Regulation of p38 MAPK by MAPK Kinases 3 and 6 in Fibroblast-Like Synoviocytes
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免疫学杂志 2005年第7期
Abstract
The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF- production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [-32P]ATP and GST-activating transcription factor-2 as substrate. IL-1 rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.
Introduction
Mitogen-activated protein kinases are serine/threonine kinases that mediate signal transduction and orchestrate cellular responses to environmental stress. In mammalian cells, three principle MAPK pathways have been identified, including ERK, JNK, and p38 MAPK (1, 2). The p38 MAPK is especially relevant to human inflammatory disease because it serves as a key regulator of cytokines as well as proteases that participate in extracellular remodeling. p38, among the four known isoforms (, , , and ), appears to account for many inflammation-related activities (3, 4). Several functions of p38 are mediated by its substrates activating transcription factor-2 (ATF-2)3 (5) and MAPK-activated protein kinase-2 (MAPKAPK-2), which alters cytokine production through posttranscriptional mechanisms (6). Pyridinyl imidazole p38 inhibitors, such as SB203580, exert anti-inflammatory effects by inhibiting p38 and (7). Multiple MAPK pathways can be simultaneously activated, and their relative balance is determined by upstream kinase cascades known as MAPK kinase (MKKs) (1, 2).
p38 has been implicated in rheumatoid arthritis (RA) based on the observation that it is activated in the rheumatoid synovium (8) and that selective inhibitors are effective in animal models of arthritis (9). These studies suggest that p38 blockade might be effective in RA, and several compounds are currently being investigated in clinical trials (10, 11). An alternative approach might be to target the upstream kinases that regulate p38. For instance, MKK3 and MKK6 are two closely related dual-specificity protein kinases that phosphorylate p38 at the Thr-Gly-Tyr site, but do not activate ERK1/2 or JNK (12, 13). Distinct profiles of cellular stress and cytokines can separately activate MKK3 and MKK6 (14) through phosphorylation of serine and threonine residues at sites Ser189/Thr193 for MKK3 and Ser207/Thr211 for MKK6 (15, 16).
Although many regulatory functions have been defined for MKK3 and MKK6 in murine knockout cells or tumor cell lines, little is known about the pharmacology of MKK inhibition in primary human cells. Our previous studies demonstrated that both MKK3 and MKK6 can phosphorylate p38 in cultured fibroblast-like synoviocytes (FLS) and are activated in RA synovium (17). However, the relative contributions of these two kinases to p38 activation in synoviocytes and the downstream effects of their blockade on cytokine and metalloproteinase production were not examined. In the present study, we used dominant-negative (DN) MKK3 and MKK6 constructs to dissect their respective roles in synoviocyte function. Our data demonstrate for the first time that full activation of p38 requires both MKK3 and MKK6 and that these MKKs control production of IL-6, IL-8, and matrix metalloproteinase (MMP) production in IL-1-stimulated FLS.
Materials and Methods
Fibroblast-like synoviocytes
FLS were isolated from synovial tissues obtained from patients with RA at the time of joint replacement, as described previously (18). The diagnosis of RA conformed to the American College of Rheumatology 1987 revised criteria (19). Synovial tissues were minced and incubated with 1 mg/ml collagenase in serum-free DMEM (Life Technologies) for 2 h as 37°C, filtered through a nylon mesh, extensively washed, and cultured in DMEM supplemented with 10% FCS (endotoxin content <0.006 ng/ml; Invitrogen Life Technologies), penicillin, streptomycin, and L-glutamine in a humidified atmosphere containing 5% CO2. After overnight culture, nonadherent cells were removed, and adherent cells were trypsinized, split at a 1:3 ratio, and cultured in medium. Synoviocytes were used from passage 4 through 9 in these experiments, when they are a homogeneous population of FLS (<1% CD11b, <1% phagocytic, and <1% FcR II positive).
Abs and reagents
Affinity-purified rabbit polyclonal anti-MKK3 Abs, goat polyclonal anti-MKK6 Abs, and secondary Abs were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-phospho-MKK3/6, anti-phosho-p38 MAPK (Thr180/Tyr182), and anti-p38 MAPK Abs and GST-ATF-2 were purchased from Cell Signaling Technology. Human rIL-1 (rhIL-1) was purchased from R&D Systems. The p38 inhibitor, SB203580, was purchased from Calbiochem. Previous studies in our laboratory showed that the concentrations used in these experiments completely block p38, but do not significantly inhibit JNK in synoviocytes (20). DN MKK3- and MKK6-expressing N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C (FLAG) tags were constructed by replacing Ser189 and Thr193 with Ala in MKK3 and Ser207 and Thr211 with Ala in MKK6, as previously described (12). The constructs were subcloned into the expression vector pcDNA3.1 and were kindly provided by Dr. R. Davis (University of Massachusetts, Worcester, MA) (Invitrogen Life Technologies), and empty vector was used as a negative control. The accession numbers of the parent sequences are: NM_002756 (MKK3) and NM_002758 (MKK6).
FLS transfection
Using the Amaxa Human Dermal Fibroblast Nucleofector kit (NHDF-adult) with program U-23, 2–10 x 105 cells were transfected with 20 μg of pcDNA3.1, 10 μg of pcDNA3.1 plus 10 μg of FLAG-DN MKK3, 10 μg of pcDNA plus 10 μg of FLAG-DN MKK6, or 10 μg of FLAG-DN MKK3 plus 10 μg of FLAG-DN MKK6, according to the manufacturer’s protocol. Briefly, FLS suspended in 100 μl of Amaxa Nucleofector solution were added to the DNA solution, and the mixture was transferred into the electroporation cuvette. Immediately after electroporation, the cells were suspended in 500 μl of cell culture medium and transferred to culture dishes or plates. Cell viability after transfection was 55–80% by trypan blue dye exclusion, and cell growth was not affected by transfection for at least 48 h.
Western blot analysis
After transfection, FLS (5 x 105 cells/dish) were cultured in DMEM with 20% FCS in 60-mm dishes for 24 h, and were synchronized in DMEM with 0.1% FCS for 48 h. FLS were then treated with medium or rhIL-1 (2 ng/ml) for 15 min. Cells were washed twice with ice-cold PBS, and protein was extracted using modified radioimmunoprecipitation assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1.5 mM ethylenediamine-tetra-acetic acid (EDTA, pH 8.0), 20 mM -glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 1 μM pepstatin A, and 1 mM PMSF). The protein concentrations in the extracts were determined using the micro bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Whole cell lysates (10 μg) were fractionated on Tris-glycine-buffered 12% SDS-PAGE and transferred to nitrocellulose membrane (PerkinElmer Life Sciences). The membranes were blocked with TBS and 0.1% Tween 20 (TBST) containing 5% nonfat milk for 1 h at room temperature, followed by incubation with Ab to MKK3, MKK6, phospho-MKK3/6, p38, phospho-p38, FLAG, or actin at 4°C overnight. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary Ab for 1 h at room temperature. Immunoreactive protein was detected with chemiluminescence and autoradiography (Eastman Kodak).
Immunoprecipitation and kinase assays
To measure the kinase activity of p38, FLS (5 x 105 cells/dish) were cultured in DMEM with 20% FCS in 60-mm dishes for 24 h after transfection and subsequently serum starved (0.1% FCS) for 48 h. FLS were then treated with either medium or rhIL-1 for 15 min. Cells were washed twice with ice-cold PBS and lysed in modified radioimmunoprecipitation assay buffer. Lysates were centrifuged at 15,000 x g for 10 min. Protein concentrations in the supernatant were determined using the micro bicinchoninic acid protein assay reagent. Then lysates were incubated with 2.5 μg of anti-p38 mAb for 4 h, followed by additional incubation with protein G-Sepharose overnight. The immunoprecipitates were washed three times with immunoprecipitation buffer and once with kinase buffer (50 mM HEPES, pH 7.4, 1 mM MgCl2, 20 mM -glycerophosphate, 1 mM Na3VO4, 0.2 mM DTT, 10 μg/ml aprotinin, 1 μM pepstatin A, and 1 mM PMSF), and resuspended in 25 μl of kinase buffer containing 5 mCi of [-32P]ATP, 100 mM ATP, and 4 μg of GST-ATF-2 (Cell Signaling Technology), and incubated at 37°C for 30 min. As a positive control for p38 inhibition, immunoprecipitates were preincubated with p38 inhibitor (SB203580, 3 μM) at 37°C for 30 min. Reactions were stopped by addition of SDS sample buffer (100 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% 2-ME, 0.25% bromphenol blue). After electrophoresis, the gel was analyzed using NIH Image (version 1.61).
MMP-3, IL-6, and IL-8 protein production
After transfection, FLS (1 x 105 cells/well) were seeded in 12-well plates and cultured in DMEM with 20% FCS at 37°C for 24 h. The supernatants were aspirated and replaced with fresh medium with 10% FCS for 24 h. FLS were then treated with medium or rhIL-1 (2 ng/ml) for 24 h, and the supernatants were harvested. Samples were assayed for IL-6, IL-8, and MMP-3 by ELISA, as per the manufacturer’s instructions (R&D Systems).
MMP-3, IL-6, and IL-8 mRNA levels
To determine mRNA levels, FLS were cultured as described for protein assays. The FLS were then harvested and cDNA was prepared, as previously described (21). Quantitative real-time PCR was performed to determine relative mRNA levels using the GeneAmp 5700 Sequence Detection System (Applied Biosystems). Predeveloped sequence detection reagents specific for human IL-6, IL-8, MMP-3, and GAPDH (Applied Biosystems) were used. PCR was performed with TaqMan Universal PCR Master Mix by using the following protocol: initial activation of AmpliTaq Gold DNA polymerase at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The fluorescent signal was plotted vs cycle number, and the cycle threshold (Ct) was determined. The standard curve method was used to determine relative gene expression. Standard curves for IL-6, IL-8, MMP-3, and GAPDH were generated, as previously described (21), and the sample Ct values were used to calculate the number of cell equivalents for IL-6, IL-8, MMP-3, and GAPDH in the test samples. The data were then normalized to GAPDH, which is a commonly used reference gene in cultured FLS that is not altered by gene transfection (data not shown), to calculate the relative cell equivalents. The details of determining relative cell equivalents are described previously (21). Each PCR run also included nontemplate controls containing all reagents except cDNA. These controls generated a Ct greater than 40 in all experiments.
Statistical analysis
Data are expressed as mean ± SEM. Comparisons between two groups were performed by Student’s t test, and multiple analysis was done by Tukey’s test. A comparison was considered statistically significant if p < 0.05.
Results
Transfection efficiency of plasmids into FLS
Initial studies were performed after optimizing transfection protocols to confirm the transfection efficiency (22). FLAG-DN MKK3 and -DN MKK6 as well as empty vector (pcDNA3.1) were transfected into RA FLS, as described in Materials and Methods. FLS were then incubated with anti-FLAG Ab, and immunohistochemistry was performed. Fig. 1 shows a representative experiment using control cells (pcDNA3.1) or various combinations of DN MKK3 or DN MKK6 plasmids. The MKK transgenes were detected in 90% of cells. This high degree of transfection efficiency allowed us to examine the effect of DN gene expression on FLS function in subsequent studies.
FIGURE 1. Transfection efficiency of FLAG-tagged DN MKK3 and DN MKK6. FLS were transfected with FLAG-DN MKK3 and -DN MKK6 plasmids, as described in Materials and Methods. The presence of the transgene was assessed by immunohistochemistry for FLAG, and the percentage of positive cells is indicated on the figure. A, Empty vector (pcDNA3.1, 20 μg)-transfected cells. B, DN MKK3 (empty vector, 10 μg; 3DN, 10 μg)-transfected cells. C, DN MKK6 (empty vector, 10 μg; 6DN, 10 μg)-transfected cells. D, Cotransfection with both DN MKK3- and DN MKK6 (DN MKK3, 10 μg; DN MKK6, 10 μg)-transfected cells. Transfection efficiency is shown as mean percentage ± SE.
Modulation of intracellular signaling in RA FLS by the DN MKK3 and DN MKK6 transfection
To analyze the relative contribution of MKK3 and MKK6 to IL-1-stimulated p38 activation in RA FLS, the expression of phospho-p38, phospho-MKK3, and phospho-MKK6 as well as the transduced protein and -actin were determined by Western blot analysis. As shown in Fig. 2A, immunoreactive MKK3 and MKK6 protein expression were markedly greater in cells transfected with DN plasmids. The identity of the bands as the respective DN MKK proteins was confirmed using anti-FLAG Ab. Fig. 2 also shows that p38 phosphorylation is induced in IL-1-stimulated cells. The DN MKK3 and DN MKK6 clones each modestly decreased phospho-p38 levels by 24.7 ± 1.4% and 24.7 ± 12.7%, respectively, in the cells exposed to cytokine (Fig. 2, A and B, for normalized results). However, cotransfection with both DN constructs was synergistic with respect to p38 inhibition (72.3 ± 6.1% inhibition; p < 0.01 compared with control and p < 0.05 compared with individual DN MKKs) (Fig. 2, A and B). Of interest, a similar pattern of inhibition was observed for phospho-MKK3/6 in the cells transfected with the DN clones (Fig. 2C), suggesting the pathway involves either autocrine activation or autophosphorylation of the upstream kinases.
FIGURE 2. Effect of DN MKK3 and DN MKK6 on p38 phosphorylation. Cultured FLS were stimulated with medium or IL-1 (2 ng/ml) for 15 min. Total proteins were extracted and evaluated by Western blot analysis. A, Representative blot of independent experiments (n = 4). Note marked increase in MKK3 and MKK6 expression after transfection with the indicated construct. Coexpression of both proteins is readily demonstrated with the anti-FLAG Ab (higher m.w. band = MKK6). B, Actin-normalized phospho-p38 levels in transfected cells (n = 4). The combination of DN MKK3 and DN MKK6 markedly decreased p38 phosphorylation. Data are presented as percentage of IL-1-induced expression for each individual cell line. 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, p < 0.05 compared with empty vector. C, Actin-normalized phospho-MKK3/6 levels in transfected cells (n = 3). The combination of DN MKK3 and DN MKK6 markedly decreased MKK3/6 phosphorylation. Data are presented as percentage of IL-1-induced expression for each individual cell line. 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, **, p < 0.05 and p < 0.01, respectively, compared with empty vector.
Inhibition of in vitro p38 MAPK activity by MKK3 and MKK6
To further investigate the functional profile of MKK3 and MKK6, in vitro p38 kinase assays were performed using GST-ATF-2 as substrate. As shown in Fig. 3 and Table I, kinase activity was significantly increased in anti-p38 Ab immunoprecipitates from IL-1-stimulated FLS. Kinase activity was decreased by DN MKK3, DN MKK6, or the combination of both DN constructs compared with empty vector-transfected cells (32.3, 53.2, or 63.1% inhibition, respectively). In contrast to the Western blot studies, the suppressive effect of DN MKK6 was modestly greater than DN MKK3 (p < 0.05). Similar suppression was observed if a saturating concentration of the p38 inhibitor SB203580 (3 μM) was added to the kinase reaction as a positive control (56.2% inhibition).
FIGURE 3. Inhibition of p38 MKK activity by DN MKK3 and DN MKK6. The functional state of p38 was determined using in vitro kinase assays. Cultured FLS were stimulated with medium or IL-1 (2 ng/ml) for 15 min. Total proteins were extracted, and p38 kinase activity was measured by immune complex kinase assay using GST-ATF-2 as the substrate. SB, SB203580 (3 μM). Three separate experiments are shown. Quantification is provided in Table I.
Table I. Inhibition of p38 MAPK activity by DN MKK3 and DN MKK6a
Regulation of MMP-3, IL-6, and IL-8 protein by MKK3 and MKK6
The preceding studies suggest that both MKK3 and MKK6 can contribute to p38 phosphorylation and kinase activity. To determine whether these kinases also regulate production of inflammatory mediators, cells were transfected and then activated with IL-1. Culture supernatants were collected after 24 h and assayed by ELISA. As shown in Fig. 4, IL-1 induced MMP-3, IL-6, and IL-8 protein production, but this activity was suppressed in DN MKK3-, DN MKK6-, and DN MKK3 + DN MKK6-transfected cells. Production was most effectively suppressed in combination DN-cotransfected cells, but the effect of DN MKK3 was greater than DN MKK6 (p < 0.05). Exogenous SB203580 was comparable to MKK blockade, indicating that the p38 was effectively blocked. DN MKK3 and the combination of DN MKK3 and DN MKK6 also inhibited IL-6 production by FLS stimulated with TNF- (10 ng/ml) as much as SB203580 (n = 2; data not shown).
FIGURE 4. Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 production. Cultured FLS were stimulated with or without rhIL-1 (2 ng/ml) for 24 h; culture supernatants were collected; and the protein level of MMP-3, IL-6, and IL-8 was measured by ELISA. Mean ± SE of six independent experiments is shown. Data are presented as percentage of IL-1-induced expression for each individual cell line. SB, SB203580 (10 μM). 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, p < 0.05 and p < 0.01, respectively, compared with empty vector. ##, p < 0.01 compared with empty vector.
Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 mRNA expression
p38 regulates mediator production through several mechanisms that vary with cell type and method of stimulation. To determine whether suppression of cytokine and MMP in FLS is due to pretranslational events, we performed real-time PCR to quantify mRNA in transfected cells. MMP-3, IL-6, and IL-8 mRNA expression was induced by IL-1. Overall, effects of the DN clones or SB203580 were not statistically significant (Fig. 5). These data indicate that cytokine and MMP regulation by p38 and MKKs is independent of mRNA levels and is most likely related to translational mechanisms.
FIGURE 5. Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 gene expression. Cultured FLS were stimulated with or without rhIL-1 (2 ng/ml) for 24 h; total RNA was extracted; and the level of MMP-3, IL-6, and IL-8 mRNA was measured by real time-PCR, as described in Materials and Methods. Mean ± SE of six independent experiments is shown. Data are presented as percentage of IL-1-induced expression for each individual cell line. SB, SB203580 (10 μM). *, p < 0.05 compared with empty vector.
Discussion
RA is a chronic inflammatory disease marked by synovial hyperplasia and local invasion into the extracellular matrix. Synovitis is regulated by cytokines such as IL-1 and TNF- that activate a broad array of cell signaling mechanisms (23). MAPKs are especially important because they control the production of MMPs and cytokines that participate in the rheumatoid process. The three MAPK families (ERK, JNK, and p38) accomplish this by phosphorylating numerous key transcription factors, such as AP-1 and ATF-1/2 (1, 2). In addition to engaging transcription factors directly, some p38 functions are also regulated through downstream kinases such as MAPKAPK-2 (4, 6, 11).
Several MAPK members are activated in the rheumatoid synovium and have been implicated in the pathogenesis of RA. p38 is thought to be crucial because selective p38 inhibitors block joint inflammation and destruction in several animal models of arthritis. Four p38 isoforms (p38, -, -, and -) have been characterized. The best-studied subtype is p38, which can be phosphorylated in many inflammatory cell lineages and regulates cytokine production by macrophages (3). The pyridinylimidazole compounds, exemplified by SB203580, were originally identified as cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38. SB203580 specifically inhibits p38 and - forms by competing for the ATP-binding pocket and blocking kinase activity (7, 10).
The present study was designed to evaluate the potential role of two main upstream kinases of p38, MKK3 and MKK6, on the activation of RA FLS. We previously demonstrated that both MKKs are phosphorylated in rheumatoid synovium and are constitutively expressed in cultured FLS (17). MKK3 and MKK6 each form stable complexes with p38 in FLS that can phosphorylate ATF-2 after cytokine exposure. Western blot studies using stimulated FLS suggested that MKK3 is preferentially phosphorylated. However, functional studies to assess the relative contribution of MKK3 and MKK6 were not possible due to difficulty transfecting primary synoviocytes and the lack of selective small molecule inhibitors. More recently, protocols have been developed that permit high transfection efficiency in FLS (90%). These advances allowed us to study the functional effects of the MKK3 and/or MKK6 inhibition using well-characterized DN constructs (12).
Our studies are the first to assess MKK3 and MKK6 function in primary human cells and demonstrate that both MKK3 and MKK6 contribute to the phosphorylation of p38, induction of p38 kinase activity, and release of key inflammatory gene products. The DN MKK constructs also inhibited phosphorylation of MKK3 and MKK6, suggesting that either autophosphorylation or positive feedback loops from p38 contribute to MKK activation. A similar scenario has been observed with selective p38 inhibitors, which can decrease p38 phosphorylation even though the compounds do not block the phosphorylation sites on the kinase (24). We observed subtle differences in the relative contributions of the two kinases to the p38 pathway. For instance, MKK6 appears to be more active in the kinase assays, whereas MKK3 had a greater contribution to the release of cytokines and MMPs. Because production of inflammatory mediators is probably the most important aspect with respect to a therapeutic target, these data suggest that either a selective MKK3 or, more likely, a combined MKK3/6 inhibitor would be the most effective in a disease such as RA.
The effects of DN MKK3 and DN MKK6 have not been studied in primary human cells, but their combined or individual effects have been examined in tumor cells and rodent cell lines. These results differ in some respects from FLS. For instance, DN MKK3 rather than DN MKK6 blocks v integrin-mediated p38 activation in invasive breast cancer cells (25). Similarly, activin A and hepatocyte growth factor-mediated p38 activation is inhibited by DN MKK3, but not by DN MKK6 in pancreatic cancer cells (26). Hypoxia-induced endoglin expression in mouse endothelial cell line is also inhibited by DN MKK3 (27). In bovine capillary endothelium, fibroblast growth factor-2-induced p38 activation is decreased by both DN MKK3 and 6 (28). Finally, pervanadate-induced p38 phosphorylation is inhibited by DN MKK3 as well as DN MKK6 in rat vascular smooth muscle cells (29). MKK function has also been evaluated in genetically engineered mice, in which TNF--induced cytokine expression in fibroblasts is decreased in MKK3–/– animals (30). Defective IL-12 production has also been noted in macrophages and dendritic cells from MKK3–/– mice (31). Both MKK3 and MKK6 are essential for TNF--stimulated p38 activation in vivo (32).
In addition to cell lineage-specific hierarchy of MKKs, the types of cellular stresses that activate MKK3 compared with MKK6 also vary widely. Our data suggest that, unlike some forms of stimulation, both MKK3 and MKK6 are rapidly activated in FLS by IL-1, which is a key cytokine implicated in RA. However, MKK6 is the primary pathway to p38 in murine lymphoma cells and human epithelial cells after osmotic stress (33), while MKK3 is required for full activation of p38 in murine embryonic fibroblasts (30). MKK6 is the principal activator of p38 in human epithelial cells stimulated with IL-1 (34), but MKK3 appears to be an important activator of p38 in LPS-stimulated murine peritoneal macrophages (31). In contrast, both the p38 and isoforms are activated by MKK3 in murine mesangial cells stimulated by TGF-1 (35).
The mechanisms by which p38 regulates gene expression are equally complex. In cytokine-stimulated synoviocytes, p38 inhibition by DN MKK3/6 or SB203580 had little effect on inflammatory gene expression despite significant decreases in the encoded proteins. Previous studies have indicated that this method of suppression is related to MAPKAPK-2-mediated translational effects. For instance, inhibition of TNF- and IL-1 protein synthesis by p38 blockade is not accompanied by a decrease in the corresponding steady state mRNA levels (36, 37). MAPKAPK-2 knockout mice exhibit impaired TNF- protein synthesis, with no discernible change in TNF- mRNA transcription or stability (38, 39). IL-6 and IL-8 mRNA translation is also regulated by p38, but extent of inhibition of protein production varies with cell type (40, 41, 42). These data are consistent with our studies on cytokine regulation by MKK3 and MKK6 in FLS, indicating that suppression of the p38 pathway is complex, but that downstream effects involve posttranslational mechanism.
The regulation of MMPs by p38 can involve several mechanisms. For example, wild-type MKK3 overexpression in human skin fibroblasts increases MMP-1 and MMP-3 mRNA expression, in part by stabilizing their respective RNA transcripts (4). The mRNA t1/2 appears to be prolonged due to decreased deadenylation of AU-rich tails (11). Our observation that DN MKK3 and DN MKK6 modestly decreased MMP-3 mRNA is consistent with an effect on gene transcription or mRNA stability. However, the changes in MMP-3 mRNA are relatively small compared with the decrease in MMP-3 protein levels. Hence, MMP regulation in FLS by p38 probably results from combination of mechanisms. The differences between our data and the dermal fibroblast study (4) are probably related to the distinct biology of wild-type overexpression compared with inhibition of the endogenous kinase.
In conclusion, our studies demonstrate that, unlike many tumor cells or physical stresses, MKK3 and MKK6 are key p38 regulators in cytokine-stimulated FLS. Both MKKs contribute to p38 activation, but MKK3 might be more important when attempting to suppress cytokine production. However, both must be blocked to interrupt p38 as effectively as small molecule p38 inhibitors in FLS. Because p38 pathway may participate in joint destruction and inflammation, selective MKK3, MKK6, or combined inhibitors have therapeutic potential for RA. Distinct activation pathways in different cell lineages suggests that the safety and efficacy profile might differ from competitive p38 inhibitors.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Institutes of Health.
2 Address correspondence and reprint requests to Dr. Gary S. Firestein, Division of Rheumatology, Allergy, and Immunology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656. E-mail address: gfirestein@ucsd.edu
3 Abbreviations used in this paper: ATF, activating transcription factor; Ct, cycle threshold; DN, dominant negative; FLS, fibroblast-like synoviocyte; MAPKAPK, MAPK-activated protein kinase; MKK, MAPK kinase; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; rhIL, human rIL; FLAG, N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C.
Received for publication October 27, 2004. Accepted for publication January 19, 2005.
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The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF- production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [-32P]ATP and GST-activating transcription factor-2 as substrate. IL-1 rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.
Introduction
Mitogen-activated protein kinases are serine/threonine kinases that mediate signal transduction and orchestrate cellular responses to environmental stress. In mammalian cells, three principle MAPK pathways have been identified, including ERK, JNK, and p38 MAPK (1, 2). The p38 MAPK is especially relevant to human inflammatory disease because it serves as a key regulator of cytokines as well as proteases that participate in extracellular remodeling. p38, among the four known isoforms (, , , and ), appears to account for many inflammation-related activities (3, 4). Several functions of p38 are mediated by its substrates activating transcription factor-2 (ATF-2)3 (5) and MAPK-activated protein kinase-2 (MAPKAPK-2), which alters cytokine production through posttranscriptional mechanisms (6). Pyridinyl imidazole p38 inhibitors, such as SB203580, exert anti-inflammatory effects by inhibiting p38 and (7). Multiple MAPK pathways can be simultaneously activated, and their relative balance is determined by upstream kinase cascades known as MAPK kinase (MKKs) (1, 2).
p38 has been implicated in rheumatoid arthritis (RA) based on the observation that it is activated in the rheumatoid synovium (8) and that selective inhibitors are effective in animal models of arthritis (9). These studies suggest that p38 blockade might be effective in RA, and several compounds are currently being investigated in clinical trials (10, 11). An alternative approach might be to target the upstream kinases that regulate p38. For instance, MKK3 and MKK6 are two closely related dual-specificity protein kinases that phosphorylate p38 at the Thr-Gly-Tyr site, but do not activate ERK1/2 or JNK (12, 13). Distinct profiles of cellular stress and cytokines can separately activate MKK3 and MKK6 (14) through phosphorylation of serine and threonine residues at sites Ser189/Thr193 for MKK3 and Ser207/Thr211 for MKK6 (15, 16).
Although many regulatory functions have been defined for MKK3 and MKK6 in murine knockout cells or tumor cell lines, little is known about the pharmacology of MKK inhibition in primary human cells. Our previous studies demonstrated that both MKK3 and MKK6 can phosphorylate p38 in cultured fibroblast-like synoviocytes (FLS) and are activated in RA synovium (17). However, the relative contributions of these two kinases to p38 activation in synoviocytes and the downstream effects of their blockade on cytokine and metalloproteinase production were not examined. In the present study, we used dominant-negative (DN) MKK3 and MKK6 constructs to dissect their respective roles in synoviocyte function. Our data demonstrate for the first time that full activation of p38 requires both MKK3 and MKK6 and that these MKKs control production of IL-6, IL-8, and matrix metalloproteinase (MMP) production in IL-1-stimulated FLS.
Materials and Methods
Fibroblast-like synoviocytes
FLS were isolated from synovial tissues obtained from patients with RA at the time of joint replacement, as described previously (18). The diagnosis of RA conformed to the American College of Rheumatology 1987 revised criteria (19). Synovial tissues were minced and incubated with 1 mg/ml collagenase in serum-free DMEM (Life Technologies) for 2 h as 37°C, filtered through a nylon mesh, extensively washed, and cultured in DMEM supplemented with 10% FCS (endotoxin content <0.006 ng/ml; Invitrogen Life Technologies), penicillin, streptomycin, and L-glutamine in a humidified atmosphere containing 5% CO2. After overnight culture, nonadherent cells were removed, and adherent cells were trypsinized, split at a 1:3 ratio, and cultured in medium. Synoviocytes were used from passage 4 through 9 in these experiments, when they are a homogeneous population of FLS (<1% CD11b, <1% phagocytic, and <1% FcR II positive).
Abs and reagents
Affinity-purified rabbit polyclonal anti-MKK3 Abs, goat polyclonal anti-MKK6 Abs, and secondary Abs were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-phospho-MKK3/6, anti-phosho-p38 MAPK (Thr180/Tyr182), and anti-p38 MAPK Abs and GST-ATF-2 were purchased from Cell Signaling Technology. Human rIL-1 (rhIL-1) was purchased from R&D Systems. The p38 inhibitor, SB203580, was purchased from Calbiochem. Previous studies in our laboratory showed that the concentrations used in these experiments completely block p38, but do not significantly inhibit JNK in synoviocytes (20). DN MKK3- and MKK6-expressing N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C (FLAG) tags were constructed by replacing Ser189 and Thr193 with Ala in MKK3 and Ser207 and Thr211 with Ala in MKK6, as previously described (12). The constructs were subcloned into the expression vector pcDNA3.1 and were kindly provided by Dr. R. Davis (University of Massachusetts, Worcester, MA) (Invitrogen Life Technologies), and empty vector was used as a negative control. The accession numbers of the parent sequences are: NM_002756 (MKK3) and NM_002758 (MKK6).
FLS transfection
Using the Amaxa Human Dermal Fibroblast Nucleofector kit (NHDF-adult) with program U-23, 2–10 x 105 cells were transfected with 20 μg of pcDNA3.1, 10 μg of pcDNA3.1 plus 10 μg of FLAG-DN MKK3, 10 μg of pcDNA plus 10 μg of FLAG-DN MKK6, or 10 μg of FLAG-DN MKK3 plus 10 μg of FLAG-DN MKK6, according to the manufacturer’s protocol. Briefly, FLS suspended in 100 μl of Amaxa Nucleofector solution were added to the DNA solution, and the mixture was transferred into the electroporation cuvette. Immediately after electroporation, the cells were suspended in 500 μl of cell culture medium and transferred to culture dishes or plates. Cell viability after transfection was 55–80% by trypan blue dye exclusion, and cell growth was not affected by transfection for at least 48 h.
Western blot analysis
After transfection, FLS (5 x 105 cells/dish) were cultured in DMEM with 20% FCS in 60-mm dishes for 24 h, and were synchronized in DMEM with 0.1% FCS for 48 h. FLS were then treated with medium or rhIL-1 (2 ng/ml) for 15 min. Cells were washed twice with ice-cold PBS, and protein was extracted using modified radioimmunoprecipitation assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1.5 mM ethylenediamine-tetra-acetic acid (EDTA, pH 8.0), 20 mM -glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 1 μM pepstatin A, and 1 mM PMSF). The protein concentrations in the extracts were determined using the micro bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Whole cell lysates (10 μg) were fractionated on Tris-glycine-buffered 12% SDS-PAGE and transferred to nitrocellulose membrane (PerkinElmer Life Sciences). The membranes were blocked with TBS and 0.1% Tween 20 (TBST) containing 5% nonfat milk for 1 h at room temperature, followed by incubation with Ab to MKK3, MKK6, phospho-MKK3/6, p38, phospho-p38, FLAG, or actin at 4°C overnight. After washing three times with TBST, the membrane was incubated with HRP-conjugated secondary Ab for 1 h at room temperature. Immunoreactive protein was detected with chemiluminescence and autoradiography (Eastman Kodak).
Immunoprecipitation and kinase assays
To measure the kinase activity of p38, FLS (5 x 105 cells/dish) were cultured in DMEM with 20% FCS in 60-mm dishes for 24 h after transfection and subsequently serum starved (0.1% FCS) for 48 h. FLS were then treated with either medium or rhIL-1 for 15 min. Cells were washed twice with ice-cold PBS and lysed in modified radioimmunoprecipitation assay buffer. Lysates were centrifuged at 15,000 x g for 10 min. Protein concentrations in the supernatant were determined using the micro bicinchoninic acid protein assay reagent. Then lysates were incubated with 2.5 μg of anti-p38 mAb for 4 h, followed by additional incubation with protein G-Sepharose overnight. The immunoprecipitates were washed three times with immunoprecipitation buffer and once with kinase buffer (50 mM HEPES, pH 7.4, 1 mM MgCl2, 20 mM -glycerophosphate, 1 mM Na3VO4, 0.2 mM DTT, 10 μg/ml aprotinin, 1 μM pepstatin A, and 1 mM PMSF), and resuspended in 25 μl of kinase buffer containing 5 mCi of [-32P]ATP, 100 mM ATP, and 4 μg of GST-ATF-2 (Cell Signaling Technology), and incubated at 37°C for 30 min. As a positive control for p38 inhibition, immunoprecipitates were preincubated with p38 inhibitor (SB203580, 3 μM) at 37°C for 30 min. Reactions were stopped by addition of SDS sample buffer (100 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5% 2-ME, 0.25% bromphenol blue). After electrophoresis, the gel was analyzed using NIH Image (version 1.61).
MMP-3, IL-6, and IL-8 protein production
After transfection, FLS (1 x 105 cells/well) were seeded in 12-well plates and cultured in DMEM with 20% FCS at 37°C for 24 h. The supernatants were aspirated and replaced with fresh medium with 10% FCS for 24 h. FLS were then treated with medium or rhIL-1 (2 ng/ml) for 24 h, and the supernatants were harvested. Samples were assayed for IL-6, IL-8, and MMP-3 by ELISA, as per the manufacturer’s instructions (R&D Systems).
MMP-3, IL-6, and IL-8 mRNA levels
To determine mRNA levels, FLS were cultured as described for protein assays. The FLS were then harvested and cDNA was prepared, as previously described (21). Quantitative real-time PCR was performed to determine relative mRNA levels using the GeneAmp 5700 Sequence Detection System (Applied Biosystems). Predeveloped sequence detection reagents specific for human IL-6, IL-8, MMP-3, and GAPDH (Applied Biosystems) were used. PCR was performed with TaqMan Universal PCR Master Mix by using the following protocol: initial activation of AmpliTaq Gold DNA polymerase at 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The fluorescent signal was plotted vs cycle number, and the cycle threshold (Ct) was determined. The standard curve method was used to determine relative gene expression. Standard curves for IL-6, IL-8, MMP-3, and GAPDH were generated, as previously described (21), and the sample Ct values were used to calculate the number of cell equivalents for IL-6, IL-8, MMP-3, and GAPDH in the test samples. The data were then normalized to GAPDH, which is a commonly used reference gene in cultured FLS that is not altered by gene transfection (data not shown), to calculate the relative cell equivalents. The details of determining relative cell equivalents are described previously (21). Each PCR run also included nontemplate controls containing all reagents except cDNA. These controls generated a Ct greater than 40 in all experiments.
Statistical analysis
Data are expressed as mean ± SEM. Comparisons between two groups were performed by Student’s t test, and multiple analysis was done by Tukey’s test. A comparison was considered statistically significant if p < 0.05.
Results
Transfection efficiency of plasmids into FLS
Initial studies were performed after optimizing transfection protocols to confirm the transfection efficiency (22). FLAG-DN MKK3 and -DN MKK6 as well as empty vector (pcDNA3.1) were transfected into RA FLS, as described in Materials and Methods. FLS were then incubated with anti-FLAG Ab, and immunohistochemistry was performed. Fig. 1 shows a representative experiment using control cells (pcDNA3.1) or various combinations of DN MKK3 or DN MKK6 plasmids. The MKK transgenes were detected in 90% of cells. This high degree of transfection efficiency allowed us to examine the effect of DN gene expression on FLS function in subsequent studies.
FIGURE 1. Transfection efficiency of FLAG-tagged DN MKK3 and DN MKK6. FLS were transfected with FLAG-DN MKK3 and -DN MKK6 plasmids, as described in Materials and Methods. The presence of the transgene was assessed by immunohistochemistry for FLAG, and the percentage of positive cells is indicated on the figure. A, Empty vector (pcDNA3.1, 20 μg)-transfected cells. B, DN MKK3 (empty vector, 10 μg; 3DN, 10 μg)-transfected cells. C, DN MKK6 (empty vector, 10 μg; 6DN, 10 μg)-transfected cells. D, Cotransfection with both DN MKK3- and DN MKK6 (DN MKK3, 10 μg; DN MKK6, 10 μg)-transfected cells. Transfection efficiency is shown as mean percentage ± SE.
Modulation of intracellular signaling in RA FLS by the DN MKK3 and DN MKK6 transfection
To analyze the relative contribution of MKK3 and MKK6 to IL-1-stimulated p38 activation in RA FLS, the expression of phospho-p38, phospho-MKK3, and phospho-MKK6 as well as the transduced protein and -actin were determined by Western blot analysis. As shown in Fig. 2A, immunoreactive MKK3 and MKK6 protein expression were markedly greater in cells transfected with DN plasmids. The identity of the bands as the respective DN MKK proteins was confirmed using anti-FLAG Ab. Fig. 2 also shows that p38 phosphorylation is induced in IL-1-stimulated cells. The DN MKK3 and DN MKK6 clones each modestly decreased phospho-p38 levels by 24.7 ± 1.4% and 24.7 ± 12.7%, respectively, in the cells exposed to cytokine (Fig. 2, A and B, for normalized results). However, cotransfection with both DN constructs was synergistic with respect to p38 inhibition (72.3 ± 6.1% inhibition; p < 0.01 compared with control and p < 0.05 compared with individual DN MKKs) (Fig. 2, A and B). Of interest, a similar pattern of inhibition was observed for phospho-MKK3/6 in the cells transfected with the DN clones (Fig. 2C), suggesting the pathway involves either autocrine activation or autophosphorylation of the upstream kinases.
FIGURE 2. Effect of DN MKK3 and DN MKK6 on p38 phosphorylation. Cultured FLS were stimulated with medium or IL-1 (2 ng/ml) for 15 min. Total proteins were extracted and evaluated by Western blot analysis. A, Representative blot of independent experiments (n = 4). Note marked increase in MKK3 and MKK6 expression after transfection with the indicated construct. Coexpression of both proteins is readily demonstrated with the anti-FLAG Ab (higher m.w. band = MKK6). B, Actin-normalized phospho-p38 levels in transfected cells (n = 4). The combination of DN MKK3 and DN MKK6 markedly decreased p38 phosphorylation. Data are presented as percentage of IL-1-induced expression for each individual cell line. 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, p < 0.05 compared with empty vector. C, Actin-normalized phospho-MKK3/6 levels in transfected cells (n = 3). The combination of DN MKK3 and DN MKK6 markedly decreased MKK3/6 phosphorylation. Data are presented as percentage of IL-1-induced expression for each individual cell line. 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, **, p < 0.05 and p < 0.01, respectively, compared with empty vector.
Inhibition of in vitro p38 MAPK activity by MKK3 and MKK6
To further investigate the functional profile of MKK3 and MKK6, in vitro p38 kinase assays were performed using GST-ATF-2 as substrate. As shown in Fig. 3 and Table I, kinase activity was significantly increased in anti-p38 Ab immunoprecipitates from IL-1-stimulated FLS. Kinase activity was decreased by DN MKK3, DN MKK6, or the combination of both DN constructs compared with empty vector-transfected cells (32.3, 53.2, or 63.1% inhibition, respectively). In contrast to the Western blot studies, the suppressive effect of DN MKK6 was modestly greater than DN MKK3 (p < 0.05). Similar suppression was observed if a saturating concentration of the p38 inhibitor SB203580 (3 μM) was added to the kinase reaction as a positive control (56.2% inhibition).
FIGURE 3. Inhibition of p38 MKK activity by DN MKK3 and DN MKK6. The functional state of p38 was determined using in vitro kinase assays. Cultured FLS were stimulated with medium or IL-1 (2 ng/ml) for 15 min. Total proteins were extracted, and p38 kinase activity was measured by immune complex kinase assay using GST-ATF-2 as the substrate. SB, SB203580 (3 μM). Three separate experiments are shown. Quantification is provided in Table I.
Table I. Inhibition of p38 MAPK activity by DN MKK3 and DN MKK6a
Regulation of MMP-3, IL-6, and IL-8 protein by MKK3 and MKK6
The preceding studies suggest that both MKK3 and MKK6 can contribute to p38 phosphorylation and kinase activity. To determine whether these kinases also regulate production of inflammatory mediators, cells were transfected and then activated with IL-1. Culture supernatants were collected after 24 h and assayed by ELISA. As shown in Fig. 4, IL-1 induced MMP-3, IL-6, and IL-8 protein production, but this activity was suppressed in DN MKK3-, DN MKK6-, and DN MKK3 + DN MKK6-transfected cells. Production was most effectively suppressed in combination DN-cotransfected cells, but the effect of DN MKK3 was greater than DN MKK6 (p < 0.05). Exogenous SB203580 was comparable to MKK blockade, indicating that the p38 was effectively blocked. DN MKK3 and the combination of DN MKK3 and DN MKK6 also inhibited IL-6 production by FLS stimulated with TNF- (10 ng/ml) as much as SB203580 (n = 2; data not shown).
FIGURE 4. Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 production. Cultured FLS were stimulated with or without rhIL-1 (2 ng/ml) for 24 h; culture supernatants were collected; and the protein level of MMP-3, IL-6, and IL-8 was measured by ELISA. Mean ± SE of six independent experiments is shown. Data are presented as percentage of IL-1-induced expression for each individual cell line. SB, SB203580 (10 μM). 3DN = DN MKK3; 6DN = DN MKK6; 3/6DN = cotransfection with DN MKK3 and DN MKK6. *, p < 0.05 and p < 0.01, respectively, compared with empty vector. ##, p < 0.01 compared with empty vector.
Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 mRNA expression
p38 regulates mediator production through several mechanisms that vary with cell type and method of stimulation. To determine whether suppression of cytokine and MMP in FLS is due to pretranslational events, we performed real-time PCR to quantify mRNA in transfected cells. MMP-3, IL-6, and IL-8 mRNA expression was induced by IL-1. Overall, effects of the DN clones or SB203580 were not statistically significant (Fig. 5). These data indicate that cytokine and MMP regulation by p38 and MKKs is independent of mRNA levels and is most likely related to translational mechanisms.
FIGURE 5. Effect of DN MKK3 and DN MKK6 on MMP-3, IL-6, and IL-8 gene expression. Cultured FLS were stimulated with or without rhIL-1 (2 ng/ml) for 24 h; total RNA was extracted; and the level of MMP-3, IL-6, and IL-8 mRNA was measured by real time-PCR, as described in Materials and Methods. Mean ± SE of six independent experiments is shown. Data are presented as percentage of IL-1-induced expression for each individual cell line. SB, SB203580 (10 μM). *, p < 0.05 compared with empty vector.
Discussion
RA is a chronic inflammatory disease marked by synovial hyperplasia and local invasion into the extracellular matrix. Synovitis is regulated by cytokines such as IL-1 and TNF- that activate a broad array of cell signaling mechanisms (23). MAPKs are especially important because they control the production of MMPs and cytokines that participate in the rheumatoid process. The three MAPK families (ERK, JNK, and p38) accomplish this by phosphorylating numerous key transcription factors, such as AP-1 and ATF-1/2 (1, 2). In addition to engaging transcription factors directly, some p38 functions are also regulated through downstream kinases such as MAPKAPK-2 (4, 6, 11).
Several MAPK members are activated in the rheumatoid synovium and have been implicated in the pathogenesis of RA. p38 is thought to be crucial because selective p38 inhibitors block joint inflammation and destruction in several animal models of arthritis. Four p38 isoforms (p38, -, -, and -) have been characterized. The best-studied subtype is p38, which can be phosphorylated in many inflammatory cell lineages and regulates cytokine production by macrophages (3). The pyridinylimidazole compounds, exemplified by SB203580, were originally identified as cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38. SB203580 specifically inhibits p38 and - forms by competing for the ATP-binding pocket and blocking kinase activity (7, 10).
The present study was designed to evaluate the potential role of two main upstream kinases of p38, MKK3 and MKK6, on the activation of RA FLS. We previously demonstrated that both MKKs are phosphorylated in rheumatoid synovium and are constitutively expressed in cultured FLS (17). MKK3 and MKK6 each form stable complexes with p38 in FLS that can phosphorylate ATF-2 after cytokine exposure. Western blot studies using stimulated FLS suggested that MKK3 is preferentially phosphorylated. However, functional studies to assess the relative contribution of MKK3 and MKK6 were not possible due to difficulty transfecting primary synoviocytes and the lack of selective small molecule inhibitors. More recently, protocols have been developed that permit high transfection efficiency in FLS (90%). These advances allowed us to study the functional effects of the MKK3 and/or MKK6 inhibition using well-characterized DN constructs (12).
Our studies are the first to assess MKK3 and MKK6 function in primary human cells and demonstrate that both MKK3 and MKK6 contribute to the phosphorylation of p38, induction of p38 kinase activity, and release of key inflammatory gene products. The DN MKK constructs also inhibited phosphorylation of MKK3 and MKK6, suggesting that either autophosphorylation or positive feedback loops from p38 contribute to MKK activation. A similar scenario has been observed with selective p38 inhibitors, which can decrease p38 phosphorylation even though the compounds do not block the phosphorylation sites on the kinase (24). We observed subtle differences in the relative contributions of the two kinases to the p38 pathway. For instance, MKK6 appears to be more active in the kinase assays, whereas MKK3 had a greater contribution to the release of cytokines and MMPs. Because production of inflammatory mediators is probably the most important aspect with respect to a therapeutic target, these data suggest that either a selective MKK3 or, more likely, a combined MKK3/6 inhibitor would be the most effective in a disease such as RA.
The effects of DN MKK3 and DN MKK6 have not been studied in primary human cells, but their combined or individual effects have been examined in tumor cells and rodent cell lines. These results differ in some respects from FLS. For instance, DN MKK3 rather than DN MKK6 blocks v integrin-mediated p38 activation in invasive breast cancer cells (25). Similarly, activin A and hepatocyte growth factor-mediated p38 activation is inhibited by DN MKK3, but not by DN MKK6 in pancreatic cancer cells (26). Hypoxia-induced endoglin expression in mouse endothelial cell line is also inhibited by DN MKK3 (27). In bovine capillary endothelium, fibroblast growth factor-2-induced p38 activation is decreased by both DN MKK3 and 6 (28). Finally, pervanadate-induced p38 phosphorylation is inhibited by DN MKK3 as well as DN MKK6 in rat vascular smooth muscle cells (29). MKK function has also been evaluated in genetically engineered mice, in which TNF--induced cytokine expression in fibroblasts is decreased in MKK3–/– animals (30). Defective IL-12 production has also been noted in macrophages and dendritic cells from MKK3–/– mice (31). Both MKK3 and MKK6 are essential for TNF--stimulated p38 activation in vivo (32).
In addition to cell lineage-specific hierarchy of MKKs, the types of cellular stresses that activate MKK3 compared with MKK6 also vary widely. Our data suggest that, unlike some forms of stimulation, both MKK3 and MKK6 are rapidly activated in FLS by IL-1, which is a key cytokine implicated in RA. However, MKK6 is the primary pathway to p38 in murine lymphoma cells and human epithelial cells after osmotic stress (33), while MKK3 is required for full activation of p38 in murine embryonic fibroblasts (30). MKK6 is the principal activator of p38 in human epithelial cells stimulated with IL-1 (34), but MKK3 appears to be an important activator of p38 in LPS-stimulated murine peritoneal macrophages (31). In contrast, both the p38 and isoforms are activated by MKK3 in murine mesangial cells stimulated by TGF-1 (35).
The mechanisms by which p38 regulates gene expression are equally complex. In cytokine-stimulated synoviocytes, p38 inhibition by DN MKK3/6 or SB203580 had little effect on inflammatory gene expression despite significant decreases in the encoded proteins. Previous studies have indicated that this method of suppression is related to MAPKAPK-2-mediated translational effects. For instance, inhibition of TNF- and IL-1 protein synthesis by p38 blockade is not accompanied by a decrease in the corresponding steady state mRNA levels (36, 37). MAPKAPK-2 knockout mice exhibit impaired TNF- protein synthesis, with no discernible change in TNF- mRNA transcription or stability (38, 39). IL-6 and IL-8 mRNA translation is also regulated by p38, but extent of inhibition of protein production varies with cell type (40, 41, 42). These data are consistent with our studies on cytokine regulation by MKK3 and MKK6 in FLS, indicating that suppression of the p38 pathway is complex, but that downstream effects involve posttranslational mechanism.
The regulation of MMPs by p38 can involve several mechanisms. For example, wild-type MKK3 overexpression in human skin fibroblasts increases MMP-1 and MMP-3 mRNA expression, in part by stabilizing their respective RNA transcripts (4). The mRNA t1/2 appears to be prolonged due to decreased deadenylation of AU-rich tails (11). Our observation that DN MKK3 and DN MKK6 modestly decreased MMP-3 mRNA is consistent with an effect on gene transcription or mRNA stability. However, the changes in MMP-3 mRNA are relatively small compared with the decrease in MMP-3 protein levels. Hence, MMP regulation in FLS by p38 probably results from combination of mechanisms. The differences between our data and the dermal fibroblast study (4) are probably related to the distinct biology of wild-type overexpression compared with inhibition of the endogenous kinase.
In conclusion, our studies demonstrate that, unlike many tumor cells or physical stresses, MKK3 and MKK6 are key p38 regulators in cytokine-stimulated FLS. Both MKKs contribute to p38 activation, but MKK3 might be more important when attempting to suppress cytokine production. However, both must be blocked to interrupt p38 as effectively as small molecule p38 inhibitors in FLS. Because p38 pathway may participate in joint destruction and inflammation, selective MKK3, MKK6, or combined inhibitors have therapeutic potential for RA. Distinct activation pathways in different cell lineages suggests that the safety and efficacy profile might differ from competitive p38 inhibitors.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the National Institutes of Health.
2 Address correspondence and reprint requests to Dr. Gary S. Firestein, Division of Rheumatology, Allergy, and Immunology, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656. E-mail address: gfirestein@ucsd.edu
3 Abbreviations used in this paper: ATF, activating transcription factor; Ct, cycle threshold; DN, dominant negative; FLS, fibroblast-like synoviocyte; MAPKAPK, MAPK-activated protein kinase; MKK, MAPK kinase; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; rhIL, human rIL; FLAG, N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C.
Received for publication October 27, 2004. Accepted for publication January 19, 2005.
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