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Critical role of NF-B and stress-activated protein kinases in steroid unresponsiveness¹

HEIKE BANTEL^*,, M. LIENHARD SCHMITZ, ARMIN RAIBLE, MICHAEL GREGOR and KLAUS SCHULZE-OSTHOFF^*,2

^* Institute of Molecular Medicine, University of Düsseldorf, Germany;
Department of Immunology and Cell Biology, University of Münster, Germany;
Institute for Chemistry and Biochemistry, University of Bern, Switzerland; and
Department of Internal Medicine, University of Tübingen, Germany

²Correspondence: Institute of Molecular Medicine, University of Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany. E-mail: kso{at}uni-duesseldorf.de

SPECIFIC AIMS

Glucocorticoid resistance (GR) is a serious clinical problem since many patients with chronic inflammatory diseases, such as Crohn’s disease, fail to respond to steroid treatment. However, the molecular mechanisms underlying this steroid unresponsiveness are unknown. Because the effects of steroids are largely mediated by interference of the glucocorticoid receptor with proinflammatory transcription factors, we investigated the role of NF-B and AP-1 as well as that of upstream kinases p38 and JNK in steroid resistance of Crohn’s disease.

PRINCIPAL FINDINGS

1. Distinct pattern of NF-B activation in steroid-resistant and -sensitive Crohn’s disease patients
We analyzed NF-B activation in tissue biopsies of inflamed ileocecal and colonic mucosa from steroid-resistant (n=19) and -sensitive (n=17) patients with Crohn’s disease. All patients had severe disease activity (Crohn’s disease activity index > 150). Steroid-resistant patients failed to respond to steroids (60 mg prednisolone equivalent/day), whereas treatment of sensitive patients resulted in complete remission according to usual clinical criteria within 4–5 wk.

To analyze NF-B activity, we used a monoclonal antibody selective for the active form of the NF-B RelA subunit. The antibody was raised against the nuclear localization sequence (NLS) of RelA, which in unstimulated cells is masked by its inhibitor, IB. Steroid-sensitive patients showed several NF-B-positive cells in the lamina propria of the inflamed mucosa (Fig. 1 A). Double staining demonstrated that cells containing activated NF-B were mainly macrophages (data not shown).

View larger version (158K): [in this window] [in a new window]   Figure 1. Different patterns of NF-B activation in mucosal biopsies of steroid-resistant and -sensitive Crohn’s disease patients. A–C) NF-B activation was investigated in tissue biopsies from steroid-sensitive (A) and -resistant (B) patients by the indirect immunoperoxidase method using an antibody selective for the active form of NF-B RelA. Sensitive patients displayed NF-B activation exclusively in macrophages of the lamina propria whereas most resistant patients expressed active NF-B predominantly in epithelial cells. In healthy colon tissue (C), no immunoreactivity was evident. D, E) Specificity control: a biopsy from the same resistant patient was incubated with the antibody in the absence (D) or presence (E) of the RelA NLS peptide used for generating the antibody. Preabsorption of the primary antibody with the peptide abolished immunoreactivity, indicating that staining was specific for NF-B.

Most steroid-resistant patients revealed a remarkably different staining pattern. Unlike sensitive patients, high expression of active NF-B was found in epithelial cells (Fig. 1B ). In contrast to patients with Crohn’s disease, no staining was observed in uninflamed mucosal tissue obtained from control individuals (Fig. 1C ). The immune reaction was specific for NF-B, since experiments including omission or preabsorption of the primary antibody with the RelA NLS peptide abolished the staining (Fig. 1D, E ).

To allow a semiquantitative analysis of the number of NF-B activated cells, positively stained cells were counted by light microscopy. The percentage of positive cells was taken from three regions of the affected mucosa to evaluate the average of NF-B activity. More than 52% (10 from 19) of the steroid-resistant patients showed NF-B activation in epithelial cells, whereas in sensitive patients NF-B activation was detected mainly in macrophages of the lamina propria but never in epithelial cells (Fig. 2 A). Active NF-B in steroid-resistant patients was found in > 50% of the epithelial cells (Fig. 2B ).

View larger version (17K): [in this window] [in a new window]   Figure 2. Quantification of patients with epithelial NF-B activation (A) and of the number of NF-B-positive epithelial cells in the two patient cohorts (B). For semiquantitative analysis, positively stained cells were counted by light microscopy with a 400-fold magnification in 4 microscopic fields. The % of positive cells was taken from 3 different regions of the affected mucosa to evaluate the average of NF-B activity; > 52% (10 from 19) of steroid-resistant patients showed NF-B activation predominantly localized in epithelial cells, whereas no epithelial NF-B activation was detected in steroid-sensitive patients.

2. Comparison of AP-1, p38, and JNK activity in steroid-resistant and -sensitive patients
In addition to NF-B, the glucocorticoid receptor binds to and antagonizes transcription factor AP-1, and so we investigated activation of AP-1 c-Jun using an activation-specific antibody that recognizes phosphorylated Ser-63 of c-Jun. We included activation-specific antibodies against the phosphorylated forms of the stress-activated protein kinases p38 and JNK1/2 in our analysis. JNK directly phosphorylates and thereby activates c-Jun. Kinases such as p38 have been reported to induce NF-B activity.

Comparing steroid-resistant and -sensitive patients, a different expression pattern was obtained for mediators of the MAP kinase pathway. Resistant patients showed p38 activation mainly in the nucleus of epithelial cells and, in some cases, in cells of the lamina propria (data not shown). In contrast, sensitive patients showed p38 activity exclusively in macrophages of the lamina propria (data not shown). Similarly, steroid resistance was strongly associated with perinuclear expression of active JNK in epithelial cells, whereas in sensitive patients JNK activity was detected in macrophages of the lamina propria (data not shown). In the resistant patients c-Jun activity was localized predominantly in the nucleus of epithelial cells, whereas in sensitive patients c-Jun activation was found in lamina propria macrophages. Epithelial expression of the active MAP kinases p38 and JNK as well as of active c-Jun did not correlate with the inflammatory infiltrate in steroid-resistant patients. In sensitive patients, however, activation of these mediators correlated with cell infiltration of the lamina propria. Thus, similar to NF-B, epithelial cells revealed high activation of stress-activated kinases observed in steroid-resistant but not in sensitive patients.

3. Distinct cellular expression of TNF- in steroid-resistant and -sensitive patients
To further verify our earlier findings of a different cellular activation pattern of the proinflammatory mediators, we investigated the functional expression of an NF-B target gene. TNF-, an important proinflammatory mediator in Crohn’s disease, is transcriptionally controlled by NF-B and AP-1. p38 and JNK have been implicated in transcriptional and post-transcriptional events of TNF- expression. Corresponding to the previous results, in steroid-sensitive patients TNF- was strongly expressed in inflammatory macrophages as detected by a TNF--specific antibody (data not shown). No staining was obtained in control biopsies from healthy individuals. Most important, however, in steroid-resistant patients epithelial cells strongly expressed TNF- (data not shown). Thus, steroid-sensitive and -resistant patients differed not only in the activation pattern of proinflammatory kinases and transcription factors, but also in functional target gene expression.

4. Inhibition of GR transcriptional activity by NF-B, JNK, and p38
Immunohistochemical results led us to speculate that increased epithelial activation of NF-B and mediators of the MAP kinase pathway might be involved in steroid unresponsiveness by antagonizing GR-dependent gene expression. To investigate this possibility, we performed GR reporter gene assays in the colonic epithelial cell line CaCo2. Cells were transfected with different amounts of plasmids encoding the NF-B trans-activating subunit RelA, JNK1, or p38 along with an expression plasmid for GR and a luciferase construct controlled by two GREs. After transfection, we treated cells with dexamethasone and analyzed GR transcriptional activity. Incubation of the colonic epithelial cells with dexamethasone strongly induced expression of the GRE-controlled luciferase reporter gene (data not shown). As expected, steroid-induced and GR-dependent reporter gene activity was significantly inhibited on expression of NF-B RelA. Likewise, transfection of the GR expression plasmid inhibited activity of a NF-B-dependent luciferase construct in NF-B RelA-expressing cells. Expression of JNK1 and p38 was also able to inhibit GR-controlled gene expression in a dose-dependent manner. The inhibitory effect was most pronounced after expression of p38, whereas JNK1 inhibited the GR less efficiently. These differences, however, were not caused by different expression levels of the kinases. Thus, the results indicate that not only NF-B, but also JNK1 and p38, are able to functionally antagonize the GR.

CONCLUSIONS AND SIGNIFICANCE

Glucocorticoid resistance poses a challenging clinical problem particularly in asthma, rheumatoid arthritis, and chronic inflammatory bowel disease such as Crohn’s disease. Even though many patients do not respond effectively to steroids, the molecular mechanisms underlying this clinical important phenomenon are virtually unknown. The effects of glucocorticoids result largely from interference with proinflammatory transcription factors such as NF-B and AP-1. Both transcription factors play a pivotal role in the immune response of chronic inflammatory bowel disease. Our present results obtained in transfected cells and tissue biopsies suggest that excessive and constitutive epithelial activation of NF-B and AP-1 as well as of the upstream MAP-kinases JNK and p38 might be involved in steroid unresponsiveness of Crohn’s disease. Whereas in steroid-sensitive patients, active NF-B, AP-1, JNK, and p38 were found predominantly in macrophages of the lamina propria, > 50% of steroid-resistant patients revealed a very different staining pattern: exaggerated activation of these proinflammatory mediators was localized in epithelial cells. Our experiments demonstrate that not only proinflammatory transcription factors and kinases revealed a distinct activation pattern, but also target genes such as TNF-. NF-B, and AP-1 as well as p38 and JNK tightly control TNF- expression by transcriptional and post-transcriptional events. Although TNF- is produced mainly by monocytes, epithelial cells are also able to secrete this cytokine. Based on the therapeutic success of anti-TNF- therapy, it is well established that TNF- plays a key pathogenic role in Crohn’s disease.

It has been demonstrated that TNF- can directly impair epithelial barrier function. Patients with inflammatory bowel disease have an increased mucosal permeability that may lead to a chronically enhanced immune response to microbial antigens and abrogated self-tolerance. This might explain the high constitutive expression of transcription factors and MAP kinases in steroid-resistant Crohn’s disease patients. As NF-B and AP-1 are strongly activated by TNF-, which itself is a target of both transcription factors, this scenario may constitute an autoamplification loop. We demonstrate that glucocorticoid resistance is associated with high constitutive epithelial activation of NF-B in concert with other proinflammatory mediators that all inhibit GR activity. Therefore, exaggerated activation of these mediators may compete with the limited number of GR molecules and prevent their DNA-binding.

Our histological findings were supported by functional GR reporter gene assays. Indeed, transfection of the colonic epithelial cell line CaCo2 with NF-B, JNK1, or p38 encoding plasmids led to a dose-dependent inhibition of the GR activity. Thus, our in vitro and in vivo data strongly indicate that exaggerated activation of proinflammatory transcription factors and kinases functionally represses GR antiinflammatory activity.

Therefore, detection of the activation state of mediators of the NF-B and MAP kinase pathway may serve as a diagnostic tool for early recognition of steroid resistance, protecting patients from the undesired severe side effects of prolonged and ineffective steroid treatment.

View larger version (27K): [in this window] [in a new window]   Figure 3. Schematic model of the role of NF-B, AP-1 and stress-activated protein kinases in steroid resistance of Crohn’s disease. Epithelial barrier dysfunction or other defects may cause a constitutive activation of proinflammatory mediators resulting in transrepression of GR activity. JNK and p38 may also directly inhibit GR transcriptional activity. These events will lead to a competition for a limited number of GR molecules and thus to an imbalance of the GR and proinflammatory NF-B and AP-1 response, which might cause steroid resistance.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0223fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0223fje

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