Inhibition of NF-κB and AP-1 by dimethylfumarate correlates with down-regulated IL-6 secretion and proliferation in human lung fibroblasts

QUESTION UNDER STUDY/PRINCIPLES: Dimethylfumarate (DMF) had been reported to reduce asthma symptoms and to improve the quality of life of asthma patients. Therefore, we assessed the anti-inflammatory and anti-remodeling effect of DMF on isolated lung fibroblasts, which are relevant to inflammatory lung diseases. We determined the effect of DMF on platelet derived growth factor (PDGF)-BB induced proliferation, as well as on PDGFBB and tumor necrosis factor (TNF)-α induced interleukin (IL)-6 secretion and on activation of activated protein (AP)-1 and nuclear factor kappaB (NF-ĸB). METHODS: Confluent lung fibroblasts were incubated with DMF (0.1–100 μM) 1 hour before stimulation with PDGF-BB or TNF-α (both 10 ng/ml). IL-6 secretion was measured by ELISA. NF-ĸB and AP-1 activation was determined by immuno-blotting and EMSA. Cell proliferation was assessed by [3H]-thymidine incorporation in subconfluent fibroblasts. RESULTS: PDGF-BB but not TNF-α induced fibroblast proliferation. This was dose dependently reduced by DMF in a concentration range of 10–100 μM. PDGF-BB and TNF-α induced IL-6 secretion by lung fibroblasts and this was inhibited by DMF in a dose-dependent manner. However, PDGF-BB induced IL-6 secretion did not correlate with NF-ĸB activity, while TNF-α did. DMF inhibited the TNF-α induced NF-ĸB–DNA binding, but had neither an inhibitory effect on NF-ĸB nuclear entry nor on the degradation of IκB-α. PDGF-BB and TNF-α activated AP-1, which was also inhibited by DMF. CONCLUSIONS: Our data suggest that DMF downregulates TNF-α-induced IL-6 secretion and proliferation by inhibiting NF-ĸB and AP-1 activity, indicating its potential beneficial use for the treatment of inflammatory lung diseases.


Introduction
Fumaric acid esters (FAE) are used to treat psoriasis and in Germany, a mixture of dimethylfumarate (DMF) is registered for the management of severe psoriasis [1].The clinical efficacy and safety of an oral FAE formulation (BG00012), which contains only DMF is documented in multiple sclerosis [2].Occasional reports indicated that DMF reduced symptoms and improved the quality of life of asthma patients.
Airway inflammation and remodeling in inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) is a multi-level process involving T-cells, neutrophils and eosinophils [3].In addition to these classical inflammatory cells, activated lung fibroblasts and airway smooth muscle cells contribute to the pathology of inflammatory lung diseases by secreting a wide range of pro-inflammatory factors [4][5][6].Today, inhaled glucocorticoids (GC) and long acting β 2 -agonists are the most frequently applied drugs for inflammatory lung diseases [7].However, in chronic inflammatory lung diseases, airway inflammation and remodeling are not well controlled by any of the available therapeutic strategies [8].Therefore, new therapeutic alternatives are needed to control airway inflammation in inflammatory lung diseases.
Interleukin (IL)-6 is considered to contribute to the severity of inflammatory lung diseases [9].The IL-6 promoter is regulated by the activator protein (AP)-1, and nuclear factor (NF)-ĸB [10].Both, NF-ĸB and AP-1, are considered as new targets for pharmacological approaches in asthma therapy [11].In human keratinocytes DMF inhibited the activation of NF-ĸB and subsequently the expression of IL-8 and IL-20 [12].In human T-cells, DMF reduced the binding of active NF-ĸB to its corresponding DNA consensus sequence [13].The nuclear accumulation of NF-ĸB p50 and the subsequent expression of ICAM-1 were inhibited by DMF in human dermal fibroblasts [14].Furthermore, DMF inhibited the TNF-α induced nuclear entry of NF-ĸB p65 and p50 thereby reducing the expression of tissue factor mRNA and protein by human endothelial cells [15].In primary human airway smooth muscle cells DMF inhibited the NF-ĸB signaling cascade on multiple levels thereby down-regulating the secretion of asthma relevant eotaxin, RANTES and IL-6.Interestingly, our data indicated that DMF directly interacts with NF-κB thereby reducing its binding capacity to the corresponding DNA consensus sequence [6].
Based on histo-pathological studies fibroblasts play a role in airway remodeling in chronic inflammatory diseases and represent the major producer of extra-cellular matrix which increases airway wall thickness [16][17][18].Airway remodeling often occurs before any sign of inflammation can be detected and may trigger inflammation [17,19,20].By secreting thymus-and activation-regulated chemokine (TARC) fibroblasts recruit Th2 cells to the inflamed section of the lung [21].
The aim of this study was to determine the effect of DMF on PDGF-BB and TNF-α induced NF-ĸB and AP-1 activation, as well as on IL-6 secretion and fibroblast proliferation.This study design was approved by the local ethics committee (University Hospital Basel, Switzerland) and written consent was obtained from all participants.

Methods
Isolation of primary human lung fibroblasts: Fibroblasts were established from disease free lung parenchyma tissue (as defined by a pathologist) which was obtained from therapeutically resected lung tissue.Lung resections were performed to remove lung metastases of primary tumors in other organs.Tissue pieces (1-2 mm 3 ) were placed into 25 cm 2 flasks containing 1 ml of growth medium (RPMI1640, 8 mM stabilized L-glutamine, 20 mM Hepes, 5% fetal calf serum, 1% MEM vitamin mix; all Gibco/ BRL, Switzerland) and were kept at standard cell culture conditions (37 °C, 100% humidity, 5% CO 2, 95% air).All experiments were performed with fibroblasts between passages 3 to 6. Cells were serum deprived for 24 hours prior drug treatment and cytokine stimulation to prevent activation of various pro-inflammatory and pro-proliferative pathways by serum itself.
Drug preparation: DMF (Sigma, Buchs, Switzerland) was dissolved in dimethysulfoxide (DMSO; Sigma) and was further diluted in serum free medium.
IL-6 secretion by primary human lung cells: Confluent fibroblasts were serum starved for 24 hours before any treatment.DMF (0.1-100 μM) was added 1 hour prior to PDGF-BB or TNF-α stimulation (R&D Systems, Minneapolis, USA).After 24 hours cell culture medium samples were collected and IL-6 protein level was measured by enzyme-linked immunosorbent assay (R&D Systems).
Isolation of nuclear and cytosolic proteins: NE-PER extraction kit (Pierce/Thermo Fisher Sci, Rockford, USA) was used to isolate nuclear and cytosolic proteins as follows: trypsinized cells were collected (3,000 rpm, 3 min) and incubated on ice with CER I buffer (10 min), than CER II buffer was added (1 min).The nuclei were centrifuged (13.000 rpm, 4 °C, 5 min), and the supernatant was collected as cytosolic protein fraction.The nuclei pellet was re-suspended in NER buffer (40 min, 4 o C), than the cell debris was removed (13,000 rpm, 4 °C, 10 min) and the supernatant was collected as nuclear protein fraction.
PDGF-BB (10 ng/ml) but not TNF-α at any concentration, strongly induced fibroblast proliferation within 24 hours by 155 ± 3.6%, and this effect was significantly decreased by DMF in a concentration range of 10 to 100 µM (p <0.05 unpaired student t-test; fig.1E).
Lung fibroblasts that were pre-incubated with increasing concentrations of DMF (1-100 µM), or the drug vehicle DMSO (0.1%), or stimulated with PDGF-BB, or TNF-α (10 ng/ml) did not show any cytotoxic effect during 24 hours (data not shown).
PDGF-BB as well as TNF-α induced the activation and binding of AP-1 to a specific DNA oligonucleotide within 30-60 minutes (fig.3A) and 30-120 minutes (fig.3B), respectively.Both PDGF-BB and TNF-α induced AP-1 activation was inhibited when the fibroblasts had been pre-incubated for 1 hour with 100 µM DMF (fig.3A, 3B).EMSA have been performed in a total of 3 fibroblast lines which yielded comparable results.The densitometric analysis of the AP-1 bands of the depicted representative EMSA are displayed in the small inserts in figure 3A and 3B.The bands representing the AP-1/DNA complex are not very

Original article
Swiss Med Wkly.2010;140:w13132 Swiss Medical Weekly • PDF of the online version • www.smw.chdefined, a phenomenon which we have observed in a previous publication [23] and which might be explained by the large number of different protein complexes of AP-1.

Discussion
In this study we provide evidence that DMF inhibits PDGF-BB and TNF-α induced IL-6 secretion as well as PDGF-BB induced proliferation in human primary lung fibroblasts.We further show that this anti-inflammatory and anti-proliferative effect of DMF correlated with a reduced activity of NF-ĸB and AP-1.We therefore propose that the inhibitory effect of DMF on NF-ĸB and AP-1 results in the reduction of IL-6 secretion and fibroblast proliferation.
PDGF-BB stimulated remodeling and inflammation in the lung [22,24], and stimulated proliferation and IL-6 synthesis via AP-1 and C/EBP-β in human lung mesenchymal cells [5,[25][26][27][28].In rat airway smooth muscle cells PDGF-BB induced inflammation was mediated by NF-ĸB [29] and in human skin fibroblasts [20], but not in human vascular and airway smooth muscle cells [5,31].In our experiments, PDGF-BB neither degraded IĸB-α, nor stimulated NF-ĸB nuclear entry, nor NF-ĸB binding to its DNA consensus sequence, suggesting cell type or species specificity.Since the human IL-6 promoter contains an AP-1 binding site [10], this may explain our observation that DMF inhibited IL-6 secretion by PDGF-BB and TNF-α as well as proliferation, which also involves AP-1 [28].TNF-α is elevated in the airways of patients with chronic inflammatory lung diseases and correlated with increased airways responsiveness [32].Furthermore, TNF-α stimulates the secretion of pro-inflammatory cytokines by airway smooth muscle cells [7].In our study in lung fibroblasts, TNF-α induced: (i) the degradation of IĸB-α, (ii) the nuclear entry of the NF-ĸB subunits p50 and p65, and (iii) NF-ĸB/DNA complex formation.DMF was reported to inhibit the nuclear entry and the binding of NF-ĸB to the DNA without inhibiting the degradation of IĸB in human airway smooth muscle [6].Here we have shown that TNFα induced NF-ĸB/DNA binding in lung fibroblasts was inhibited by DMF treatment.However, the nuclear entry of the NF-ĸB subunits p50 and p65 was not inhibited and IĸBα re-synthesis was delayed by DMF after TNF-α stimulation.The delay in IĸB-α re-synthesis might be explained by the fact that the IĸB-α gene itself is regulated by NF-ĸB [33].Thus, NF-ĸB inhibition may delay IĸB-α re-synthesis as it was previously described in mouse embryo fibroblasts [34].This DMF cell type specificity might be important for its use as an anti-inflammatory and anti-remodeling drug in chronic inflammatory lung.
We have previously shown that DMF directly inhibits binding of active NF-ĸB to its corresponding DNA consensus sequence [6].Helenalin, another inhibitor of active NF-ĸB, directly alkylates NF-ĸB p65, thereby inhibiting NF-ĸB binding to the respective DNA sequence [35].Even we have no experimental evidence, the fact that both DMF and Helenalin contain electrophilic α, β-unsaturated carbonyl structures makes it likely that the modification of NF-κB which inhibits its DNA binding depends on alkylation.For Helenalin, it has been shown electrophilic α, βunsaturated carbonyl structures react with nucleophilic protein sulfhydryl groups via a Michael-type addition [36,37].The NF-ĸB p65 and p50 contain a number of cysteine residues, therefore it is possible that DMF suppresses NF-ĸB/DNA binding by a direct interaction with sulfhydryl groups located in the DNA binding domain [38,39].

Conclusion
In summary, our data show that DMF down-regulates PDGF-BB and TNF-α induced IL-6 secretion by human lung fibroblasts and their proliferation.Both effects may be mediated through the inhibition of NF-ĸB and AP-1 activity.The clinical efficacy of DMF and its long term safety profile in psoriasis makes DMF an interesting drug that could help to control inflammation and airway wall remodeling in inflammatory lung diseases.

Original article
Swiss Med Wkly.2010;140:w13132 Swiss Medical Weekly • PDF of the online version • www.smw.ch

Figure 3 DMF
Figure 3 DMF (100 µM) inhibits PDGF-BB (A) or TNF-α (B) induced AP-1/ DNA binding in lung fibroblasts (n = 3).The AP-1/DNA complex formation is shown as a representative EMSA which has been analyzed by densitometry, the result of which are shown as additional bar charts.Similar results were obtained in two additional experiments.Pre-incubation of extracts with a 100-fold excess of unlabeled oligonucleotides competed off the specific AP-1/DNA complexes (C: competition).