotting and the membrane was serially stripped and reprobed for three phosphorylated MAPKs, with total p38 MAPK as a loading control. The result is representative of five separate experiments. B, Regulation of p38 by IL-1 in Anx-1–/– cells. Cells were treated with IL-1beta (1 ng/ml) over 3 h. Activation of p38 MAPK was measured by Western blot analysis, the membrane was serially stripped and reprobed for other phosphorylated MAPKs, and total p38 was used as a loading control. Phosphorylated and total p38 in WT and Anx-1–/– cells treated with IL-1 and the relative densitometry values are shown. Results of the mean of three independent experiments are expressed as arbitrary units (AU).

 
Requirement for p38 MAPK for IL-6 overexpression in Anx-1–/– cells

To study the role of p38 MAPK in the regulation of IL-6, we used the inhibitor SB203580, which blocks the ability of activated p38 to phosphorylate downstream substrates, and studied IL-6 protein and mRNA expression. Cells were pretreated with SB203580 for 1 h and then incubated with IL-1 for an additional 3 or 24 h. IL-6 protein was detected in 24-h culture supernatants. Inhibition of p38 MAPK significantly inhibited IL-1-induced IL-6 protein in WT cells (p < 0.01) (Fig. 3A). p38 MAPK inhibition significantly reduced basal IL-6 in Anx-1–/– cells (p < 0.01), indicating that the increased IL-6 release by these cells is dependent on phospho-p38 MAPK. p38 MAPK inhibition also significantly decreased IL-6 in IL-1-treated Anx-1–/– cells (p < 0.01) (Fig. 3A).


Figure 3
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  		FIGURE 3. Requirement of p38 MAPK for IL-6 expression. A, Increased IL-6 protein in Anx-1–/– cells requires p38 activation. Cells were preincubated with SB203580 (SB, 5 µM) for 1 h, then treated with or without IL-1beta (1 ng/ml) for 24 h. Supernatant IL-6 was measured by ELISA. Results are expressed as mean ± SEM of three experiments. *, p < 0.01, SB203580 treatment vs control or IL-1beta/SB203580 vs IL-1. B, IL-1beta-induced IL-6 mRNA does not require p38 activation in Anx-1–/– cells. Cells were preincubated with SB203580 (5 µM) for 1 h, then treated with IL-1beta (1 ng/ml) for 3 h. IL-6 expression was measured by real-time PCR. Results are expressed as the mean ± SEM of three experiments in each group.

 
Increased IL-6 mRNA stability in Anx-1–/– cells

To investigate the mechanism of p38 MAPK-dependent control of IL-6 by Anx-1, we measured IL-6 mRNA in these cells. Inhibition of p38 MAPK reduced IL-1-induced IL-6 mRNA in WT cells (Fig. 3B). In contrast, p38 MAPK inhibition failed to reduce IL-6 mRNA in Anx-1–/– cells. Given the difference between the effects of p38 MAPK inhibition on IL-6 protein and mRNA, the effects of p38 inhibition on IL-6 mRNA steady-state levels was investigated. Actinomycin D was added to cells with or without SB203580 after 3 h of IL-1 stimulation. Actinomycin D was associated with a constitutive loss of IL-6 mRNA over time in WT cells (Fig. 4A). Actinomycin D-induced IL-6 mRNA loss was significantly less in Anx-1–/– cells (Fig. 4A). Inhibition of p38 MAPK increased the rate of loss of IL-6 mRNA in Anx-1–/– cells to a level similar to that of untreated WT cells. These data suggest p38 MAPK-dependent effects of Anx-1 on IL-6 mRNA stability.


Figure 4
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  		FIGURE 4. p38 increases the stability of IL-6 mRNA in Anx-1–/– cells. WT and Anx-1–/– cells were preincubated with IL-1beta (1 ng/ml) for 3 h, then treated with actinomycin D for an additional 4 h with or without SB203580 as indicated. Total RNA was isolated at time points indicated, and IL-6 mRNA levels were evaluated with real-time PCR. The results, based on a ratio of IL-6 to beta-actin mRNA amplification, are presented as the percentage of the control (0 h). The results are expressed as the mean ± SEM of three separate experiments in duplicate. *, p < 0.01 Anx-1–/– cells vs WT cells.

 
We next measured the rate of IL-6 mRNA degradation in the context of dexamethasone treatment. Actinomycin D was added to cells with or without dexamethasone after 3 h of IL-1 stimulation. Dexamethasone significantly destabilized IL-6 mRNA in IL-1-treated WT cells at 30 min, but this effect of dexamethasone was impaired in Anx-1–/– cells (Fig. 4B). The half-life of IL-6 mRNA was increased from 6 min in control cells to 48 min in Anx-1–/– cells exposed to dexamethasone/IL-1beta for 1 h.

Regulation of MKP-1 by dexamethasone is dependent on Anx-1

MKP-1 is a glucocorticoid-inducible negative regulator of MAPK activity. We investigated whether the increase in MAPK phosphorylation in the absence of Anx-1 was associated with changes in MKP-1 expression. WT cells treated with dexamethasone exhibited a rapid and sustained increase of MKP-1 mRNA expression (Fig. 5A). The up-regulation of MKP-1 transcripts by dexamethasone was detected as early as 30 min and remained elevated for 24 h in WT cells. In contrast, the ability of dexamethasone to induce MKP-1 mRNA was significantly impaired in Anx-1–/– cells. Analysis of MKP-1 protein confirmed similar differences in response to dexamethasone between WT and Anx-1–/– cells (Fig. 5B). Sustained dexamethasone-induced MKP-1 protein was observed in WT cells, but this was reduced in Anx-1–/– cells.


Figure 5
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  		FIGURE 5. Effect of dexamethasone on MKP-1 in Anx-1–/– cells. A, Dexamethasone up-regulates MKP-1 mRNA. Fibroblasts were treated with dexamethasone 10–7 M over 6 h. MKP-1 mRNA was measured by real-time PCR. The results, based on a ratio of MKP-1 mRNA to beta-actin amplification, are presented as the fold induction in MKP-1 mRNA expression relative to control samples. B, Dexamethasone up-regulates MKP-1 protein. Cells were treated with dexamethasone 10–7 M over 24 h, and MKP-1 protein was measured by Western blot analysis. Densitometry of MKP-1 values were normalized to total p38 content and are expressed in arbitrary units (AU). All results are representative of three independent experiments and results are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
To confirm the effect of Anx-1 on the regulation of MKP-1, a bioactive N-terminal Anx-1 peptide was used. The effect of Anx-1 Ac2–26 has been demonstrated to be similar to that of full-length protein, acting through the formyl peptide receptor family (23). Anx-1 Ac2–26 had no effect on MKP-1 expression in WT cells (Fig. 6A). Treatment of Anx-1–/– cells with Anx-1 Ac2–26 for 3 h significantly increased MKP-1 mRNA up to 3.7-fold (Fig. 6A). Significant increases in MKP-1 protein were also observed in Anx-1–/– cells in response to Anx-1 Ac2–26 (Fig. 6B). Restoration of Anx-1 in Anx-1–/– cells by administration of Anx-1 Ac2–26 also significantly inhibited phospho-p38 MAPK in these cells (Fig. 6C).


Figure 6
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  		FIGURE 6. Effect of Anx-1 restoration on MKP-1 expression and MAPK activation. A and B, Effect of Anx-1 on MKP-1 expression. Cells were treated with Anx-1 N-terminal peptide 2–26 (Ac2–26) for 0.5 and 1 h. MKP-1 expression was measured by real-time PCR (A) and Western blotting (B). PCR results based on a ratio of MKP-1 mRNA to beta-actin amplification are presented as the fold induction in MKP-1 mRNA expression relative to control samples. Results in A are expressed as the mean ± SEM of five separate experiments. Western blotting results in B are shown with densitometry values expressed as the mean ± SEM of five separate experiments. Treatment with Anx-1 N-terminal peptide Ac2–26 significantly induced MKP-1 mRNA and protein in Anx-1–/– cells. *, p < 0.05; **, p < 0.01. C, Regulation of p38 MAPK by Anx-1 N-terminal peptide Ac2–26. Anx-1–/– cells were treated with Anx-1 N-terminal peptide Ac2–26, (100 µg/ml) over 3 h. Activation of p38 MAPK was measured by Western blotting with beta-actin used as a loading control. Phosphorylated p38 and relative densitometry values (representative of five separate experiments) are shown. Treatment with Anx-1 N-terminal peptide Ac2–26 significantly reduced phospho-p38 in Anx-1–/– cells at 1 and 3 h. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The current results demonstrate that Anx-1 is a negative regulator of IL-6 production via effects on the p38 MAPK signal transduction pathway. The absence of Anx-1 significantly increased IL-6 mRNA and protein expression, with increased protein levels up to 200-fold. We previously reported that the exacerbation of a model of rheumatoid arthritis in Anx-1–/– mice is paralleled by increased expression of IL-1beta, TNF-{alpha}, IL-6, and macrophage inhibitory factor mRNA in the synovium (13). Similarly, Anx-1–/– mice exhibited exaggerated leukocyte emigration and IL-1beta generation in an acute inflammatory model (12), and accelerated lethality within 48 h of endotoxin administration, accompanied by deregulated cytokine expression (14). Together with the current results, therefore, the evidence suggests that Anx-1 is a key negative regulator of IL-6.

Control of the expression of IL-6 is mediated by intracellular signal transduction cascades involving a number of protein kinases. The phenomenon of increased IL-6 in Anx-1–/– cells was accompanied by increased basal MAPK activity and was prevented by inhibition of p38 MAPK, suggesting the effects of Anx-1 on IL-6 are dependent on MAPK activation. We also observed a greater and more sustained increase of the activation of p38 MAPK by IL-1 in the absence of Anx-1. p38 MAPK is a key regulator of the expression of inflammatory cytokines involved in human diseases (24), and modulation of mRNA stability by p38 MAPK has been identified for several proinflammatory mRNA (25). It has been reported that IL-6 mRNA stability is regulated via a signal transduction pathway that requires p38 (26, 27).

As well as effects on basal and IL-1-induced IL-6, Anx-1 influences the effects of glucocorticoids. The current results show that dexamethasone inhibited IL-6 release equally in WT and Anx-1–/– cells, suggesting that Anx-1 is not required for dexamethasone inhibition of IL-6 release. In contrast, reduced sensitivity of IL-6 mRNA expression to inhibition by dexamethasone was observed in Anx-1–/– cells, suggesting Anx-1–/–-dependent regulation of glucocorticoid sensitivity of IL-6 transcription. This suggestion was confirmed in studies of mRNA stability, in which dexamethasone failed to reduce RNA stability in Anx-1–/– cells.

Sensitivity to glucocorticoids in acute and chronic inflammation in vivo is impaired in the absence of Anx-1 (12, 13). Glucocorticoid sensitivity has been reported to be associated with reduced glucocorticoid receptor nuclear translocation (28), increased expression of glucocorticoid receptor beta (29), or the effects of macrophage migration inhibitory factor (30). Recently, the phosphatase MKP-1 has also been identified as a molecule that plays an important role in the mediation of the anti-inflammatory action of glucocorticoids (31). MKP-1 is required for glucocorticoid inhibition of p38, JNK, and ERK1/2 (32) and is a constitutive inhibitory regulator of p38 MAPK responses to TLR ligation (33). We report that dexamethasone induction of MKP-1 was impaired in Anx-1–/– cells, and in contrast, restoration of Anx-1 in Anx-1–/– cells significantly increased MKP-1, accompanied by inhibition of p38 MAPK phosphorylation. These data suggest that Anx-1 is a key regulator of MKP-1 expression. Glucocorticoid receptor activation is required for the induction of MKP-1 mRNA expression (34), and as Anx-1 affects the transcriptional activity of glucocorticoid receptors (35), this influence may provide a mechanism for this observation.

A link between Anx-1 and MAPK has been reported in stably transfected RAW 264.7 cells (36). The present results differ somewhat from findings reported in the RAW 264.7 macrophage cell line, in which Anx-1 regulated the activity of the ERK MAPK cascade but had no effect on LPS-induced JNK, p38 MAPK, or NF-{kappa}B activation (36). Differences in signal transduction pathway use between different cell types and stimuli are well described and may account for the discrepancy between these two studies.

In summary, Anx-1 exerts a powerful tonic inhibitory effect on cellular IL-6 expression. The increase in IL-6 is reflected in mRNA stability and protein levels, dependent on the increase in p38 MAPK activation seen in the absence of Anx-1. Anx-1 is also required for glucocorticoid inhibition of IL-6 mRNA expression and for glucocorticoid induction of MKP-1 expression. These data provide insight into a range of observations in relation to Anx-1, including its role in endogenous and glucocorticoid-mediated regulation of inflammation. These data suggest a potential role for Anx-1-based therapies in the treatment of inflammatory diseases in which IL-6 is known to play a role.


   Acknowledgments
 
We thank Prof. Rod Flower and Dr. Jamie Croxtall, Department of Biochemical Pharmacology, William Harvey Research Institute (London, U.K.), for providing lung fibroblasts from Anx-1–/– and WT mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by the National Health and Medical Research Council, Australia. Back

2 Address correspondence and reprint requests to Prof. Eric F. Morand, Department of Medicine, Centre for Inflammatory Diseases, Monash University Monash Medical Centre, Locked Bag 29 Clayton, Victoria 3168, Australia. E-mail address: eric.morand{at}med.monash.edu.au Back

3 Abbreviations used in this paper: Anx-1, annexin 1; MKP-1, MAPK phosphatase-1; WT, wild type. Back

Received for publication March 9, 2006. Accepted for publication September 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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