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The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-κB and the expression of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α). B. burgdorferi-induced TNF-α production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38α MAP kinase regulates the phosphorylation of NF-κB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-κB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-α in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-α production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-κB.
Lyme disease, the most common arthropod-borne disease in the United States, is caused by infection with the pathogenic spirochete Borrelia burgdorferi. In the eastern United States, B. burgdorferi is transmitted primarily during engorgement of the tick Ixodes scapularis on mammalian hosts. Recently, a shift in environmental conditions favoring the life cycle of the spirochete has augmented the number of Lyme disease diagnoses (31, 49). Typically, the earliest hallmark of infection with B. burgdorferi is the development of a characteristic skin rash, erythema migrans, which often occurs during the first week of infection and is usually accompanied by flu-like symptoms (52). However, more severe pathological consequences can manifest from infection with the spirochete, including conduction system abnormalities, meningitis, and acute arthritis, which appears in 60% of untreated individuals in the United States. Some untreated individuals become persistently infected with the spirochete, which can result in chronic inflammation (52). Treatment-resistant chronic pathologies resulting from B. burgdorferi infection have been reported previously (20, 47, 48).
The development of inflammation arising from infection with B. burgdorferi is dependent on several factors, including spirochetal virulence, the number of bacteria in the affected tissues, and the host immune response generated against B. burgdorferi, none of which appear to be mutually exclusive (3). Recent studies using mice that are deficient for Toll-like receptors (TLRs) or the TLR adaptor molecule MyD88 have shed light on the importance of cells that compromise the innate immune system, including phagocytic cells, for the clearance of infection and resolution of disease (9, 29, 54). Infection of these mice resulted in increased spirochetal burden at the peak of infection, although the impact of their deficiency on pathology varied among the studies. Those studies underscore the pressing significance of the early innate immune response for maintaining a level of infection that can be cleared by the more specific adaptive immune system.
B. burgdorferi induces the production of proinflammatory cytokines in different cell types (38, 39, 43, 44). Although it has been shown that B. burgdorferi stimulation of monocytes, mast cells, and other cell types results in increased expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and gamma interferon (IFN-γ), the specific mechanisms leading to this increased expression have not yet been completely elucidated (13, 30, 39, 51). B. burgdorferi contains lipidated outer surface proteins that activate the transcription factor NF-κB and subsequently upregulate the transcription of genes encoding chemokines and adhesion molecules in endothelial cells and fibroblasts (15, 34, 50). Indeed, the lipoproteins comprising the outer membrane of B. burgdorferi are the major immunogens of this spirochete, and as such, lipidated OspA, a model cell surface lipoprotein of B. burgdorferi, induces the translocation of NF-κB and the transcription of target genes in phagocytic cells following TLR engagement (55).
Activation of the p38 mitogen-activated protein (MAP) kinase pathway is critical for many immune response-related functions, including T-cell differentiation and death, macrophage and neutrophil effector functions (e.g., respiratory burst, granular exocytosis, etc.) (4), and the production of proinflammatory cytokines including TNF-α and IFN-γ (2, 22). Specific upstream activators of p38 MAP kinase include MKK3 and MKK6 (17), which catalyze the transfer of a phosphate group to threonine and tyrosine residues within the activation loop kinase subdomain VIII. Four isoforms of p38 MAP kinase (p38α, p38β, p38δ, and p38γ) have been identified, and each one is encoded by a different gene. The expression and function of the p38 MAP kinase family members are tissue specific, although some members have demonstrated overlapping substrate specificities (33, 46).
Members of the p38 MAP kinase family are selected targets for pyridinyl imidazole compounds (28). Pyridinyl imidazoles act by competitively inhibiting the binding of ATP to the ATP binding cleft of protein kinases and therefore prevent the catalytic transfer of a phosphate molecule from ATP to hydroxyls on serine, threonine, and tyrosine residues on the target substrate (18). SB203580, a member of this class of protein kinase inhibitors, specifically inhibits p38α and p38β MAP kinase function because it binds to a threonine side chain located in the ATP binding cleft (18). Other MAP kinases, including p38δ and p38γ, are not inhibited by SB203580 due to the presence of a larger side chain, such as methionine or glutamine (18, 19, 21).
p38 MAP kinase phosphorylates and activates transcription factors and other kinases, including the nuclear kinases mitogen- and stress-activated protein kinase 1 (MSK1) and MSK2. MSK1 activation constitutes a convergence point for the p38 MAP kinase and extracellular signal-regulated kinase (ERK) pathways, which are activated by different stimuli (12). The role of MSK1 in proinflammatory responses is associated with its ability to phosphorylate RelA at Ser276 and the subsequent increase in the transcriptional activity of the transcription factor NF-κB (53). Other substrates for MSK1 have been defined, including the transcription factor ATF-1 and the cyclic AMP response element binding (CREB) protein. CREB is a transcription factor involved in the regulation of several genes in different cell types including IL-2 during T-cell activation. However, since naïve CD4+ T cells express low or no levels of p38 MAP kinase (40), MSK1 is probably a substrate for the MAP kinase ERK. The regulation of CREB activity through MSK1 may be relevant in stress conditions, such as during oxidative stress (41), but it seems not to regulate IL-2 production during T-cell activation under nonstress conditions (41). In other cell types, MSK-dependent CREB/ATF-1 activation also regulates the expression of several proinflammatory genes, including IL-6 (25) and Cox-2 (16, 36). Therefore, MSK-dependent activation of both CREB/ATF-1 and NF-κB regulate the expression of several proinflammatory factors.
Here, we report the p38 MAP kinase signaling pathway required for the production of TNF-α by phagocytic cells in response to B. burgdorferi lysates. Our results show that p38 MAP kinase signaling directs the production of TNF-α by phosphorylating the transcriptionally active subunit of NF-κB, an event that is mediated by MSK1.
Mice that express the luciferase gene under NF-κB control (3 × NF-κB, NF-κB-luciferase) have been described previously (32). The transgenic mice were screened by PCR using primers specific to the reporter system (42). Age-matched littermates (B10.BR) were used as controls. All the experiments involving animals were approved by the institutional animal care and use committees at the University of North Carolina at Charlotte and the University of Massachusetts at Amherst.
RAW264.7 cells were purchased from the ATCC (Manassas, VA) and propagated in RPMI medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum, 3.7 g/liter sodium bicarbonate, and 5.96 g/liter HEPES at 37°C and 5% CO2.
Low-passage B. burgdorferi N40 cultures were grown in Barbour-Stoenner-Kelly II medium at 33°C. B. burgdorferi extracts were obtained from mid-log-phase cultures by washing the cells in phosphate-buffered saline, followed by sonication and clearance by centrifugation. The protein content of the bacterial extracts was quantified by the Bradford method (Bio-Rad, Hercules, CA).
In vitro stimulations were performed using a B. burgdorferi sonicate. All stimulations were performed using 10 μg/ml of the B. burgdorferi lysates, which correspond to the stimulation of cells with 106 live spirochetes per ml (Fig. (Fig.1).1). Where indicated, the cells were pretreated with increasing concentrations of the compound SB203580 (EMD Biosciences, San Diego, CA) (1 to 10 μM) 1 h prior to their stimulation.
Transfections were carried out in 3 × 106 RAW264.7 cells with 0.5 μg of plasmid in serum-free medium using Lipofectamine (10 μg/ml; Invitrogen, Carlsbad, CA) or DEAE dextran (Promega, Madison, WI) according to the manufacturers' instructions. Four hours (Lipofectamine) or 45 min (DEAE) after transfection, the cells were washed, and medium containing 10% fetal calf serum was replaced. The stimulations of the cells were carried out 36 to 48 h posttransfection.
Small interfering RNA (siRNA) transfections were carried out using 3 × 106 RAW264.7 cells with 10 nM of siRNA targeting p38α (SuperArray Bioscience, Frederick, MD) or MSK1 (New England Biolabs, Beverly, MA) and siPORT Amine (Ambion Inc., Austin, TX) in reduced-serum medium (Opti-MEM I; Invitrogen) according to the manufacturer's protocol. The cells were maintained in Opti-MEM medium for 16 h, washed, and incubated with serum-supplemented RPMI. Cell stimulations were performed 72 h posttransfection to ensure protein turnover.
CD11b+ cells were purified from the spleens of NF-κB-luc mice by positive selection, incubation with biotin anti-CD11b (BD Biosciences, San Jose, CA) followed by anti-biotin microbeads (Miltenyi Biotec, Auburn, CA), and separation over a magnetic column (Miltenyi Biotec). Purified CD11b+ cells were stimulated with B. burgdorferi cells for 48 h. The cells were then lysed with reporter lysis buffer and incubated with luciferase substrate (Promega, Madison, WI), and the reaction was measured using a TD20/20 luminometer (Turner Biosystems, Sunnyvale, CA). Similar experiments were performed using RAW264.7 cells transfected with the NF-κB reporter system or cells doubly transfected with the reporter and a plasmid expressing the dominant negative form of p38 MAP kinase (dnp38).
Analysis of I-κBα degradation was performed by Western blot analysis on RAW264.7 cells. Three to five million cells were stimulated with a B. burgdorferi lysate, as described above, in the absence or presence of SB203580 (5 μM). Alternatively, the cells were transfected with the dnp38 MAP kinase construct and stimulated as described above. At different time points, the cells were lysed, subjected to polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. The membranes were then probed with specific antibodies for I-κBα (Cell Signaling Technologies, Beverly, MA).
The nuclear translocation and phosphorylation of the RelA subunit of NF-κB as well as the presence of MSK1 were analyzed by Western blot analysis of nuclear extracts obtained from stimulated and treated RAW264.7 cells. The extracts were probed with antibodies specific for RelA (Santa Cruz Biotechnology, Santa Cruz, CA) and its phospho-Ser276 form (Cell Signaling Technologies) or antibodies specific for MSK1 (Santa Cruz Biotechnology) or phospho-MSK1 Ser360 (Cell Signaling Technologies).
Nuclear extracts from stimulated and treated cells were obtained as described above and subjected to an electrophoretic mobility shift assay (EMSA). The binding reaction was conducted using 1 μg of nuclear proteins and 105 cpm of 32P-end-labeled double-stranded NF-κB oligonucleotides derived from the mouse B intronic enhancer (5′-GAT CAG AGG GGA CTT TCC GAG-3′). The complexes were resolved in a 6% acrylamide gel. The gels were dried and exposed to X-ray film (Kodak, Rochester, NY) at −80°C.
Total RNA was extracted from RAW264.7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, treated with DNase I (Promega, Madison, WI), and reverse transcribed using Random primers (Invitrogen) and SuperScript II reverse transcriptase (RT) (Invitrogen). The cDNA was amplified using specific primers and Taq DNA polymerase (Promega). The primers used were as follows: msk1 Forward (5′-CGA CAA GGC AGT TGA GTG-3′) and msk1 Reverse (5′-GCGAAGTTACTCACATCTAACT-3′) (350 bp). The identity of the PCR-amplified fragment was verified by size comparison with DNA standards. All samples were normalized based on the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (forward, 5′-CAC AGT CTT CTG GGT GGC AGT GAT-3′; reverse, 5′-GAG CGA GGA CCT TCT ACC ACT ACC-3′ [347 bp]).
B. burgdorferi-induced proinflammatory cytokine production by phagocytic cells is dependent on the activation of p38 MAP kinase (2). However, the intracellular signaling pathways leading to the expression of proinflammatory cytokines in response to B. burgdorferi remain unsolved. Others have reported previously that p38 MAP kinase signaling is required for NF-κB activation in response to different stimuli (10, 26, 36, 53). Furthermore, NF-κB has been identified as the transcriptional regulator of TNF-α in murine macrophages (1, 11, 14). Thus, to begin exploring the pathways underlying spirochete-induced proinflammatory cytokine production, we assessed the effect of the p38 MAP kinase inhibitor SB203580 on NF-κB activity in phagocytic cells following stimulation with B. burgdorferi. Purified splenic CD11b+ cells (3 × 106 cells) from NF-κB-luciferase-transgenic mice were stimulated in vitro with 10 μg/ml of a B. burgdorferi lysate that corresponds to approximately 106 spirochetes/ml (Fig. (Fig.1)1) in the presence of increasing concentrations of SB203580 (0, 1, 5, and 10 μM). Luciferase activity was measured 48 h later. The presence of increasing concentrations of the inhibitor caused a reduction in the luciferase activity (Fig. (Fig.2A),2A), suggesting that p38 MAP kinase is required for NF-κB transcriptional activation following stimulation with B. burgdorferi. Similar results were obtained when RAW264.7 cells that overexpressed a dominant negative form of p38 MAP kinase were stimulated with B. burgdorferi lysates (Fig. (Fig.2B).2B). RAW264.7 cells were cotransfected with plasmids containing the luciferase gene under three repeats of NF-κB consensus binding sequences and dnp38 MAP kinase. Forty hours later, the cells were stimulated with 10 μg/ml of a B. burgdorferi lysate. Overexpression of the dominant negative form of p38 MAP kinase resulted in decreased luciferase activity (Fig. (Fig.2B).2B). Taken together, these data suggested that p38 MAP kinase is a necessary component of the signaling pathway leading to the transcription of NF-κB target genes in phagocytic cells following stimulation with extracts of B. burgdorferi.
We argued that our previous results could be due to a direct effect of p38 MAP kinase on NF-κB activation (10, 26, 36, 53) or an indirect effect due to decreased p38 MAP kinase-mediated TNF-α production (2), which also activates NF-κB (53). In order to dissociate these two alternatives, we first sought to determine whether p38 MAP kinase inhibition during stimulation of macrophages with the spirochete affected the degradation of I-κBα, the inhibitory subunit of the NF-κB complex at early time points, in which no TNF-α is yet produced (data not shown). RAW264.7 cells (3 × 106 cells) were stimulated with 10 μg/ml of a B. burgdorferi extract in the absence or presence of 5 μM SB203580 for 0, 5, 10, 20, 30, and 60 min. Total cell extracts were analyzed for I-κBα by Western blotting. The inhibition of p38 MAP kinase did not prevent the degradation of I-κBα in response to B. burgdorferi antigens (Fig. (Fig.3A).3A). Similarly, Western blot analysis of the DNA binding subunit (p50) and the transcriptionally active subunit (RelA) showed that the inhibition of p38 MAP kinase had no effect on the nuclear translocation of these proteins (data not shown and see Fig. Fig.3C3C for RelA), suggesting that p38 MAP kinase-mediated control of NF-κB activation occurred downstream of the translocation of the transcription factor to the nucleus. To assess whether the inhibition of p38 MAP kinase activity during stimulation with B. burgdorferi lysates had affected the DNA binding activity of nuclear NF-κB, we performed EMSA using nuclear extracts of RAW264.7 cells that had been stimulated for 30 min with 10 μg/ml of a spirochetal lysate in the absence or presence of 5 μM SB203580. One microgram of nuclear protein was tested for its ability to form complexes with double-stranded oligonucleotides corresponding to the NF-κB consensus binding sequence. The presence of the inhibitor during stimulation of the macrophages did not affect the binding of NF-κB to its consensus binding sequence (Fig. (Fig.3B).3B). Overall, these data indicate that p38 MAP kinase activity does not regulate the translocation of NF-κB to the nucleus or NF-κB binding to its target sequence upon stimulation.
Our data suggested that p38 MAP kinase affects the transcriptional activation of NF-κB but not its ability to translocate to the nucleus and bind target sequences. Some reports previously suggested that p38 MAP kinase is involved in the phosphorylation of RelA, the transcriptionally active subunit of the NF-κB complex at Ser276, leading to the full activation of the transcription factor. Thus, we tested whether the inhibition of p38 MAP kinase activity during stimulation with spirochetal extracts affected the phosphorylation of RelA at this particular residue. RAW264.7 cells were activated for 0, 5, 10, 15, 30, and 60 min as described above, and nuclear extracts were immunoblotted with an anti-phospho-Ser276 RelA antibody. The inhibition of p38 MAP kinase during stimulation resulted in reduced RelA phosphorylation (Fig. (Fig.3C),3C), indicating that the contribution of p38 MAP kinase-mediated signaling to the activation of NF-κB occurred through the phosphorylation of the transcriptionally active subunit of the NF-κB complex. Similar results were obtained when RAW264.7 cells were transfected with plasmid dnp38 and analyzed as described above (data not shown). The decreased RelA phosphorylation in the presence of SB203580 in response to a B. burgdorferi lysate was also observed in purified splenic CD11b+ cells, confirming that the effect occurs in primary phagocytic cells (data not shown). Thus, activation of p38 MAP kinase is a necessary precursor to RelA phosphorylation and transcription of NF-κB target genes. p38α is the major isoform involved in the production of proinflammatory cytokines by macrophages (6). Therefore, we tested the contribution of the α isoform of p38 MAP kinase to the phosphorylation of RelA. RAW264.7 cells (3 × 106 cells) were transfected with an siRNA mixture targeting p38α MAP kinase. The transfection resulted in the efficient inhibition of p38α MAP kinase expression, as demonstrated by RT-PCR (Fig. (Fig.4A).4A). The siRNA-transfected (and control transfected) cells were then stimulated with B. burgdorferi lysates for 30 min, and the phosphorylation status of RelA at Ser276 was determined as described above. The silencing of the p38α MAP kinase gene resulted in diminished RelA phosphorylation following spirochetal stimulation compared to siRNA-transfected controls (Fig. (Fig.4B).4B). Collectively, these results indicate that p38 MAP kinase signaling is required for the transcriptional activation of NF-κB through the phosphorylation of RelA. Moreover, our results suggest that p38α MAP kinase is the specific isoform involved in the signaling pathway leading to the phosphorylation of RelA in response to antigens of B. burgdorferi.
p38 MAP kinase controls several cellular processes through the phosphorylation of an array of substrates. These include transcription factors, such as ATF-2, and the nuclear kinases mitogen-activated protein kinase-interacting kinase 1 (MNK1), MNK2, mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2, MSK1, and MSK2 (35). Upon activation, nuclear kinases can phosphorylate specific residues on multiple substrates. For example, MSK1 phosphorylates CREB at Ser133 and ATF-1 at Ser63 and has been associated with the transcriptional activation of NF-κB through the phosphorylation of RelA at Ser276 (4, 8). Thus, we explored the possibility that MSK1 is involved in B. burgdorferi-induced and p38 MAP kinase-directed activation of NF-κB in phagocytic cells. RAW264.7 cells (3 × 106 cells) were stimulated for 30 min with a B. burgdorferi lysate (10 μg/ml) in the presence or absence of SB203580 (5 μM). Stimulation of the phagocytic cells in the absence of the inhibitor resulted in the phosphorylation of MSK1 at Ser360; however, the presence of the inhibitor reduced this phosphorylation (Fig. (Fig.5).5). The effect was also observed in purified splenic CD11b+ cells (data not shown). Based on these results, we hypothesized that MSK1, a downstream target of p38 MAP kinase, is the direct activator of NF-κB in stimulated phagocytic cells.
We thus tested the contribution of MSK1 to p38 MAP kinase-directed phosphorylation of RelA. RAW264.7 cells (3 × 106 cells) were transfected with an oligonucleotide mixture that targets the MSK1 mRNA. Seventy-two hours posttransfection, the cells were stimulated for 30 min with 10 μg/ml of a B. burgdorferi lysate, and the phosphorylation status of RelA at Ser276 was determined as described above. Silencing of the MSK1 gene disrupted the signaling pathway that leads to RelA phosphorylation following spirochetal stimulation, indicating that this downstream target of p38 MAP kinase is involved in the activation of NF-κB in response to antigens of B. burgdorferi. (Fig. (Fig.6A).6A). To further substantiate the role of MSK1 activity in proinflammatory cytokine production in response to B. burgdorferi lysates, we also analyzed the capacity of RAW264.7 cells lacking MSK1 to produce TNF-α. siRNA-transfected cells were stimulated with a B. burgdorferi lysate, and the supernatants were analyzed for TNF-α production. As expected, cells transfected with siRNA for MSK1 produced significantly lower amounts of TNF-α in response to B. burgdorferi antigens than transfected controls (P < 0.001) (Fig. (Fig.6B).6B). In order to verify the repression of MSK1 expression, the cells were harvested, and RT-PCR for MSK1 was performed. The siRNA efficiently repressed the expression of the msk1 gene (Fig. (Fig.6C),6C), which corresponded with lower protein levels, compared to the controls (Fig. (Fig.6A).6A). Collectively, these results indicate that B. burgdorferi antigen-induced p38 MAP kinase signaling in phagocytic cells controls proinflammatory cytokine production through its activation of MSK1, the direct activator of NF-κB. Thus, p38 MAP kinase has a central role in the activation of phagocytic cells following stimulation with antigens of B. burgdorferi.
The response of phagocytic cells to B. burgdorferi is, for the most part, attributed to TLR-mediated signaling that results in the production of cytokines and other proinflammatory factors (9, 45, 54, 56). Impaired TLR signaling results in higher spirochetal burden and various levels of inflammation in mice at the peak of infection with B. burgdorferi (7, 9, 29, 54, 56). Recently, Behera and colleagues identified a TLR-independent mechanism by which B. burgdorferi interacts with human chondrocytes, resulting in the production of proinflammatory cytokines and matrix metalloproteinases (8). The increased bacterial burden and continued interaction of the spirochete with phagocytic cells through this alternative mechanism may at least partially account for the persistent inflammation that coincides with higher spirochetal numbers in TLR2- and MyD88-deficient mice (7, 9, 29, 54, 56). Thus, the binding of an unknown ligand(s) of B. burgdorferi to the integrin α3β1 leads to proinflammatory cytokine production, which seems to be independent of p38 MAP kinase activity (8). However, the exact role of this interaction in the immune response to the spirochete and the development of acute inflammation remains unsolved.
Our results demonstrate a central role for p38 MAP kinase activity in the generation of the proinflammatory response to the spirochete by regulating the activation of NF-κB in an MSK1-dependent fashion. We show that in response to B. burgdorferi antigens, MSK1, a nuclear substrate for p38 MAP kinase, is critical for the expression of TNF-α through the control of the transcriptional activation of NF-κB by RelA phosphorylation at Ser276. Since integrin-mediated signaling does not seem to depend on the activation of p38 MAP kinase (8), our results suggest that the response that we report is mediated through the interaction of B. burgdorferi antigens with TLRs and are independent, at least initially, from the probable synergistic effect induced by TNF-α, which also signals through p38 MAP kinase (2). These results are in agreement with previous reports indicating that p38 MAP kinase is necessary for the transactivation of NF-κB in response to TLR ligands such as lipopolysaccharides (24), microorganisms such as Mycobacterium avium (36), and cytokines, including TNF-α (53) and IL-1β (24). Our results also confirm in vivo observations of mice deficient in MKK3, an upstream activator of p38 MAP kinase, that were infected with B. burgdorferi (2). These mice had lower levels of TNF-α in sera than their littermate infected controls (2). The results presented in this report clarify the mechanisms of this reduction.
Our data do not argue against a potential contribution of transcription factors other than NF-κB, located downstream of MSK1, in B. burgdorferi-induced expression of proinflammatory factors. Indeed, MSK1 has been shown to regulate the transcriptional activation of CREB, which is also involved in the expression of, for example, the cox-2 gene (36). Moreover, MSK1 and MSK2 are involved in the phosphorylation of histone H3, which potentially further implicates MSK1 in the expression of proinflammatory genes (27). However, our results clarify the contribution of NF-κB to TNF-α production in response to B. burgdorferi antigens, which requires the activation of p38 MAP kinase-induced MSK1 activity. However, the incomplete inhibition of RelA phosphorylation under conditions of repressed or absent p38 MAP kinase activity suggests that other kinases may be involved in this response.
MSK1 is also a downstream target of ERK, which has been proposed to be activated following TLR engagement (5, 37). The relative contribution of ERK and p38 MAP kinase to the activation of MSK1 is unclear, although the high levels of inhibition reached by the presence of the specific p38 MAP kinase inhibitor, or the presence of the dominant negative form of the kinase, in NF-κB transcriptional activity suggest that the p38 MAP kinase-MSK1 pathway is predominant in our system.
Other substrates of p38 MAP kinase, including MAPKAP-2, have been involved in the generation of proinflammatory factors and noninfectious arthritis (23). The participation of this downstream kinase in the production of these factors seems to be at the posttranscriptional level (23). Therefore, the predominant and precedent role of MSK1 in the transcription of proinflammatory genes suggests that this nuclear kinase may be an excellent target for the generation of specific therapies that target inflammation, including arthritis induced by infection with B. burgdorferi. The relatively narrow activity of the kinase specifically related to proinflammatory factor production and its position as a convergence point of proinflammatory signals makes it a better target than its upstream activators, p38 MAP kinase and ERK. Furthermore, as opposed to p38 MAP kinase, the role of MSK1 in acquired immune responses, namely, T-cell function, seems to be restricted to stress conditions in which the ERK-MSK1 pathway may contribute to IL-2 production during T-cell activation (41). Even though the inhibition of p38 MAP kinase in B. burgdorferi-specific effector T cells results in decreased IFN-γ production and does not seem to result in a less efficient clearance of bacteria (2), a narrower target should provide a better safety profile.
In addition to clarifying the contribution of the p38 MAP kinase signaling pathway to the upregulation of proinflammatory cytokines by phagocytic cells responding to antigens of B. burgdorferi, our results also provide insight into the signaling pathways that occur downstream of TLR engagement with B. burgdorferi lipoproteins. In response to TLR engagement, two distinct but converging signaling pathways are activated, resulting in the transcriptional activation of NF-κB. The p38 MAP kinase-independent signaling pathway leads to the translocation and binding of the NF-κB complex to its target DNA, while the p38 MAP kinase-dependent pathway results in the phosphorylation of RelA and thus the activation of NF-κB and the subsequent transcription of its target genes. This latter pathway is dependent on the phosphorylation and activation of MSK1 (Fig. (Fig.7).7). Moreover, our results suggest that the α isoform of p38 MAP kinase regulates the transcriptional activation of NF-κB following stimulation with antigens of B. burgdorferi.
This work was supported by NIH grant AR 048265 and an award from the American Heart Association to Chris M. Olson.
We are grateful to Mercedes Rincón for providing the NF-κB-luciferase mice and the dnp38 plasmid.
Editor: D. L. Burns
Published ahead of print on 30 October 2006.