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Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia

Background T cells play a dominant role in the pathogenesis of asthma. Costimulation of T cells is necessary to fully activate them. An inducible costimulator (ICOS) of T cells is predominantly expressed on Th2 cells. Therefore, interference of signaling pathways precipitated by ICOS may present new therapeutic options for Th2 dominated diseases such as asthma. However, these signaling pathways are poorly characterized in vitro and in vivo. Methods Human primary CD4+ T cells from blood were activated by beads with defined combinations of surface receptor stimulating antibodies and costimulatory receptor ligands. Real-time RT-PCR was used for measuring the production of cytokines from activated T cells. Activation of mitogen activated protein kinase (MAPK) signaling pathways leading to cytokine synthesis were investigated by western blot analysis and by specific inhibitors. The effect of inhibitors in vivo was tested in a murine asthma model of late phase eosinophilia. Lung inflammation was assessed by differential cell count of the bronchoalveolar lavage, determination of serum IgE and lung histology. Results We showed in vitro that ICOS and CD28 are stimulatory members of an expanding family of co-receptors, whereas PD1 ligands failed to co-stimulate T cells. ICOS and CD28 activated different MAPK signaling cascades necessary for cytokine activation. By means of specific inhibitors we showed that p38 and ERK act downstream of CD28 and that ERK and JNK act downstream of ICOS leading to the induction of various T cell derived cytokines. Using a murine asthma model of late phase eosinophilia, we demonstrated that the ERK inhibitor U0126 and the JNK inhibitor SP600125 inhibited lung inflammation in vivo. This inhibition correlated with the inhibition of Th2 cytokines in the BAL fluid. Despite acting on different signaling cascades, we could not detect synergistic action of any combination of MAPK inhibitors. In contrast, we found that the p38 inhibitor SB203580 antagonizes the action of the ERK inhibitor U0126 in vitro and in vivo. Conclusion These results demonstrate that the MAPKs ERK and JNK may be suitable targets for anti-inflammatory therapy of asthma, whereas inhibition of p38 seems to be an unlikely target.

Background
Asthma is a chronic inflammatory disease of the airways. The inflammation characterizing asthma is complex and involves multiple cells and mediators. The cells involved include well-recognized immune and inflammatory cells, lymphocytes, macrophages, eosinophils, mast cells and neutrophils as well as resident lung cells [1,2]. The importance of allergen-specific CD4 + Th2 cells has been demonstrated. Th2-associated cytokines such as IL-4, IL-5, IL-9 and IL-13 are known to be involved in IgE production, airway eosinophilia, and airway hyperresponsiveness [3]. Consequently, the inhibition or modulation of allergenspecific Th2 cells and their cytokines has become an attractive target for novel therapeutic intervention strategies [4][5][6]. Downregulation of cytokine production has been achieved by glucocorticoids and immunosuppressants both in vitro and in vivo. As these agents suppressed a broad spectrum of immune function, more specific regulatory pathways of T cell need to be addressed.
In recent years, considerable effort has been mounted to dissect the signaling events in T cells. Full activation of T cells requires signaling through both the TCR/CD3 complex and the CD28 costimulatory receptor [7][8][9][10]. CD28 engagement by B7-1 and B7-2 on resting T cells provides a primary costimulatory signal critical for initial cell cycle progression, interleukin 2 production and clonal expansion [11]. Engagement of CTLA-4 by the same B7-1 or B7-2 ligands results in attenuation of T cell responses. Recently, molecular homologues of CD28 and CTLA-4 coreceptors and their B7-like ligands have been identified. These homologues presumably play an essential role in the acquisition of effector function and/or tolerance induction. One of the CD28-like molecules is induced during activation of T cells, thus it is referred as an inducible costimulator (ICOS) and has a unique B7-like ligand (B7-H2, B7 h or B7RP-1). PD-1 is an inhibitory CD28-like receptor, with two B7-like ligands (PD-L1 and PD-L2) [12,13]. It has been shown that the ICOS/B7-H2 pathway controls T cell dependent immune responses [14][15][16][17].
Several protein kinases such as the family of MAPKs are involved in the transmission of extracellular signals into the nucleus. [18]. MAPKs are serine/threonine kinases that include extracellular signal-regulated kinases (ERKs) [19], Jun NH2-terminal kinases (JNKs) [20] and p38 MAPK [21]. CD3 signaling alone has been shown to activate ERK, and the combination of CD3 and CD28 signaling can synergistically activate JNK in T cell lines and clones [22,23]. In these studies, CD28 signaling alone was shown to minimally activate ERK and JNK, while others have reported much more significant ERK activation [24]. Activation of p38 has also been observed in Jurkat cells and in mouse T cells treated with CD3/CD28 [25,26]. While it has become clear that productive T cell activation requires the cooperation of multiple signaling pathways, the composition and the relative contribution of these pathways to the activation process and cytokine production remain yet to be fully deciphered.
The predominant expression of ICOS on Th2 cells and its effect on the development of Th 2 cells has been reported [15,27]. Therefore, interference of signaling pathways precipitated by ICOS may present new therapeutic options for Th2 dominated diseases such as asthma. However, these signaling pathways are poorly characterized. Recently, we reported that the ERK pathway plays a pivotal role in regulating IL-13 production in human CD4 + T cells after CD28 costimulation [28]. In the present study, we studied the activation of different MAP kinases in human CD4 + T cells stimulated by α-CD3/α-ICOS, their function in the production of cytokines from these cells and compared these results to α-CD3/α-CD28 stimulated T cells. Our results demonstrate, that in human CD4 + T cells, p42/p44 ERK, p38 and JNK differentially regulate the production of IL-2, IL-4, IL-13 and IFN-γ in response to α-CD3/α-CD28 or α-CD3/α-ICOS. Furthermore, inhibitors able to inhibit ICOS induced signaling proved to be effective in the murine asthma model of late phase eosinophilia.

Enzyme-linked immunosorbent assay
Cytokine measurements in culture supernatants were done by sandwich ELISA using matched antibody pairs (BD Pharmingen, Heidelberg, Germany). ELISA plates (Maxisorb, Nunc) were coated overnight with anticytokine mAb in 0.1 M carbonate buffer, pH 9.5. After being washed, plates were blocked with Assay Diluent (Pharmingen, Heidelberg, Germany) for 1 h and washed again. Appropriately diluted supernatant samples and standards were distributed in duplicates and the plates were incubated for 2 h at room temperature. Plates were washed, incubated for 1 h with working detector (biotinylated anti-cytokine Ab and Avidin-horseradish peroxidase conjugate). After washing, substrate (TMB and hydrogen peroxide) was added. The reaction was stopped by addition of 1 M H 3 PO 4 . Plates were read at 450 nm (reference 570 nm) in a microplate reader (Dynatech). The results were expressed as a percentage of the control level of cytokines production by cells stimulated in the presence of the vehicle of the corresponding compound.

Determination of serum levels of total and OVA-specific IgE
Serum levels of OVA-specific IgE were measured by IgE ELISA (OptEIA, BD Pharmingen). Briefly, 96-well plates (Maxisorb, Nunc) were coated with OVA (100 µg/ml). After addition of serum samples, a sheep anti-IgE Ab was added to individual wells and its binding was detected with peroxidase-conjugated anti-sheep IgG. IgE concentrations were calculated by comparison with commercial mouse IgE standards (BD Pharmingen).

Analysis of cytokine mRNA expression by real-time RT-PCR
RNA was prepared from frozen lysates using Rneasy (QIA-GEN, Hilden, Germany). One-tube RT-PCR was performed using Quantitect Probe RT-PCR Kit from QIAGEN (Hilden, Germany). Expression of cytokines were determined in relation to beta-actin by real time RT-PCR using TaqMan assay on a ABI Prism 7900. Human primers and probes have been described [6]. Murine primers and probes were Assay-On-Demand purchased from Applied Biosystems (Darmstadt, Germany). Quantity of mRNA was calculated using the ∆∆C T method (PE Applied Biosystems User Bulletin #2; ABI PRISM 7700 Sequence Detection System, 1997). For each RT-PCR the threshold cycle (C T ) was determined, being defined as the cycle at which the fluorescence exceeds 10 times the standard deviation of the mean baseline emission for cycles 3 to 10. Cytokine mRNA levels were normalized to the housekeeping gene β-actin according to the following formula: ∆C T = C T β-actin -C T cytokine . Subsequently, respective cytokine mRNA levels were calculated using the ∆∆C T method, i.e., ∆C T values representing mRNA from cells treated with stimulus in combination with a test compound were set in relation to the ∆C T value representing mRNA levels from cells treated with stimulus alone according to the following formula: ∆∆C T = ∆C T (drug) -∆C T (vehicle). The relative mRNA level for the respective test compound was calculated as 2 -∆∆C T * 100% based on the results of control experiments with an efficiency of the PCR reaction of approximately 100%.

Western Blotting analysis of the MAPKs
Human CD4 + T cells were stimulated with Dynabeads CD3/CD28 T Cell Expander (Dynal Biotech GmbH, Hamburg, Germany) or with Dynal beads coated with α-CD3/ B7-H2 Fc for 0, 5, 15, 60 or 120 min. After stimulation, cells were washed with ice-cold PBS and lysed in cell lysis buffer (Tris 50 mM, pH 7.2, containing 10 mM EDTA, 150 mM NaCl, 1% NaDOC, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitors (1 mM PMSF, 5 µg/µl Aprotinin and 5 µg/µl Leupeptin). Cells were left on ice for 20 min and then centrifuged at 14.000 rpm for 10 min. The supernatants were stored at -80°C until measurement of the MAPKs. Equal amounts of protein were resolved in 10% SDS-polyacrylamid gel electrophoresis and transferred onto PVDF membrane. Membranes were blocked overnight at 4°C with 0.2 % I-block (Applied Biosystems, Darmstadt, Germany) in TBS/T buffer and probed with rabbit primary antibodies specific for the dually phosphorylated active forms of ERK, p38 and JNK (Promega GmbH, Mannheim, Germany) for 2 h at room temperature. Blots were washed, incubated 1 h at room temperature with second antibody (donkey anti-rabbit horseradish peroxidase conjugated) (Promega GmbH, Mannheim, Germany). Immunoreactive bands were visualized by enhanced chemiluminiscence (ECL) according to the manufacturer's recommendations (Amersham Pharmacia Biotech, Freiburg, Germany). After detection of the active form of the MAPK, membranes were stripped and reprobed with the corresponding antibody that recognize both active and inactive forms of p38, JNK (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and ERK (Promega GmbH, Mannheim, Germany).

ELISA detection of activated JNK
The FACE (Fast Activated Cell-based ELISA) kit (Active Motif, Rixensart, Belgium) was used according to the provider's protocol. CD4 + T cells were cultured in precoated (poly-L-lysine) (Sigma, Deisenhofen, Germany) 96-well plates at a concentration of 1 × 10 6 /ml and were stimulated with α-CD3/α-CD28 and α-CD3/B7-H2 Fc for different time points (1 min, 5 min, 15 min, 60 min and 120 min) to induce the JNK pathway. Following stimulation the cells were fixed in order to preserve protein modifications, including phosphorylation. Corresponding wells were then incubated with a primary antibody specific for pJNK and total JNK respectively. Subsequent incubation with secondary HRP-conjugated antibody and developing solution was followed by a colorimetric detection. The signals were then normalized for cell number using Crystal Violet.

Murine model of late phase eosinophilia
Male Balb/c mice weighing 22-25 g were used. Animals were purchased from Harlan (Borchen, Germany). The animals were kept under constant environmental conditions (temperature: 18 ± 2°C, humidity: 40-60 %, light cycle: 7 am -7 pm). They had free access to standardized food pellets (purchased from Altromin, Lage, Germany) and tap water. All animal studies were performed in accordance with the national animal protection rules and permitted by the local governmental authority (Regierung von Mittelfranken, Germany). The sensitization strategy was adapted from previously described mouse model [29]. Briefly, all Balb/c mice were actively immunized and boosted by intraperitoneal injections with 200 µl of a solution containing 100 µg ovalbumin (grade V, Sigma, Taufkirchen, Germany) adsorbed on 4 mg of an aqueous solution of aluminium hydroxide and magnesium hydroxide (Alum) (Perbio Science, Bonn, Germany) on days 1, 14 and 20. On day 26, the animals were used for experiments. Compounds, as suspension in 5% DMSO, were given intraperitoneally two hours prior to challenge once a day. Control animals (negative and positive) received only vehicle solution. Mice were challenged twice a day for 4 days by intranasal application of 50 µl of 2 µg/ µl ovalbumin solution in NaCl to provoke an influx of inflammatory cells into the airways. Negative control mice were sensitized intraperitoneally with OVA/Alum and were challenged intranasally with saline solution. Positive control animals were sensitized as mentioned above and challenged intranasally with ovalbumin. On day 30 animals were sacrificed by putting them into a CO 2 rich atmosphere and a bronchoalveolar lavage (BAL) was performed by flushing the lungs and the airways six times with 0.5 ml Hank's balanced solution (Gibco, Karlsruhe, Germany) supplemented with Na-EDTA and HEPES (Sigma, Taufkirchen, Germany). The number of eosinophils as well as the total cell number from the pooled BAL samples of one animal was counted using a haemocytometer (Sysmex microcellcounter F-300, Norderstedt, Germany). BAL cells were spun onto glass slides using a cytospin centrifuge and stained with Diff-Quik (Dade-Behring, Marburg, Germany). The percentage and number of eosinophils were determined microscopically using standard cytological (morphological and staining) criteria by counting 400 cells/slide. Each group of animals treated with compounds was compared with saline challenged (negative control) and vehicle-treated ovalbumin challenged (positive control) groups. For RNA analysis cells recovered from broncheoalveolar lavage were sedimented by centrifugation and RNA was prepared from the cell pellet.

Histological examination of lungs
Lungs removed from the chest were instilled intratracheally with 0.8 ml 4% buffered formalin solution and immersed in this fixative for 48 hours. Tissues were embedded in paraffin and sections of 5 µm were cut. Slices were stained with May-Grünwald Giemsa for examining inflammation and eosinophilic infiltration and periodic acid-Schiffs stain was done for measuring mucus production under the light microscope. To determine the severity of peribronchial inflammation a semiquantitatively score described by Myou et al. [30] was determined. Briefly, this score describes followingcategories: 0, no inflammation; 1, few inflammatory cells; 2, a ring of inflammatorycells 1 cell layer deep; 3, a ring of inflammatory cells 2-4 cells deep; 4, a ring of inflammatory cells of >4 cells deep. The numerical scores for the abundance of PAS-positive mucus-containingcell in each airway were adapted from the same reference as follows: 0, <0.5%PASpositive cells; 1, 5-25%; 2, 25-50%; 3, 50-75%;4, >75%.

Data analysis
Data are expressed as means ± s.e.mean. Significant differences were statistically analyzed by the unpaired Student's t-test and by ANOVA. IC 50 and ED 50 values were calculated using the computer program PRISM 3.0 (GraphPad Software Inc., San Diego, CA, U.S.A).

Influence of coreceptors on TCR induced cytokine expression in CD4 + T cells
The stimulation of human CD4 + T cells by beads coated with α-CD3 antibodies and various ligands for coreceptors was compared. For our studies, we developed a realtime RT-PCR method for accurate quantization of T cell cytokines. Cells were stimulated with beads coated with α-CD3 alone, α-CD3 plus α-CD28, α-CD3 plus B7-2 Fc fusion protein, α-CD3 plus B7-H2 Fc fusion protein, α-CD3 plus PD-1 ligand Fc fusion protein (PD-L1 or B7-H1) or α-CD3 plus B7-H3 Fc fusion protein. Naked beads alone were included as a negative control and did not activate CD4 + T cells. IL-5 mRNA was not induced by any stimulation condition. In contrast, IL-2, IL-4, IL-10, IL-13 and IFNγ mRNA expression was already stimulated by α-CD3 activation alone and maximally stimulated by α-CD3/α-CD28 activation, which was included as a positive control ( Figure 1A). B7-2 and B7-H2 binding to their respective counterreceptors costimulated cytokine gene expression to elevated levels compared to α-CD3 activation alone. Costimulation strength by ICOS ligation was comparable to CD28 costimulation. In contrast, B7-H1 and B7-H3 mediated costimulation did not augment α-CD3 induced cytokine mRNA levels ( Figure 1A). To substantiate these results, cytokine protein concentration of the prototypic cytokines IL-2, IL-4 and IFN was determined in stimulated CD4 + T cell supernatants ( Figure 1B). The pattern was similar to the induction on the mRNA level. B7-2 and B7-H2 costimulation produced more cytokines than α-CD3 stimulation alone. B7-2 was more effective in costimulation that B7-H2 ( Figure 1B). Similarly, B7-H1 and B7-H3 mediated costimulation did not augment α-CD3 induced cytokine protein levels (data not shown).

Time course of cytokine induction in CD4 + T cells
Since CD28 and ICOS were the only stimulating coreceptors, they were chosen to study the time course of cytokine mRNA induction. As can be seen from figure 2A, α-CD3/ B7-2 stimulation induced cytokine mRNA levels rapidly till two hours. Whereas IL-13 mRNA levels started to decline from that time point on, other cytokine mRNA levels still increased. Similar observations were made after α-CD3/B7-H2 stimulation ( Figure 2B). Most remarkable, IL-13 and IFNγ were more strongly induced as compared to α-CD3/B7-2 stimulation, whereas IL-2 was much weaker induced. The induction of IL-10 was weak for both costimulatory receptors.

Activation of MAPKs by costimulatory receptors
We speculated that differential activation of MAPK family members by CD28 and ICOS may contribute to their different cytokine production patterns. To this end, we investigated the activation of the MAPK family members as downstream signaling molecules. T cell receptor and B7-2 ( Figure 3A) or B7-H2 ( Figure 3B) cross-linking elicited the phosphorylation of ERK and p38 indicating the up-regulation of kinase activity. The activation reached a maximum 15 min after B7-2 costimulation and 2 min after B7-H2 costimulation for both kinases. It persisted over a period of two hours. Since we could not detect JNK by western blot, we analyzed JNK activation by a recently available ELISA method. A cell-based ELISA method was used to determine the JNK phosphorylation relative to the total JNK protein found in CD4 + T cells. Stimulation with α-CD3/α-CD28 and α-CD3/B7-H2 for different time points was assessed to induce the JNK pathway. No effect could be observed in cell costimulated with α-CD3/α-CD28. In CD4 + T cells costimulated with α-CD3/B7-H2 an increase in the ratio of phosphorylated JNK to total JNK was observed ( Figure 3C).

Effect of MAPK inhibitors on costimulated CD4 + T cells
These results indicated a role of MAPKs in the signaling cascade precipitated from the costimulatory receptors CD28 and ICOS leading to cytokine expression. To elucidate which MAPK is important for cytokine induction, we first analyzed whether selective MAPK inhibitors were able to inhibit α-CD3/B7-2 induced gene expression. To this end, CD4 + T cells were preincubated with three selective MAPK inhibitors: SP600125 (selective inhibitor of c-Jun N-terminal kinase (JNK)), U0126 (selective inhibitor of MEK-1 and MEK2) and SB203580 (selective inhibitor of p38 MAPK). These compounds were chosen based on their pharmacological profile (high potency and specificity) for these kinases [31][32][33]. Since the cytokine induction behaved similar on the protein and mRNA level (see above), only mRNA was taken into consideration for determination of IC 50 values for inhibition of cytokines. The JNK inhibitor did not affect induction of any cytokine analyzed. The p38 inhibitor inhibited IL-2, IL-13 and IFNγ with a similar IC 50 of 3-7 µM, whereas IL-4 gene expression was not affected ( Table 1). The MEK inhibitor U0126 inhibited IL-2 and IFNγ gene expression with IC 50 of about 1 µM. IL-4 gene expression was not affected, whereas IL-13 was potently inhibited with an IC 50 of 0.18 µM (Table 1). Since ICOS costimulation also induced the secretion of various cytokines and activated MAPKs, we further analyzed whether this induction is influenced by different pharmacological inhibitors of MAPKs in the same manner as for α-CD3/B7-2 activation. The selective γ Induction of cytokines in CD4+ T cells by different costimulatory receptors Figure 1 Induction of cytokines in CD4+ T cells by different costimulatory receptors. Human CD4 + T cells were stimulated with various stimuli. (A) Cytokine mRNA level were determined after eight hours stimulation using real-time RT-PCR. mRNA level were normalized to β-actin and CD3 stimulated cells were set to 1. Each column represents mean ± s.e.mean of three different donors. (B) Cytokine protein level was determined in the supernatant by ELISA after twenty-four hours of stimulation. Each column represents mean ± s.e.mean of three different volunteers. *P < 0.05 (versus CD3 stimulated samples). Figure 2 Time course of cytokine induction by costimulatory receptors. Human CD4 + T cells were stimulated by α-CD3/B7-2 (A) and α-CD3/B7-H2 (B) respectively and harvested after different time points. Cytokine mRNA level were determined using real-time RT-PCR. Levels were normalized to β-actin and unstimulated cells were set to 1. Each point and bar represents mean ± s.e.mean of duplicates measurements of one donor. Similar results were obtained from three independent donors.  Table  1). The ERK inhibitor U0126 inhibited IL-2, IL-13 and IFNγ dose dependently at similar concentrations of 0.1 to 0.4 µM whereas IL-4 was inhibited only at ten-fold higher doses.

Effects of MAPK inhibitors in vivo
The finding that the MAPKs inhibitors very effectively inhibited several cytokines in CD4 + T cells costimulated through CD28 and ICOS, prompted us to investigate whether these selective MAPKs inhibitors may be active in an animal model of allergic asthma. BALB/c mice were actively sensitized to ovalbumin and challenged by intranasal application of ovalbumin solution. Different doses of the three selective MAPKs inhibitors SP600125, U0126, SB203580 were given intraperitoneally 2 hours before challenge. After 4 days of challenge mice were killed and bronchoalveolar lavage (BAL) was performed. Antigen challenge caused a strong eosinophilic inflammation of the lung and this was reflected in the cellular composition of the lung lavage fluid. Compared with saline challenged animals, the allergen challenge in sensitized animals led to an increase in all leukocyte populations especially of eosinophils in the lavage fluid ( Figure 4). On average, the total amount of leucocytes in the BAL of the positive control animals was 2.26 × 10 6 cells /ml (n = 7) and eosinophils accounted for 60.16 % of the total cell count, whereas in the saline challenged animals 0.16 × 10 6 cells /ml (n = 7) could be detected, which contained no granulocytes or lymphocytes. The treatment of the animals with SB203580 hardly affected leukocyte and eosinophils numbers in the lavage fluid, even at very high doses (20 mg/kg) ( Figure 4A). In contrast, the administration of U0126 and SP600125 inhibited recruitment of the total cell number in the bronchoalveolar lavage dose dependently with an ED 50 value of 2.5 mg/kg and 9 mg/kg respectively. Absolute eosinophil and lymphocyte numbers were also dose dependently inhibited by the ERK and JNK inhibitors, whereas neutrophil numbers were not affected. There were minor but not significant relative changes in the different cell populations. The cytokine protein levels in BAL fluid were below the detection limit of the ELISA kits used. Therefore cytokine mRNA levels were determined from BAL cells. SB203580 lacked also any effect on the cytokine mRNA induced in BAL cells ( Figure 5A-C). In contrast, SP600125 and U0126 dose dependently inhibited IL-5 mRNA induced in BAL cells (Fig. 5D, G). Both compounds inhibited also IL-13 and IFNγmRNA at higher doses (Fig. 5 E, F, H, I).

Effects of MAPK inhibitors on serum OVA-specific IgE levels
We next determined whether MAPK inhibitors could modify an ongoing OVA-specificTh2 response in vivo by analyzing OVA-specific IgE levels. Serum was collected24 h after the last OVA challenge. Levels of OVA-specfic IgE, was determined using ELISA. Substantial elevation OVAspecific IgE was observed in serum from OVA-sensitized and -challengedmice as compared with untreated mice (Fig. 6). U0126 and SP600125 significantly lowered total  1.17 ± 0.97 no effect* 0.18 ± 0.14 0.72 ± 0.39 SB 203580

Effects of MAPK inhibitors on lung inflammation in vivo
On separate experiments, lung tissue was collected 24 hours after the last challenge. Ovalbumin challenge induced marked infiltration of inflammatory cells into the peribronchial and perivasular tissue as compared with saline challenge (Figure 7 A, B). The majority of the infiltrated inflammatory cells were eosinophils as detected in the May-Günwald Giemsa stain (data not shown). MAPK inhibitors had different effects on peribronchial inflammation. The inflammatory score (calculated as described in Methods) decreased significantly after administration of 20 mg/kg ERK or JNK inhibitor, whereas inhibition of p38 had no influence on peribronchial inflammation ( Figure 7G). The allergen challenge induced mucus secretion was detected with periodic acid-Schiff stain counterstained with hematoxylin ( Figure 7A, B). The percentage of PAS-positive mucus-containing epithelial cells decreased as an effect of ERK or JNK inhibition ( Figure  7D, E, G). The p38 inhibitor had no effect on mucus production ( Figure 7C, G).

Effects of combined administration of MAPK inhibitors
Since the three MAPKs are part of different signaling cascades with different upstream and downstream mediators, we asked whether the combination of two MAPK inhibitors may have synergistic effects on the induction of cytokines in CD4+T cells. The simultaneous addition of SB203580 (5 µM) and SP600125 (3 µM) did not cause any synergistic effect. Unexpectedly, the simultaneous Effects of MAPK inhibitors on BAL cytokine levels Figure 5 Effects of MAPK inhibitors on BAL cytokine levels. Cells were recovered from bronchoalveolar lavage by centrifugation and RNA was prepared from the cell pellet. Cytokine mRNA level were determined using real-time RT-PCR and were normalized to β-actin. Each column represents mean ± s.e.mean. n = 5 mice per group. n.d. -not detectable. *p < 0.05 (versus OVA challenged positive control), + p < 0.05 (negative versus positive control) administration of increasing concentrations of U0126 (0.01 µM to 30 µM) and SB 203580 to α-CD3/α-B7-2 ( Figure 8A, B) and α-CD3/B7-H2 ( Figure 8C and 8D) stimulated CD4 + T cells led to a shift to the right of the dose-response curve for U0126 compared to the administration of U 0126 alone. The dose for SB203580 was 10 µM for ICOS costimulation and 1 µM for CD28 costimulation, respectively. This was reflected by an increase in the corresponding IC 50 values (Table 2). In contrast, the addition of SP600125 (0.3 µM for ICOS costimulation and 1.5 µM for CD28 costimulation) did not change the dose response curve for U0126 (Figure 8). Since we observed for the combination SB203560 and U0126 only unforeseen effects in vitro, we analyzed this combination in vivo. The simultaneous treatment of the animals with 5 mg/kg SB203580 and 5 mg/kg U0126 (Figure 9) diminished the inhibition of late phase eosinophilia by the solely administration of the ERK inhibitor. There were minor and not significant change in the relative numbers of the populations counted in the BAL. This effect could also be observed measuring cytokine mRNA levels in the BAL cells. Treatment of the animals with SB203580 antagonized the inhibiting effect of U0126 on IL-5, IL-13 and IFNγ mRNA level ( Figure 10). The effect of the combined administration of ERK inhibitor and p38 inhibitor on lung histology had the same effect as observed in vitro, as well as on the cellular composition of the BAL. The p38 inhibitor abolished the effect of ERK inhibition when administered simultaneously compared to administration of ERK inhibitor alone ( Figure 7F, G). Furthermore, OVAspecific IgE Level in the serum of mice treated with the combination of the ERK and p38 inhibitor were higher than in mice treated with the ERK inhibitor alone ( Figure  6).

Discussion
Costimulation is essential for the full activation of CD4 + T cells. In this study, we analyzed the induction and regulation of Th 1 and Th 2 cytokines in CD4 + T cells upon engagement of different costimulatory receptors. Beads with defined combinations of surface receptor stimulating antibodies and costimulatory receptor ligands were applied to induce cytokine synthesis. Ligation of two costimulatory receptors, CD28 and ICOS, augmented TCR activation and this in part is due to MAPK activation. The effects of pharmacological inhibitors of three different MAPKs on cytokines induced by α-CD3/B7-2 or α-CD3/ B7-2 were investigated. Using these compounds, we were able to elucidate different MAPK signaling pathways leading to cytokines synthesis in dependence on the costimulatory receptor engaged. We tested these compounds in an animal model of asthma in which the ERK inhibitor U0126 and the JNK inhibitor SP600125 were able to reduce the influx of eosinophils into the lungs of sensitized and challenged mice. We found no synergistic effect of any combination of these MAPK inhibitors. Unexpectedly, SB203580 antagonized the in vitro and in vivo action of U0126.
For optimal activation, CD4 + T cells require specific antigen recognition by the TCR and additional signals (collectively called costimulatory signals), delivered by the same antigen-presenting cell [34]. In the absence of costimulation, lymphocytes fail to respond effectively and are rendered anergic [35]. CD28 is the best-characterized costimulatory receptor and constitutively present on the surface of T cells (reviewed in [12]). CD28 is activated by B7-1 and B7-2 counter-receptors on antigen-presenting cells. Its signaling contributes to the overall strength of T cell activation [36]. However, new B7-and CD28-like molecules have recently been discovered and new pathways have been delineated that seem to be important for regulating the responses of previously activated T cells [37]. Some B7 homologues have unknown receptors, indicating that other immunoregulatory pathways remain to be described. We established an in vitro test system to analyze to role of each coreceptor. To this end, we conjugated beads with defined combinations of α-CD3 and B7 counterreceptors. In line with previous reports Effects of MAPK inhibitors on OVA-specific IgE antibody lev-els in the serum Figure 6 Effects of MAPK inhibitors on OVA-specific IgE antibody levels in the serum. Mouse serum was collected 24 h after the last OVA challenge. Samples were obtained from saline-(negative) or OVA-challenged mice in the absence (positive) or presence of MAPK inhibitors: SB203580 (20 mg/ kg), U0126 (20 mg/kg), SP600125 (20 mg/kg) or of the combination of U0126 (5 mg/kg) plus SB203580 (5 mg/kg). Serum titers for OVA-specific IgE antibodies were measured by ELISA as described in methods. Each column represents mean ± s.e.mean. n = 5 mice per group. *p < 0.05 (versus OVA challenged positive control), + p < 0.05 (negative versus positive control) described above we found that CD28 ligation augments the synthesis of all T cell derived cytokines. Similar results were obtained by ligating ICOS via its counter receptor ICOS-L (B7-H2). The effects of ICOS signals on T cell proliferation and IL-2 production were reported to be modest in comparison with those of CD28 [16]. However, ICOS costimulation is equivalent to that mediated by CD28 costimulation for the production of effector cytokines like IFNγ, IL-4, IL-10 and IL-13. IL-5 was not detected. It has been shown, that efficient in vitro IL-5 production needs elevated cAMP level in addition to TCR stimulation [38]. There was no difference in the strength of induction of Th2 compared with Th1 cytokines for CD28 and ICOS costimulation. Conflicting data has been reported about the function of PD-1. The engagement of PD-1 by its specific ligands, B7-H1 (PD-L1) or B7-DC (PD-L2) was reported to inhibit T and B cell proliferation and cytokine production [39,40]. In our test system engagement of PD-1 by ligation with B7-H1 neither augmented nor inhibited cytokine synthesis. Peribronchial inflammation and the abundance of PAS-positive mucus-containing cells were determined by applying the scores as described in methods (G). The scores described in methods were applied to ten bronchi for each animal and mean and standard deviation were determined for all the animals in a group (n = 5). *P < 0.05 and **P < 0.01 compared with positive control group.

Histology of lung inflammation
Activation of MAPK family members by CD28 and ICOS may contribute to their costimulatory activity. To analyze whether different signaling cascades are precipitated by the different coreceptors, their ability to activate different MAPK pathways was determined. Since only CD28 and ICOS activation augmented cytokine synthesis we focused  our analyses on these receptors. We analyzed the signaling pathways of ERK1/2, JNK, and p38 MAP kinase, during the primary activation of human T-cells, costimulated with ICOS or CD28. ERK1/2 and p38 kinase were markedly activated by both CD28-and ICOS-mediated costimulation. In our cellular assay system the activation of ERK has a role in T-cell activation via CD28 and ICOS. The activation of ERK by CD28 is mediated by binding of SOS to the YMNM motif of the intracellular domain of CD28 [41]. The YMNM motif is not conserved in ICOS, the sequence being YMFM, and amino-acid substitutions in this motif results in a failure to associate with Grb2 [16,42]. Therefore it is currently unclear, how ICOS is able to activate ERK.

Effects of combined administration of MAPK inhibitors in vitro
Previous studies of p38 MAP kinase in human purified T cells [43] and in the CD4 + subset [7] clearly demonstrated the involvement of p38 MAP kinase in the cell activation through TCR and CD28 costimulation signal pathways.
However, little is known about this MAP kinase in ICOS costimulated T cells. In our cellular assay activation of p38 MAPK by ICOS was detected. This is in line with recent reports, in which ICOS ligation synergized with TCR signals for activation of the ERK and p38 MAP kinases [44,45].
While enhanced activityof JNK is an absolute necessity for regulation of IL-2 geneexpression in T cell lines [46], a defect in JNK signalingwas claimed to be involved in T cell differentiation, but notin T cell activation in vivo, i.e. in JNK-deficient animals [47]. This corresponds to our results with human T cells, where JNK was found not to be activated after CD28 costimulation. Conflicting reports appeared about the ability of ICOS to activate the JNK pathway. Parry et al. reported that only CD28 but not ICOS costimulation activated c-jun N-terminal kinase [48]. Arimura et al. reported that the cross-linking of ICOS induced much less phosphorylation of JNK than did the Figure 9 Effects of combined administration of MAPK inhibitors in vivo. Late phase eosinophilia was induced in mice pretreated either with 5 mg/kg U0126 and 5 mg/kg SB 203580 or with the combination thereof. BAL was performed and total cell number and differential leukocyte count was determined. OVA challenged vehicle-treated animals were taken as positive control. Each column represents mean ± s.e.mean. n = 5 mice per group. *p < 0.05 (versus OVA challenged vehicle control), + p < 0.05 (versus U0126 alone).

Effects of combined administration of MAPK inhibitors in vivo
cross-linking of CD28 [49]. In our cellular assay system we found that ICOS activated the JNK pathway. However, this was only detected by using a sensitive ELISA based assay indicating a very low expression of JNK in primary human T cells. Alltogether, whereas p38 and ERK are consistently found to be activated after CD28 and ICOS costimulation, the activation of JNK appeared only after ICOS costimulation.
In order to elucidate which of the activated MAP kinases are important for cytokine induction after T cell activation and costimulation we employed specific inhibitors of the different MAPKs in our cellular assay system. These compounds were chosen based on their pharmacological profile (high potency and specificity) for these three kinases [31][32][33]. The p38 inhibitor SB203580 and the ERK inhibitor U0126 inhibited IL-2, IL-13 and IFNγ in CD28 costimulated T-cells. IL-4 induction, in contrast, was not affected by these inhibitors. In line with our observations, that JNK does not become activated by CD28 costimulation, the JNK inhibitor did not affect cytokine induction in CD28 costimulated T cells. Only one report analyzed the effect of a p38 inhibitor in primary human CD4 + T cells. There SB203580 blocked human CD4 T cell production of IL-4, IL-5, TNF-, and IFN-, but not IL-2, in response to CD3 and CD28 stimulation [7]. This difference is most likely due to different experimental protocols and the use of a different T cell population. While U0126 was never tested to inhibit human T cell cytokine production, in the first description of the JNK inhibitor SP600125, its effect on polarized Th1 and Th2 cells was described. There Th1 cytokines and some Th2 cytokines but not IL-4 was inhibited by this compound [32]. It is tempting to speculate, that the polarization of the T cells rendered them susceptible to JNK inhibition. Different observations were made in ICOS costimulated T cells. For human T cells, it has only been reported, that U0126 blocked the proliferation of T-cells and the transcription of IL-2 following costimulation by ICOS in human T cell [45]. We found, that the p38 inhibitor was not effective, but the JNK inhibitor inhibited IL-2, IL-13 and IFNγ. The ERK inhibitor U0126 inhibited all cytokines with similar efficiency as in CD28 costimulated T cells. Together these data indicates that in human T cells ICOS precipitates MAPK signaling cascades differentially compared to murine T cells. More interestingly, our inhibitor studies revealed that not every MAPK signaling cascade activated by costimulation is necessary for cytokine synthesis.
The sensitivity of CD4 + T cells to various MAPK inhibitors prompted us to analyze these compounds in an animal model of asthma. The p38 inhibitor SB203580 did not affect airway inflammation in our model. Conflicting data about the action of p38 inhibitors in asthma models have been reported in the literature. Similar to our results, one study found no effect of SB203580 in rats [50]. In contrast, Underwood et al. demonstrated anti-allergic activity using a novel second-generation p38 MAPK inhibitor [51]. Either increased specificity of this new inhibitor or better bioavailability of this compound may explain its effect in vivo. In contrast to the p38 inhibitor, the ERK as well as the JNK inhibitor dose dependently decreased allergen induced airway eosinophilia in our model. This anti-inflammatory effect of the ERK and the JNK inhibitor Effects of combined MAPK inhibitors on BAL cytokine levels Figure 10 Effects of combined MAPK inhibitors on BAL cytokine levels. Cells were recovered from bronchoalveolar lavage by centrifugation and RNA was prepared from the cell pellet. Cytokine mRNA level were determined using real-time RT-PCR and were normalized to β-actin. Each column represents mean ± s.e.mean. n = 5 mice per group. *p < 0.05 (versus OVA challenged vehicle control), + p < 0.05 (versus U0126 alone).
was also detected by measuring peribronchial inflammation and mucus production. Elevated serum IgE is a hallmark of a Th2 immune response. Our data showed that serum levels of OVA-specific IgE were also substantially reduced by U0126 and SP600125, whereas no significant inhibition was observed by SB203580. This extends our previous findings, where 10 mg/kg of U0126 were found to be effective in a rat asthma model [28]. During the preparation of the manuscript it was reported that 30 mg/ kg SP600125 inhibited airway eosinophilia in rats [52]. This supports our finding, that targeting JNK reduces allergic airway inflammation.
The inhibitory effect could also be observed by the reduction of cytokine mRNA level. However, the inhibition was not selective for Th2 cytokines. The induction of IFNγ in BAL cells was also inhibited by U0126 and SP600125. This is in line with our findings described above, that costimulation of T cells induces Th1 as well as Th2 cytokines and that MAPK inhibitors are also effective in inhibiting IFNγ. This may be beneficial for the antiinflammatory effect in the asthma model. It has been shown, that in IFNγ knockout mice less pronounced allergic symptoms such as antigen-specificIgE, eosinophilic infiltration, and airway hyperresponsiveness occur [53]. Therefore, it seems likely that endogenous IFNγ is necessaryfor optimal IgE production also during a secondary response.
The in vivo profile of MAPK inhibitors reflects the in vitro profile of ICOS costimulated T cells: ERK and JNK inhibitors are effective whereas the p38 inhibitor is not. In contrast, the inhibitory profile of CD28 costimulated T cells is different. There p38 is effective, while the JNK inhibitor is not. In line with this, it has been shown, that ICOS plays an important role in the Th2 effector responses in the lungs, in that inhibition of ICOS suppresses lung inflammation and Th2 cytokine production [54]. Similarly, ICOS-L-Fc augments lung inflammation and the production of cytokines [55]. Together these data indicate the importance of ICOS costimulation in the allergic lung response. Therefore, targeting signaling pathways precipitated by ICOS should dampen the allergic lung inflammation.
Since the three MAPKs are part of different signaling cascades with different upstream and downstream mediators, we asked whether the combination of two MAPK inhibitors may have synergistic effects on the induction of cytokines in CD4+T cells. Our in vitro studies did not reveal any such synergistic effect regardless of the combination used. However, we could observe that the simultaneous administration of increasing concentrations of U0126 and 1 µM SB203580 to α-CD3/α-CD28 and α-CD3/α-ICOS stimulated CD4+ T cells led to a right-shift of the dose-response curve for U0126 compared to the administration of U0126 alone. We analyzed this combination also in vivo. Whereas treatment of the animals with U0126 alone inhibited late phase eosinophilia markedly as in previous experiments the concomitant treatment with SB203580 diminished the effect of U0126 almost completely. To our knowledge no in vitro or in vivo studies reported the simultaneous addition of two MAPK inhibitors in T cell activation studies or asthma models. Currently, we investigate the mechanism for this antagonistic action.

Conclusion
Different MAPK signaling pathways are activated in T cells dependent on the costimulatory receptor engaged. The MAP kinase ERK is important for both stimuli. p38 MAPK activation is important for CD28 induced cytokine synthesis, whereas JNK is important for ICOS induced cytokine synthesis. The different regulation can be exploited to find new specifically targeted drugs aimed for diseases where different costimulatory molecules play an important pathophysiological role. We could demonstrate for the first time that inhibition of the JNK cascade is a therapeutic option for asthma. The specific JNK inhibitor SP 600125 reduced the influx of eosinophils in an animal model of asthma. The development of more specific MEK-ERK and JNK-targeted drugs would support this approach to treat T cell dominated diseases such as asthma.


extfieldsofstudy: Medicine Biology

text:
Disabled-2 (Dab2) Mediates Transforming Growth Factor (cid:1) (TGF (cid:1) )-stimulated Fibronectin Synthesis through TGF (cid:1) -activated Kinase 1 and Activation of the JNK Pathway*

The multifunctional cytokine transforming growth factor (cid:1) (TGF (cid:1) ) exerts many of its effects through its regulation of extracellular matrix components, including fibronectin (FN). Although expression of both TGF (cid:1) and FN are essential for embryonic development and wound healing in the adult, overexpression leads to ex-cessive deposition of extracellular matrix observed in many fibroproliferative disorders. We previously have demonstrated that TGF (cid:1) -stimulated FN induction requires activation of the c-Jun N-terminal kinase (JNK) pathway; however, the signaling molecules that link the TGF (cid:1) receptors to the JNK pathway remain unknown. We show here that the cytosolic adaptor protein dis-abled-2 (Dab2) directly stimulates JNK activity, whereas stable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells demonstrates that its expression is required for TGF (cid:1) -mediated FN induction. We demonstrate that TGF (cid:1) treatment stimulates the association of Dab2 with the mitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellular TAK1 levels by transient double-stranded RNA oligonucleotide transfection as well as overexpression of kinase-deficient TAK1 leads to abrogation of TGF (cid:1)

The multifunctional cytokine transforming growth factor ␤ (TGF␤) exerts many of its effects through its regulation of extracellular matrix components, including fibronectin (FN). Although expression of both TGF␤ and FN are essential for embryonic development and wound healing in the adult, overexpression leads to excessive deposition of extracellular matrix observed in many fibroproliferative disorders. We previously have demonstrated that TGF␤-stimulated FN induction requires activation of the c-Jun N-terminal kinase (JNK) pathway; however, the signaling molecules that link the TGF␤ receptors to the JNK pathway remain unknown. We show here that the cytosolic adaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereas stable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells demonstrates that its expression is required for TGF␤-mediated FN induction. We demonstrate that TGF␤ treatment stimulates the association of Dab2 with the mitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellular TAK1 levels by transient double-stranded RNA oligonucleotide transfection as well as overexpression of kinase-deficient TAK1 leads to abrogation of TGF␤-stimulated FN induction. Furthermore, cell migration, another JNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We, therefore, delineate a signaling pathway proceeding from the TGF␤ receptors to Dab2 and TAK1, leading to TGF␤-stimulated JNK activation, FN expression, and cell migration.
Fibronectin (FN), 1 a widely expressed component of the extracellular matrix (ECM), is a dimeric glycoprotein whose abil-ity to promote cell adhesion and migration plays a key role in signaling events that regulate cell growth and differentiation (1)(2)(3). Fibronectin expression is essential for embryogenesis and angiogenesis and modulates wound healing, host defense, and maintenance of tissue homeostasis in the adult (2,4). The cytokine transforming growth factor ␤ (TGF␤) controls cellular growth, proliferation, adhesion, and migration in part due to its ability to modulate expression of cell surface adhesion receptors and ECM protein expression, including FN (5,6).
TGF␤, the founding member of the TGF␤ superfamily, mediates its effects through two Ser/Thr kinase receptors, termed the TGF␤ type I (T␤RI) and II (T␤RII) receptors (7). TGF␤ ligand binding to T␤RII triggers recruitment of T␤RI, thereby forming an active receptor complex. Further propagation of TGF␤ signaling can then be initiated through Smad-dependent or Smad-independent pathways, such as the mitogen-activated protein kinase (MAPK) pathway (8,9). Signaling through the Smad family of proteins is triggered by phosphorylation of the receptor-activated Smads by T␤RI. In the case of Smad2 and Smad3 and the TGF␤-and activin-specific receptor-activated Smads, this phosphorylation can be facilitated by SARA (Smad anchor for receptor activation) (10) or Disabled-2 (Dab2) (11). After phosphorylation, receptor-activated Smads dimerize with the required co-Smad, Smad4, translocate to the nucleus, and activate gene transcription by either directly binding to DNA or through cooperation with other DNA binding transcription factors such as FAST-1 (12), FAST-2 (13), c-Jun (14,15), ATF-2 (16), and TFE3 (17).
Members of the MAPK family have also been shown to be activated after TGF␤ stimulation, including the extracellular signal-regulated kinase (18,19), c-Jun N-terminal kinase (JNK) (20,21), and the p38 kinase pathways (22). We previously have demonstrated that TGF␤-mediated induction of FN is mediated through a JNK-dependent, Smad-independent pathway (21). Consistent with this observation, mouse fibroblasts derived from Smad2-, Smad3-, and Smad4-deficient embryos retain the ability to induce FN synthesis after TGF␤ stimulation (23,24). Furthermore, efficient activation of the Smad pathway may require activation of JNK, in that fibroblasts that express antisense JNK oligonucleotides show decreased TGF␤-stimulated Smad2 phosphorylation (25), whereas JNK has been shown to directly phosphorylate Smad3, leading to its activation and nuclear accumulation (26).
Activation of JNK is triggered by dual phosphorylation of a TPY motif by the upstream MAPK kinases MKK7 and MKK4, whereas activation of these MAPK kinases can be mediated through multiple MAPK kinase kinases including members of the MAPK/extracellular signal-regulated kinase kinase (MEK) kinase (MEKK), mixed-lineage kinase, and apoptosis signalregulating kinase families, Tpl2, and TGF␤-activated kinase 1 (TAK1) (27). TAK1 was initially identified as a kinase whose activity was stimulated by TGF␤ and BMP4, a TGF␤ superfamily member, resulting in phosphorylation of MKK4 (28). TAK1 has subsequently been shown to be involved in interleukin 1-and Wnt-mediated signal transduction (29 -31) and can activate the p38 pathway by phosphorylation of MKK3 and MKK6 (32). Activation of TAK1 requires interaction with TAB1 (TAK1-binding protein) (33), which also binds to the X-linked inhibitor of apoptosis (34). TAB1 has been shown to associate with the type I bone-morphogenetic protein receptor when the X-linked inhibitor of apoptosis is co-expressed, suggesting a possible link of the receptor complex to TAK1. However, this association may involve another interacting protein since yeast two-hybrid analysis failed to demonstrate a direct interaction between X-linked inhibitor of apoptosis and type I BMP receptor (34).
Dab2, a member of the Disabled gene family, is a widely expressed adaptor molecule shown to be involved in several receptor-mediated signaling pathways (11,35,36). Disabled family members possess a highly conserved N-terminal phosphotyrosine-interacting/phosphotyrosine binding domain (PID/ PTB), recently renamed the DAB homology domain (37), linked to divergent C-terminal sequences. Dab2 directly binds to several members of the lipoprotein receptor family through its PTB domain (35,36,38), whereas its association with clathrin, the clathrin adaptor protein AP2, and myosin VI, mediated by sequences in the linker and C-terminal regions of Dab2, facilitates clathrin-coated pit assembly and receptor-mediated endocytosis (35,39,40). We have recently demonstrated that Dab2 serves as an adaptor molecule to link the TGF␤ receptors to activation of the Smad pathway (11). Expression of Dab2 restored Smad-dependent responses to a TGF␤-signaling-deficient mutant cell line. In addition, Dab2 was found to augment TGF␤-stimulated FN induction, a JNK-dependent and Smadindependent response (11). We, therefore, wished to explore the possibility that Dab2 could directly mediate TGF␤-stimulated JNK activity, leading to induction of FN.
We report here that stable small interfering RNA (siRNA)mediated silencing of Dab2 in NIH3T3 mouse fibroblasts leads to decreased TGF␤-stimulated FN induction, concomitant with decreased TGF␤-mediated JNK activation. Dab2 is shown to directly stimulate JNK activity, which is blocked by expression of a kinase-deficient form of TAK1, TAKKW. We also report that abrogation of TAK1 expression or TAKKW overexpression leads to decreased TGF␤-stimulated FN protein and mRNA induction, respectively. At a biological level we provide evidence suggesting that Dab2 is required for TGF␤-stimulated cellular migration, a JNK-dependent response. Dab2, thus, provides a link between the activated TGF␤ receptors and TAK1, leading to activation of the JNK-signaling pathway.
Plasmid Construction-Construction of the FLAG-tagged human Dab2 in the RK5 plasmid has been described previously (11). The various domain constructs of human Dab2 were generated by standard PCR methods using the full-length construct as template and inserted into the RK5 vector containing a FLAG epitope. Sequences used for PCR amplification are available upon request. For construction of the stable siDab2 construct, forward and complementary reverse primers corresponding to mouse p96 (5Ј-CAAATGGAGTCACCTCCTG-3Ј) flanked by AA at the 5Ј-end and TT at the 3Ј-end were synthesized, annealed, and ligated into the pSUPER expression vector as described (41). The FLAG-tagged JNK construct was provided by Roger Davis (42), and the HA-tagged wild-type TAK1, kinase-deficient TAK1 (TAKK63W), constitutively active TAK1 (TAK1dN) (29), and Myctagged TAB1 (34) were provided by E. Nishida. The pPur vector was purchased from Clontech.
Cell Culture and Generation of Cell Lines-NIH3T3 and COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum. A10 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. To generate cells that stably express the siDab2 construct, NIH3T3 and A10 cells in 100-mm-diameter plates were transfected with the siDab2 construct (10 g) and pPur (2 g) utilizing FuGENE 6 (Roche Diagnostics) or Lipofectamine PLUS (Invitrogen), respectively, as per the manufacturer's instructions. Stable transfectants were selected in serumcontaining Dulbecco's modified Eagle's medium supplemented with 1 g/ml puromycin and maintained in the same media either as a pool (A10) or individual clones (NIH3T3). Expression levels of Dab2 were assessed by Western analysis utilizing the monoclonal ␣-p96 antibody. To generate the 3T3-TAKKW cell line, NIH3T3 cells were co-transfected with TAKKW (10 g) and pPur (2 g) constructs using FuGENE 6 and selected and maintained as a pool in Dulbecco's modified Eagle's medium containing 10% newborn calf serum and 1 g/ml puromycin. Expression of the TAKKW construct was verified by Western analysis utilizing a monoclonal ␣-HA antibody.
Preparation of Cell Lysates, Immunoprecipitation, and Protein Kinase Assays-For immunoprecipitation and Western blot analysis, cells were lysed in buffer D (20 mM Tris, pH 7.5, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM ␤-glycerophosphate, 1 mM Na 3 V0 4 , and Complete EDTA-free protease inhibitor mixture; Roche Diagnostics), and immunoprecipitation was carried out as previously described (21). For JNK assays in COS7 cells, transient transfection of the indicated constructs was performed utilizing FuGENE 6 (Roche Diagnostics) according to the manufacturer's protocols. After 48 h cells were lysed in buffer D. Equal amounts of cellular protein (250 g) were immunoprecipitated with ␣-FLAG antibody and captured on protein G-Sepharose beads. The beads were subsequently washed twice with buffer D followed by 1 wash in kinase assay buffer (25 mM HEPES, pH 7.5, 25 mM ␤-glycerophosphate, 25 mM MgCl 2 , 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , and Complete EDTA-free protease inhibitor mixture; Roche Diagnostics). Kinase reactions were performed in 30 l of kinase buffer containing 1 g GST-c-Jun (Stratagene), 50 M ATP, and 2 Ci of [␥-32 P]ATP. Reactions were incubated at 30°C for 20 min and terminated by the addition of an equal volume of 2ϫ Laemmli sample buffer. Samples were resolved on 10% SDS/PAGE gels, dried, and visualized by autoradiography. The JNK assay in NIH3T3 and 3T3-siDab2 clones was performed as described previously (32) after stimulation with 5 ng/ml TGF␤ for various times. Reaction products were resolved on 10% SDS/PAGE gels, transferred to Immobilon membranes, and subjected to Western blot analysis with ␣-phospho-c-Jun (Ser-63) antibody II.
Fibronectin Protein Assay-TGF␤-stimulated FN induction was determined as previously described (21), with the exception that in some assays gelatin-agarose beads (Sigma) were used to precipitate 35 Slabeled FN from the media of labeled cells. Samples were resolved on 6% SDS/PAGE gels, subjected to fluorography using Enhance (Amersham Biosciences), and visualized by autoradiography. The inhibitors SP600125 (10 M and 50 M), SB203580 (10 M), or PD98059 (10 M) were added simultaneously with TGF␤ (2 ng/ml) at the initiation and maintained throughout the FN assay.
Transient siRNA-mediated Silencing of TAK1-The negative control, TAK1 pool, and individual double-stranded RNA oligos to TAK1 were purchased from Dharmacon RNA Technologies (SMARTpool Plus, Q-004500-00-09) and resuspended in reagents provided by the manufacturer. NIH3T3 cells were transfected in 6-well plates (2 ϫ 10 5 cells/ well) using Oligofectamine reagent (Invitrogen) with 200 nM concentrations each of double-stranded RNA oligo per manufacturer's instructions. After 48 h of siRNA treatment, the FN assay was initiated and performed as described above. FN protein synthesis was analyzed from the media, whereas cell lysates from the same wells were analyzed for TAK1 expression by Western analysis.
Northern Blot Analysis-Total RNA was isolated from NIH3T3, 3T3-siDab2 clone 8, and 3T3-TAKKW cells after treatment with 5 ng/ml TGF␤ for the various times indicated using TRIzol reagent (Invitrogen) per the manufacturer's instructions. Total RNA (40 g) of each sample was resolved in a 1.2% formaldehyde/agarose gel, transferred to nitro-cellulose, and subjected to Northern analysis utilizing probes to FN (45) and cyclophilin (1B15) (43) as previously described (21).
Wound Closure Assay-For the wound closure assay, confluent monolayer cells in 60-mm plates were wounded by manual scraping with a pipette tip. Plates were washed several times to remove non-adherent cells and placed in complete media with or without TGF␤ (5 ng/ml). Assessment of cell migration into the wounded area was performed by microscopy after 24 h. Cell migration of NIH3T3 and 3T3-siDab2 cells was quantitated utilizing the QCM TM 24-well colorimetric cell migration assay (Chemicon International) as suggested by the manufacturer. Briefly, 2 ϫ 10 5 cells resuspended in serum-free media were added to the upper chamber and allowed to migrate through a membrane insert with an 8-m pore size for 4 h toward 10% fetal bovine serum-containing media in the absence or presence of 5 ng/ml TGF␤. After the incubation period, the cells on the upper surface of the membrane were removed, cells that had migrated through the membrane were stained, and 5 random fields were counted by microscopy at 250ϫ magnification.

Dab2 Expression Is Required for TGF␤-stimulated FN Induction-
We have previously shown that TGF␤-stimulated FN induction is greatly abrogated in a TGF␤-signaling-deficient cell line, derived from the human HT1080 fibrosarcoma cell line, which expresses a mutant form of Dab2 (11,21). We, therefore, wished to test whether Dab2 expression is required for TGF␤-mediated FN induction in two different cell types that respond to TGF␤ stimulation, namely fibroblast and smooth muscle cells. To ablate the expression of Dab2, we chose to use stable expression of a siRNA specifically targeted to decrease expression of the p96 splice-form of Dab2, which we previously have shown to restore TGF␤ signaling to a mutant cell line (11). We have previously characterized this siRNA-Dab2 vector in several cell lines with regard to its specificity for the p96 form of Dab2 (71). Stable introduction of the siDab2 construct into mouse fibroblast NIH3T3 and rat A10 aortic smooth muscle cells was performed. As shown by Western analysis in Fig. 1A, the siDab2 construct was able to decrease the p96, but not p67, form of Dab2 expression in individual clones of NIH3T3 cells (left panel) and in a pool of A10 (right panel) cells to varying extents. Analysis of these clones for induction of FN synthesis after TGF␤ stimulation reveals a dose dependence of Dab2 expression for both basal and TGF␤mediated FN induction, in that clones that express the lowest levels of Dab2 (i.e. clone 8 and 9) also demonstrate the lowest basal and TGF␤-stimulated FN protein induction (Fig. 1B). FN protein expression was also assessed in wild-type and the siDab2-expressing A10 cells after stimulation with TGF␤ for 4 and 24 h. Similarly to NIH3T3 cells, TGF␤-mediated FN induction was markedly attenuated in A10 cells with decreased Dab2 expression (Fig. 1C). These results, thus, demonstrate that Dab2 expression is required for TGF␤-mediated FN induction in two different cell types that can be physiologically stimulated to express FN after TGF␤ treatment, which are fibroblast and smooth muscle cells.
Dab2 Directly Stimulates JNK Activity-We have previously shown that TGF␤ stimulation of human HT1080 fibrosarcoma cells leads to activation of the JNK pathway and that TGF␤stimulated induction of FN could be blocked by overexpression of dominant-negative JNK and MKK4 (21). We, therefore, wished to assess whether Dab2 could directly activate the JNK pathway, since we observed that TGF␤-stimulated FN induction was dependent on expression of Dab2. In addition, we wished to assess the contribution of the individual domains of Dab2 to JNK activation, because we have previously demonstrated that restoration of TGF␤ signaling to a TGF␤-signaling-deficient cell line requires both the N-terminal PTB and C-terminal proline-rich domains of Dab2 (11). To assess this, COS7 cells were transiently transfected with JNK along with full-length Dab2 or various constructs containing the different domains of Dab2 (Fig. 2, A and C). As shown in Fig. 2B, expression of full-length Dab2 is capable of efficiently stimulating JNK activity that was assessed by the ability of JNK to phosphorylate GST-c-Jun in an in vitro kinase assay. Furthermore, the construct bearing only the C-terminal portion of Dab2, designated #323 consisting of amino acids 323-770, stimulates JNK activity to the same extent as full-length (Fig.  2B). Expression of a construct bearing the proline-rich domain of Dab2, #534, consisting of amino acids 534 -770, however, is not sufficient to stimulate JNK activity even though it is expressed at the same level as full-length Dab2 (Fig. 2C). These results demonstrate that Dab2 can directly mediate JNK activation and that this activity can be localized to the C-terminal domain of Dab2.
TGF␤-stimulated FN Induction Requires JNK Activity-The results presented above demonstrate the requirement of Dab2 in TGF␤-stimulated FN induction and suggest that Dab2 can modulate JNK activity. We next wished to assess whether TGF␤-stimulated JNK activation is affected by the level of Dab2 expression. To determine this, parental NIH3T3 and the siDab2 expressing clone #9 were stimulated with TGF␤ for various times followed by assessment of JNK activation using GST-c-Jun as a substrate. As depicted in Fig. 3A, JNK activation in response to TGF␤ is observed within 5 min in the Dab2 Mediates TGF␤-stimulated JNK Activation NIH3T3 cells, with maximal activation occurring at 2 h, and a secondary wave of activity persisting through 24 h. In contrast, JNK activation in response to TGF␤ is greatly attenuated in cells expressing the siDab2 construct (3T3-siDab2#9). This lack of JNK activation in cells deficient in Dab2 expression cannot be attributed to a decrease in expression of the p54 and p46 forms of JNK, which are equivalent in both the wild-type and siDab2-expressing cell lines (Fig. 3B).
Because TGF␤ has been shown to activate all three branches of the MAPK family in various cell lines, we wished to confirm the requirement for TGF␤-mediated JNK activation in the induction of FN synthesis. To address this, we utilized pharmacological inhibitors that have been shown to specifically inhibit JNK (SP600125 (44)), p38 (SB203580 (45)), or MEK1 (PD98059 (46)), the upstream activator of extracellular signalregulated kinase, in FN assays. As shown in Fig. 3C, treatment of NIH3T3 cells with the specific JNK inhibitor SP600125 significantly abrogates TGF␤-stimulated FN induction in a dose-dependent manner. In contrast, neither the p38-specific nor the MEK1-specific inhibitor had any significant effect on TGF␤-mediated FN induction (Fig. 3C). Similar results were obtained in the A10 cell line (data not shown). These results demonstrate that Dab2 expression is required for TGF␤-mediated JNK activation that in turn modulates induction of FN expression.
TAK1 Associates with Dab2 and Mediates Dab2-stimulated JNK Activation-We previously have demonstrated the involvement of MKK4 as an upstream activator of JNK in modulation of TGF␤-stimulated FN induction (21); however, the upstream MAPK kinase kinase that activates MKK4 has not been determined. TAK1 is a potential candidate to mediate activation of MKK4, as TAK1 is activated by TGF␤ treatment and has been shown to phosphorylate MKK4 both in vitro and in vivo (29,47). To investigate if TAK1 is involved in mediating JNK activation stimulated by Dab2, we transiently transfected COS7 cells with JNK and either wild-type (WT) or kinasedeficient (KW) forms of TAK1 in the presence or absence of Dab2 and assessed for activation of the JNK pathway. As seen in Fig. 4A, transfection of Dab2 leads to JNK activation as monitored by phosphorylation of GST-c-Jun in an in vitro JNK assay. Expression of wild-type TAK1 by itself is seen to lead to efficient JNK activation, which is further increased by the co-expression of Dab2. In contrast, transfection of the kinasedeficient form of TAK1, TAKKW, eliminates activation of JNK stimulated by Dab2 (Fig. 4A). In addition, we observe that Dab2 exists as a doublet when co-transfected with JNK, which collapses to a single band when TAKKW is co-expressed (Fig.  4B), suggesting that Dab2 itself may be phosphorylated by JNK.
To extend the observation that TAK1 is able to modulate Dab2-mediated JNK activation, we asked whether TAK1 and Dab2 associate in vivo. NIH3T3 cells were stimulated with TGF␤ for the indicated times followed by immunoprecipitation with ␣-TAK1 and subsequent Western analysis with ␣-Dab2 antibody. As shown in Fig. 4C, TGF␤ treatment results in a time-dependent association of endogenous Dab2 and TAK1. These results, thus, demonstrate a time-dependent TGF␤-stimulated association between endogenous TAK1 and Dab2 and suggest that this interaction may mediate TGF␤-stimulated JNK activation (Fig. 3).
Given the requirement of Dab2 expression for TGF␤-stimulated FN induction and the ability of TAKKW to block Dab2mediated JNK activation, we hypothesized that TAK1 may also be required for TGF␤ stimulation of FN synthesis. To test this hypothesis, we transiently transfected NIH3T3 cells with double-stranded RNA oligonucleotides targeted to specifically ablate TAK1 expression. Western analysis of the cell lysates reveals a specific diminution of TAK1 protein expression, whereas expression of Hsp90 remains constant (Fig. 5A, left  panel). Analysis of these cells for their ability to induce FN synthesis after TGF␤ stimulation reveals that TAK1 oligonucleotides, but not a negative control pool, could attenuate basal FN as well as TGF␤-stimulated FN expression (Fig. 5A, right  panel). To further confirm these observations, we established a stable pool of NIH3T3 cells that overexpresses the kinasedeficient form of TAK1, TAKKW (Fig. 5B). As analyzed by Western blot (Fig. 5B), this pool of cells (3T3-TAKKW) expresses similar levels of Dab2 to the wild-type NIH3T3 cells but expresses very high levels of the mutant form of TAK1 (Fig.  5B). The induction of FN mRNA was then assessed in NIH3T3, 3T3-siDab2#8, and 3T3-TAKKW cells after TGF␤ stimulation for 0, 8, and 24 h by Northern analysis. Consistent with previous results, we observe that TGF␤ induces FN mRNA in a time-dependent fashion in NIH3T3 cells (Fig. 5C). In contrast, both basal and TGF␤-stimulated FN mRNA levels are greatly abrogated in 3T3-siDab2#8 and 3T3TAKKW cells (Fig. 5C). These data suggest that, similarly to Dab2, TAK1 is required for TGF␤ stimulation of FN synthesis.
Expression of Dab2 Alters Cell Migration-By mediating the phosphorylation of c-Jun, JNK plays an essential role in activation of AP-1 complexes leading to gene induction; however, a key role of JNK in controlling cell migration, independent of FIG. 3. Decreased Dab2 expression leads to attenuated JNK activation by TGF␤. A, 3T3-siDab2 clones exhibit altered TGF␤-stimulated JNK activation. NIH3T3 cells and 3T3-siDab2 clone #9 were treated with 5 ng/ml TGF␤ for the various times indicated. Lysates were prepared, and JNK kinase assays were performed utilizing GST-c-Jun as substrate. Reaction products were resolved on 10% SDS/PAGE gels, transferred to Immobilon membranes, and subjected to Western analysis utilizing a phospho-specific antibody to c-Jun (Ser-63). The assay was performed three times with similar results. Shown is a representative experiment. B, the p54 and p46 isoforms of JNK are expressed equally in NIH3T3 cells and 3T3-siDab2 clone #9 as determined by immunoblotting with ␣-JNK antibody. C, JNK activity is required for stimulation of FN synthesis by TGF␤. NIH3T3 cells were untreated or treated with SP600125 (SP, 10 and 50 M), SB203580 (SB, 10 M), or PD98059 (PD, 10 M) in the absence or presence of TGF␤. The ability to induce FN after stimulation with TGF␤ was assessed by immunoprecipitation of 35 Slabeled FN from the media of metabolically labeled cells. FN is indicated by the arrow. The experiment was performed four times with similar results; depicted is an individual experiment.
FIG. 4. TAK1 associates with Dab2 and mediates Dab2-stimulated JNK activation. A and B, expression of kinase-deficient TAK1 blocks Dab2-stimulated JNK activity. COS7 cells were transiently cotransfected with FLAG-tagged JNK and either HA-tagged wild-type TAK1 (WT) or kinase-deficient TAK1 (KW) in the absence or presence of FLAG-tagged full-length Dab2. After 48 h, lysates were immunoprecipitated with ␣-FLAG antibody and subjected to a JNK assay utilizing GST-c-Jun as substrate. Reaction products were resolved on a 10% SDS/PAGE gel, and phosphorylation of GST-c-Jun (indicated by the arrow) was visualized by autoradiography. The expression of transfected FLAG-tagged JNK and Dab2 was confirmed by Western analysis utilizing ␣-FLAG antibody. The experiment was repeated four times with similar results. B, expression of HA-tagged wild-type and kinasedeficient TAK1 was demonstrated by immunoblotting analysis using ␣-HA antibody. C, the association of endogenous Dab2 and TAK1 is stimulated by TGF␤ treatment. NIH3T3 cells were stimulated with TGF␤ for the indicated times followed by immunoprecipitation (IP) with ␣-TAK1 antibody (top panel). The presence of Dab2 in the immunoprecipitates was confirmed by Western analysis with ␣-Dab2 antibody. Expression of comparable Dab2 and TAK1 levels is demonstrated by Western analysis (lower panels). Depicted is an individual time-course experiment, which was repeated twice.

Dab2 Mediates TGF␤-stimulated JNK Activation
gene transcription, has recently been demonstrated (48). We wished, therefore, to test whether Dab2 plays a role in mediating cell migration stimulated by TGF␤, since decreased Dab2 expression leads to decreased TGF␤-stimulated JNK activation. To address this, we performed a wounding assay on monolayer cells, which examines the ability of cells to migrate into an area denuded by scraping. We investigated the ability of TGF␤ to stimulate migration in parental NIH3T3 and the 3T3-siDab2#8 clone. After a 24-h treatment with TGF␤, we observed that, although NIH3T3 cells efficiently migrate into the wounded area, the 3T3-siDab2#8 clone exhibited a de-creased migratory capability (Fig. 6A). To obtain a more quantitative measure of cell motility, we assessed the ability of parental NIH3T3 and 3T3-siDab2#8 cells to migrate through a membrane containing 8-m pores in the absence or presence of TGF␤. As shown in Fig. 6B, basal migration of 3T3-siDab2#8 cells is decreased by ϳ40% as compared with the parental NIH3T3 cells, whereas the addition of TGF␤ was able to augment migration to a similar extent in both cell lines. We, thus, demonstrate by two independent methods that cell migration, a JNK-dependent response, is attenuated in cells that express low levels of Dab2. DISCUSSION Efficient tissue repair after wounding is essential for maintenance of tissue homeostasis. After injury, underlying fibroblasts proliferate, migrate into the wound site, and synthesize ECM proteins, including FN. The production of FN is potently stimulated by TGF␤, which is released from degranulating platelets at the wound site. Together, TGF␤ and FN act coordinately to induce fibroblast to myofibroblast transition, characterized by the expression of ␣-smooth muscle cell actin (␣-SMA) (5). This endows to the myofibroblast an ability to contract the ECM, thereby effecting wound closure and termination of the healing process. Here we demonstrate that Dab2 expression controls two key aspects required for efficient wound healing, TGF␤-stimulated induction of FN synthesis and fibroblast migration.
TGF␤ has previously been shown to potently stimulate FN protein synthesis, acting at both the level of gene transcription and at the level of mRNA stabilization (49 -51). We report here that stable siRNA-mediated silencing of Dab2 results in loss of TGF␤-stimulated induction of both FN mRNA and protein levels ( Fig. 1B and Fig. 5C), suggesting that Dab2 plays a role in regulation of FN gene transcription. The FN promoter contains three CRE elements (52), one of which has previously been shown to be occupied by a c-Jun/ATF-2 heterodimer (53). The JNK-signaling pathway, thus, is implicated in TGF␤-mediated up-regulation of FN, since JNK can phosphorylate both c-Jun and ATF-2, leading to their transcriptional activation (27). Indeed, we have previously demonstrated that expression of dominant-negative JNK and MKK4 abolishes TGF␤-mediated induction of FN (21), and here we show that treatment of both NIH3T3 mouse fibroblasts and A10 rat aortic smooth muscle cells with a specific pharmacological inhibitor of JNK, SP600125 (44), leads to decreased TGF␤-stimulated FN synthesis (Fig. 3C). In addition, we show that Dab2 can directly stimulate JNK activity and demonstrate that ablation of Dab2 expression in NIH3T3 cells leads to decreased TGF␤-stimulated JNK activation.
TAK1 was initially identified in a screen to identify potential MAPK kinase kinases that could mediate activation of the yeast pheromone-induced MAPK-signaling pathway (28). TAK1 activity was shown to be stimulated by TGF␤ and BMP4 treatment, whereas expression of constitutively active TAK1 resulted in transactivation of the TGF␤-responsive PAI-1 promoter (28). TAK1 can also be stimulated by treatment with TNF␣, interleukin 1, ceramide, and lipopolysaccharide and has been shown to be involved in the JNK and p38 activation elicited by these agents (47,54). Although TAK1 was found to activate both p38 and JNK in 293T cells, activation of JNK was greater and found to be dependent on MKK4 (55). Studies in Drosophila also demonstrate that TAK1 participates in JNK, but not p38 activation during development (56,57). Here we show that TAK1 and Dab2 can be found in association in vivo after treatment with TGF␤, with similar kinetics observed for TGF␤-stimulated JNK activation (Fig. 4C), suggesting a direct involvement of TAK1 in mediation of Dab2-stimulated JNK activation. In addition, down-regulation of TAK1 expression by double-stranded RNA oligonucleotides or overexpression of TAKKW in NIH3T3 cells (Fig. 5) attenuates TGF␤-stimulated FN protein and mRNA up-regulation, respectively. These results are in agreement with a previous study, which demonstrated that overexpression of the TAB1 binding domain of TAK1 led to decreased TGF␤-stimulated induction of the matrix components PAI-1, type I and type IV collagen, and FN (58). Collectively, our results suggest that TGF␤-mediated FN induction proceeds through Dab2 to TAK1, leading to MKK4 and subsequent JNK activation.
The Smads are a family of transcription factors that display relatively low affinity for their cognate DNA binding elements, and thus, interactions between the Smads and other DNAbinding proteins are necessary to achieve specific, high affinity interactions with DNA (59). The Smads have been shown to interact with c-Jun and c-Fos (15), thereby providing the potential for synergistic as well as antagonistic cross-talk between the Smad-and MAPK-signaling pathways. The interaction of Smad3 with c-Jun, which occurs off-DNA, was found to antagonize transcription of the Smad-dependent COL7A1 and PAI-1 promoters; however, this interaction proved to be synergistic at AP-1-dependent promoter sites (60). Some promoters regulated by TGF␤ contain both Smad and AP-1 binding sites, such as the c-Jun gene itself. Although TGF␤ could still mediate a 2-fold activation of the c-Jun promoter that contained a mutated Smad binding site, mutation of the AP-1/CRE site eliminated all promoter activation (61). We have previously shown that TGF␤-mediated induction of FN was independent of Smad4 but dependent on JNK activation (21). Subsequent studies in mouse embryo fibroblasts that lack Smad2, Smad3, and Smad4 have corroborated the Smad independence of TGF␤-stimulated FN up-regulation (23,24). Moreover, activation of the JNK pathway may actually be required for activation of the Smad pathway, since JNK phosphorylation of Smad3 was found to facilitate the subsequent T␤RI-mediated phosphorylation of Smad3, which led to enhanced nuclear translocation and transcriptional activity (26). In addition, fibroblasts transfected with antisense oligonucleotides to JNK1 and JNK2 exhibited loss of TGF␤-stimulated Smad2 phosphorylation (25). We previously demonstrated that the expression of Dab2 restored both Smad-dependent and Smad-independent responses to a TGF␤-signaling-deficient cell line (11). Dab2 may, thus, play the role of a scaffold molecule to bridge activation of the JNK pathway, mediated by its C-terminal domain, to activation of the Smad pathway, mediated through its Nterminal PTB domain.
Although the study of JNK signaling in mammalian cells has previously focused on transcriptional regulation of AP-1 activity, the role of JNK in mediating cell migration has recently gained attention (48). In Drosophila, the JNK pathway is responsible for the morphogenetic movement of lateral dorsal epithelia toward the dorsal midline, resulting in dorsal closure (62). This involves the JNK-mediated up-regulation of dpp, a TGF␤ superfamily member, which then acts in a paracrine fashion to promote migration of the leading edge cells (63). In mice that are deficient in MEKK1 expression or compound Jnk1Ϫ/ϪJnk2ϩ/Ϫ mutant mice, an eye-open at birth phenotype is observed (64,65). This process is also dependent on the movement of epithelial cells and, thus, resembles dorsal closure in Drosophila. The eye-open at birth phenotype is also observed in mice deficient in activin ␤B (66), again implicating the TGF␤ superfamily in regulation of cell migration. Because the process of dorsal closure is morphologically similar to mammalian wound repair, JNK has been postulated to play a role in the cell migration required for efficient wound healing (67). We demonstrate here that loss of Dab2 expression in NIH3T3 mouse fibroblasts results in decreased cell migration assayed either by a wound healing assay or a quantitative cell migration assay (Fig. 6). A previous study has recently demonstrated that mouse fibroblasts deficient in JNK expression exhibit lower basal JNK activity and show a similar failure to migrate in a wounding assay (68). Although MEKK1 deficiency leads to the migratory defect in epithelial cells observed in the eye-open at birth phenotype, MEKK1 expression is low or absent in eyelid dermis, thus suggesting that JNK-mediated cell migration in fibroblasts is regulated by another MAPK kinase kinase (48,65). We demonstrate here that TAKKW expression blocks Dab2-mediated JNK activation, whereas studies in Drosophila also implicate the Drosophila homolog of TAK (dTAK) in JNKmediated processes of dorsal closure and planar cell polarity Dab2 Mediates TGF␤-stimulated JNK Activation (56,57). Additionally, the defect we observe in cell migration may be due in part to the lack of basal and TGF␤-stimulated FN induction, which by itself has been shown to stimulate JNK activity and cell migration (69,70). Taken together, our studies delineate a signaling pathway dependent on Dab2 expression proceeding through TAK1, which leads to TGF␤-stimulated JNK activation, FN expression, and cell migration.