Originally published online as doi:10.1189/jlb.1006639 on February 8, 2007
Published online before print February 8, 2007
(Journal of Leukocyte Biology. 2007;81:860-869.)
© 2007
by Society for Leukocyte Biology
Regulation of innate immunity by MAPK dual-specificity phosphatases: knockout models reveal new tricks of old genes
Konstantin Salojin1 and
Tamas Oravecz
Lexicon Genetics Incorporated, The Woodlands, Texas, USA
1 Correspondence: Lexicon Genetics Inc., 8800 Technology Forest Place, The Woodlands, TX 77381, USA. E-mail: ksalojin{at}lexgen.com
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ABSTRACT
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Throughout evolution, mammals have developed an elaborate network of positive and negative regulatory mechanisms, which provide balance between defensive measures against bacterial and viral pathogens and protective measures against unwarranted destruction of the host by the activated immune system. Kinases and phosphatases encompassing the MAPK pathway are key players in the orderly action of pro- and anti-inflammatory processes, forming numerous promiscuous interactions. Several lines of evidence demonstrate that the phosphorylation and activation status of kinases in the MAPK system has crucial impact on the outcome of downstream events that regulate cytokine production. At least 13 members of the family of dual-specificity phosphatases (DUSP) display unique substrate specificities for MAPKs. Despite the considerable amount of information obtained about the contribution of the different DUSP to MAPK-mediated signaling and innate immunity, the interpretation of available data remains problematic. The in vitro and ex vivo findings are often complicated by functional redundancy of signaling molecules and do not always accurately predict the situation in vivo. Until recently, DUSP research has been hampered by the lack of relevant mammalian knockout (KO) models, which is a powerful tool for delineating in vivo function and redundancy in gene families. This situation changed dramatically over the last year, and this review integrates recent insights into the precise biological role of the DUSP family in innate immunity gained from a comprehensive analysis of mammalian KO models.
Key Words: transgenic • knockout mice • monocytes/macrophages • autoimmunity • inflammation • kinases/phosphatases
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INNATE IMMUNITY AND MAPK SIGNALING
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Stimulation of the innate immune system by a variety of agents induces inflammatory reactions, including production of proinflammatory cytokines such as TNF-
, IL-1ß, IL-6, IL-12, and IFN-
. The primary role of the rapid inflammatory response is to protect the host by timely activation of defense mechanisms, but a disproportionate cytokine response can also lead to detrimental effects, as is the case during sepsis and septic shock [1
2
3
]. Therefore, activation of the proinflammatory signaling cascade also triggers negative-feedback mechanisms, which can restrain and terminate the innate immune response. The MAPK pathway is a primary example of the balanced biochemical network whose elements are regulated carefully during inflammation. The initial activation of the inflammatory cascade is facilitated by the interaction between microbial molecular patterns and TLRs [2
]. The ensuing signal transduction events lead to activation of Ser/Thr protein kinases, which belong to the three MAPK subfamilies, p38 MAPK, JNK, and ERK [4
5
6
7
]. The counter-mechanism that limits innate immune responses and TLR signaling involves phosphatases, which are up-regulated by stress-induced stimuli and display promiscuous substrate specificities with respect to the different MAPKs [4
].
Several lines of evidence demonstrate that the activation status of numerous kinases in the MAPK pathway has a crucial impact on the outcome of downstream events, which regulate proinflammatory cytokine production. Activity of p38 MAPK, JNK, and ERK1/2, as well as several enzymes up- and downstream of these kinases, is regulated by their phosphorylation status (Fig. 1
). Transient activation and increased phosphorylation of MAPKs were observed in immortalized and primary murine macrophages after stimulation via TLR4 [8
]. Phosphorylation of p38 MAPK on tyrosine and threonine residues is critical for activation of multiple transcription factors (e.g., NF-
B, ATF-2, Elk-1, and C/EBP homologous protein), which control expression of proinflammatory cytokine genes [5
, 9
]. Several downstream kinases are also regulated through direct phosphorylation by p38 MAPK. In mice, targeted deletion of one of these enzymes, MAPK-activated protein kinase-2, leads to 90% reduction in the production of TNF- and renders the animals resistant to LPS/D-galactosamine-induced shock [6
]. Furthermore, deletion of MKK3, which is one of the two upstream kinases whose target is p38-MAPK, leads to defective LPS- and CD40 ligand-induced IL-12 production by macrophages and bone marrow-derived dendritic cells (DC) ex vivo, accompanied by a significant decrease in total p38 activity [10
]. However, MKK3 knockout (KO) mice still retain the ability to produce IL-6 in response to TLR4 ligation in a p38-dependent manner in vivo [11
]. Further upstream in the MAPK signaling pathway lies MKKK 3 (MEKK3), which is an essential signal transducer of the MyD88/IL-1 receptor (IL-1R)-associated protein kinase/TNF receptor (TNFR)-associated factor 6 complex, formed during TLR4 signaling [12
]. Embryonic fibroblasts from MEKK3-deficient mice exhibit deficient TLR4-induced IL-6 production and defective IL-1- and LPS-induced activation of not only p38 MAPK but also JNK and NF-B [12
]. Just like p38-MAPK, the JNK subfamily of MAPKs influences many aspects of leukocyte biology, such as stress response, cell growth, and apoptosis [13
14
15
]. LPS and the proinflammatory cytokines TNF- and IL-1 induce phosphorylation of multiple substrates of JNK, including the transcriptional regulators c-Jun, ATF2, and Elk-1 [13
14
15
]. In addition, JNK2 is required for efficient induction of Type I IFNs and IL-6 in response to viral infection or dsRNA [13
]. Besides transcriptional regulation, the JNK and p38 MAPK signaling pathways are also important contributors to the LPS-induced expression of TNF- by stabilizing the TNF- mRNA and relieving its translational silencing [9
]. Finally, the importance of the ERK subfamily of MAPKs in innate immunity was revealed by analysis of the function of tumor progression locus 2 (TPL-2), a MKKK (MAP3K8), which activates ERK-1 and ERK-2 by phosphorylating and activating two upstream MKKs, MEK-1 and MEK-2, respectively [16
, 17
]. TPL-2 is essential for activation of MEK-1, ERK-1, and ERK-2 but not JNK, p38 MAPK, and NF-B in LPS-stimulated peritoneal macrophages [17
]. TPL-2 also controls ERK-mediated signals originated from members of the TNFR superfamily, including CD40 and the TNFR1 [18
]. Once more, KO mice provided in vivo evidence for an important role of TPL-2 in LPS-induced TNF- release [17
], in agreement with studies showing a notable defect in TNF- induction in murine macrophages treated with the MEK inhibitor PD98059 [17
].
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Figure 1. Regulation of MAPK signaling pathways by dual-specificity phosphatases (DUSP). Ligation of TLR and cytokine receptors initiates the MAPK kinase kinase (MKKK)-MKK-MAPK signaling cascades, leading to phosphorylation and activation of downstream transcriptional targets. These signaling pathways play an important role in the production of cytokines and various proinflammatory proteins. Dephosphorylation and inactivation of MAPKs by DUSP are critical negative-feedback mechanisms, which limit TLR-mediated, proinflammatory cytokine production and innate immune responses. MKP-1, MAPK phosphatase-1; PAC-1, phosphatase of activated cells 1; DSP2, dual-specificity protein phosphatase 2; ATF2, activating transcription factor 2; ELK-1, ets-like protein 1; MEF-2C, myocyte enhancer factor 2C; VHR, vaccinia H1-related; HVH, homologue of vaccinia virus H1.
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REGULATION OF INNATE IMMUNITY BY DUSP
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A complex network of negative regulatory mechanisms has been developed by mammals throughout evolution to control TLR signaling and prevent deleterious effects of innate immune response elicited by bacterial and viral pathogens (reviewed in refs. [19
, 20
]). Part of this feedback mechanism is inactivation of MAPKs by phosphatases, which act on phosphotyrosine residues (HePTP/LC-PTP and PTP-SL/STEP) or on serine/threonine residues (PP1, PP2A, PP2B, and PP2C) or on both (DUSP) [21
, 22
].
Human and mouse genomic studies identified at least 30 phosphatases, which share two common features: a C-terminal catalytic domain, which contains a conserved sequence motif [VXVHCXXGXSRSXTXXXAY(L/I)M], and two N-terminal, Cdc25-like CH2 domains [23
, 24
]. These phosphatases belong to the DUSP family [21
, 24
, 25
]. At least 13 members of the family display unique substrate specificities for MAPKs (Table 1
). Several DUSP (DUSP8, DUSP9, and DUSP10) dephosphorylate their substrates in the nucleus and cytoplasm, and others are localized exclusively in the nucleus (DUSP1, DUSP2, DUSP4, and DUSP5) or in the cytoplasm (DUSP6, DUSP7, and DUSP16; reviewed in ref. [24
]). Only a few of DUSP display a restricted tissue distribution pattern: DUSP2 is expressed mainly in lymphoid tissues [24
, 33
]; DUSP8 is expressed predominantly in the adult brain, heart, and skeletal muscle [24
, 34
, 35
]; DUSP9 was detected only in placenta, kidney, and fetal liver [24
, 36
]. Although the majority of DUSP shows a ubiquitous expression pattern, their distribution and expression kinetics in hematopoietic tissues and lymphoid organs varies significantly (Table 1)
. Myeloid cells and lymphocytes express MKP-1, MKP-2, MKP-5, PYST2, and PAC-1 [27
, 37
, 38
]. In contrast, MKP-3 and DSP2 were detected only in monocytes and macrophages, and HVH3, MKP-6, MKP-7, and DSP2 show a more lymphocyte-specific expression in immune tissues. In addition, a systematic analysis of the murine thymus DUSP transcriptome revealed thymic expression of 10 DUSP, including DUSP1, -2, -4, -5, -6, -7, -10, -11, -12, and -19 [39
]. Seven of these DUSP were true MKPs, based on substrate specificity demonstrated previously.
In vitro studies indicate that p38 MAPK, JNK, and ERK1/2 are substrates of a multitude of DUSP [21
, 22
, 40
41
42
], which suggests significant enzymatic redundancy within this family. However, a certain degree of selectivity in the phosphatase-mediated regulation of MAPKs can be achieved through temporal variations in the expression kinetics and spatial compartmentalization of phosphatases during cell activation. The "sequential phosphatase" model proposes that these regulatory enzymes act sequentially, responding to different extracellular stimuli [22
]. As an example, unchallenged immune tissues do not show significant expression of MKP-5, but it is strongly induced in mouse macrophages after LPS treatment [7
]. Similarly, some DUSP, such as MKP-1, -2, -3, -5, and -7, PAC-1, HVH3, and HVH5, are silent in quiescent cells but are induced rapidly by various growth stimuli and stress signals, which act through the MAPK signaling pathways (Table 1)
. Differences in post-transcriptional stabilization of DUSP mRNAs may add an extra layer to the variation in enzyme activity. In addition, binding studies have established direct association of some phosphatases with their substrates and suggest that catalytic activity is promoted as a consequence of substrate binding. MKP-5 has been shown to interact with p38 and JNK kinases [43
, 44
]. In contrast, MKP-1 and MKP-3 preferentially bind p38-MAPK and ERK [42
, 45
], and tight association between MKP3 and ERK enhances phosphatase activity [45
].
Despite the considerable amount of information about the contribution of different DUSP to MAPK-mediated signaling and innate immunity, its interpretation remains problematic, as the overlapping substrate specificities defined in vitro may have lesser impact on physiological mechanisms operating in the whole organism [24
]. Until recently, DUSP research has been hampered by the lack of relevant mammalian KO models. The situation changed dramatically over the last year when several groups used gene targeting to elucidate the physiological roles played by MKP-1, MKP-5, and PAC-1 in the immune system. For the rest of this review, we will focus on recent insights into the precise biological role of the DUSP family of MAPK-specific phosphatases in the regulation of innate and adaptive immunity, which have been gained from a comprehensive analysis of mammalian KO models. We will also discuss recent advancements in our understanding of the specific immune functions of each member of the DUSP family, attained as part of our Genome5000 program [46
47
48
], which incorporates a large-scale, phenotypic analysis of KO mice for innate and adaptive immune responses, as well as for other physiological parameters relevant to drug development [46
, 47
]. As part of this screen, we have generated and analyzed several mouse lines, which are deficient for expression of various enzymes involved in MAPK signaling, including a number of DUSP.
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MKP-1 (DUSP1)
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MKP-1 is a phosphatase of 39.5 kDa, widely expressed in various tissues [21
, 22
, 41
, 49
]. In vitro studies suggest that MKP-1 is a nuclear phosphatase that dephosphorylates p38 MAPK and JNK, and its activity against ERK is noticeably lower [40
, 50
]. Expression of MKP-1 is strongly up-regulated by various stimuli, including growth factors, LPS, p53, osmotic and heat shock, anisomycin, UV, 12-O-tetradecanoylphorbol 13-acetate, and Ca2+ ionophores [21
, 22
, 41
, 49
]. MKP-1 is constitutively expressed in human lymphoid organs, including spleen, lymph nodes, and thymus, and its expression is induced rapidly after activation of various cell types within the immune system, including CD4- and CD8-positive T cells, B cells, neutrophils, and monocytes (Table 1)
. In LPS-stimulated mouse macrophages, MKP-1 shows a transient expression pattern with rapid induction, followed by a quick return to basal levels [28
].
MKP-1 is considered to be an important negative-feedback regulator of macrophage function and inflammatory responses to TLR signals transduced via the p38 MAPK pathway [8
, 37
, 50
51
52
]. This model is supported by the observations that activation of MAPK signaling was accompanied by induction of MKP-1-mediated MAPK dephosphorylation, and transient overexpression of MKP-1 in THP-1 cells and peritoneal macrophages induced endotoxin tolerance and down-regulation of TNF- production via inhibition of p38 MAPK phosphorylation [51
]. Furthermore, ectopic expression of MKP-1 in RAW264.7 macrophages and adenovirus-mediated MKP-1 overexpression in immortalized alveolar macrophages inhibited the production of TNF- and IL-6 by accelerating JNK and p38 inactivation [8
, 37
].
Recently, we and others [27
28
29
30
] examined the contribution of MKP-1 to the negative regulation of innate immunity, inflammatory responses, and signaling events downstream of p38 MAPK in mice with a targeted disruption of the MKP-1 gene. Although MKP-1-deficient mice did not show significant abnormalities in lymphoid and myeloid development, the genetic deletion led to markedly elevated serum levels of proinflammatory cytokines and other mediators of inflammation, such as PGE2, in response to bacterial LPS challenge, compared with the normal response. This exacerbated, inflammatory response was accompanied by a dramatically increased mortality rate in the low-dose endotoxin challenge model. The innate immune response in this inflammatory stress model mimics bacterial infection and is mediated by the release of macrophage-derived cytokines such as TNF-, IL-6, IL-12, and IL-10 and the proinflammatory chemokines CCL3, CCL4, and CXCL2, triggered by the dimerization of the TLR4 complex on macrophages [27
28
29
30
, 53
, 54
]. Thus, the hyper-responsiveness to the low-dose LPS-induced toxicity observed in MKP-1–/– mice is likely a result of uncontrolled release of proinflammatory cytokines from activated macrophages, leading to LPS-induced hepatotoxicity and shock. Consequently, MKP-1–/– mice were more susceptible to the development of septic shock syndrome, associated with hypotension, respiratory failure, increased NO production, and multiple organ failure, as revealed by significantly accelerated renal, hepatic, and pulmonary damage in MKP-1–/– mice in response to LPS challenge [27
]. In our studies, IFN- production was also significantly above the normal values in LPS-challenged MKP-1–/– mice. As this cytokine is produced by T and NK cells after activation by macrophage-derived IL-12, this observation provides a potential link between MKP-1 deficiency and enhanced T cell-mediated effector immune function, contributing to endotoxin shock [29
]. However, Hammer et al. [30
] found no significant effect of MKP-1 deficiency on systemic release of IL-12 and IFN-. Although the exact reason for these differences is unclear, we suggest that the dose of LPS (1 mg/kg vs. 10 mg/kg) used in the studies and the duration of the assays (2 h vs. 6 h) may account for this different outcome.
Ex vivo cell activation studies demonstrated that the TNF- response of MKP-1–/– macrophages after stimulation by TLR-binding agents was markedly higher than that of wild-type cells, irrespective of the stimulators’ TLR specificity. The studies examined responses to zymosan and bacterial lipoprotein, which act via TLR2 [4
, 55
], polyinosinic:polycytidylic acid [poly(I:C)], which triggers TLR3 [4
, 56
], LPS, which binds to TLR4 [57
, 58
], and flagellin and CpG, which trigger TLR5 and TLR9, respectively [4
, 56
]. LPS-stimulated MKP-1–/– splenocytes and DC exhibited a similar pattern of elevated cytokine production induced by LPS (ref. [27
] and our unpublished data). The higher cytokine response was accompanied by increased expression of CD86 and CD40 on LPS- and flagellin-activated MKP-1–/– macrophages [29
]. CD86 is a costimulatory ligand, which potentiates T cell activation via its interaction with CD28, whereas CD40 and its ligand play a critical role in the induction of CD86 expression and stimulation of proinflammatory cytokine production by macrophages. Taken together, these observations are consistent with the role of the TLR adaptor molecules MyD88 and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-ß (TRIF) in TLR-mediated MKP-1 induction [28
]. MyD88 is a critical adaptor molecule, which transduces signals from TLR1, -2, -4, -5, -6, -7, -8, and -9 [4
], whereas TRIF operates in a MyD88-independent manner downstream of TLR3 and TLR4 to promote IFN-ß production [59
, 60
]. Using MyD88-deficient and TRIF-deficient mice, Chi et al. [28
] demonstrated that both of these adaptor proteins were necessary for optimal LPS-induced MKP-1 expression.
In agreement with the ability of MKP-1 to inhibit activation of p38 MAPK in a variety of mouse and human cell types [8
, 37
, 50
51
52
], MKP-1-deficient macrophages displayed significantly elevated and sustained levels of phospho-p38 MAPK after ligation of TLR4 and TLR9 with LPS and CpG, respectively [27
, 29
, 30
, 52
]. In addition, Chi et al. [28
] observed a substantial increase of the AP-1 transcriptional factor activity in MKP-1–/– macrophages. We also found that the expression of costimulatory molecules and increased TNF- production by LPS-stimulated MKP-1–/– cells could be inhibited by the p38 MAPK-specific inhibitor SB203580, which also inhibits the production of TNF- and IL-1 selectively by LPS-stimulated human monocytes [5
, 61
, 62
]. Besides regulating proinflammatory cytokine production, p38 MAPK also controls the release of the anti-inflammatory cytokine IL-10 from LPS-stimulated macrophages, and in turn, IL-10-induced Bcl-3 is required for suppression of TNF- production in macrophages stimulated with LPS [63
64
65
66
]. Analysis of IL-10 secretion in the low-dose endotoxin challenge model and in activated macrophage cultures demonstrated significant potentiation of IL-10 production by MKP-1 deletion [27
28
29
30
]. Chi et al. [28
] also showed that chemical inhibition of MKP-1 abrogated the increased IL-10 production completely in LPS- or flagellin-stimulated MKP-1–/– macrophages. Moreover, the delayed kinetics of IL-10 induction in LPS-stimulated macrophages suggests a biphasic nature of macrophage activation: The p38 MAPK-dependent synthesis of IL-10 during the second phase of macrophage activation is required to offset, in an autocrine/paracrine manner, the production of proinflammatory cytokines observed during the initial phase of macrophage activation [27
, 28
]. Altogether, the in vivo and ex vivo KO studies strongly implicate MKP-1 as a negative regulator of the inflammatory response of macrophages via diverse TLR and p38 MAPK signaling pathways, which mediate pro- and anti-inflammatory cytokine production during activation of the innate immune system. It is important that we also showed that even in a relatively quiescent state, MKP-1-deficient macrophages maintain a higher basal level of phosphorylated p38 MAPK [29
]. This indicates that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function. Of note, Gadd45, a small p38 MAPK-binding molecule, has been demonstrated recently to serve a similar function in T cells. p38 MAPK from resting Gadd45-deficient T cells is spontaneously phosphorylated, and mice lacking Gadd45 exhibit signs of T cell hyperproliferation and lupus-like autoimmune disease [67
].
Another important negative-feedback mechanism that controls inflammation relies on the ability of stress-induced glucocorticoid (GC) hormones to up-regulate the expression of MKP-1. This has been demonstrated in a recent study by Abraham et al. [68
], where MKP-1-deficient macrophages displayed altered GC-mediated inhibition of p38 MAPK and JNK activation in response to LPS. Impaired anti-inflammatory action of GC in MKP-1-deficient mice was also demonstrated in vivo using the cutaneous air-pouch model of inflammation and leukocyte recruitment, induced by zymosan and mediated by TNF- and the chemokine CXCL1. This study also showed that MKP-1 is involved in GC-mediated suppression of several proinflammatory genes, including TNF-, cyclooxygenase 2 (COX-2), IL-1, and IL-1ß.
Whereas most of the MKP-1 KO-based reports agree that MKP-1 is an important component of p38 MAPK activation in macrophages, the role of MKP-1 in dephosphorylation of JNK is less clear. Previous studies showed that the activity of JNK may be controlled by other phosphatases, such as MKP-5 [21
, 22
] and MKP-2 [40
]. Furthermore, by using MKP-1-deficient macrophages, we and others [29
, 30
] found that deletion of MKP-1 had only a limited effect on the degree of JNK phosphorylation in LPS-activated macrophages. Conversely, Chi et al. [28
] and Zhao et al. [27
] observed delayed kinetics of JNK dephosphorylation in MKP-1–/– macrophages, suggesting that MKP-1 may also negatively regulate JNK activation. The contribution of MKP-1 to down-regulation of JNK activity may be limited by the presence of other phosphatases capable of dephosphorylating JNK in macrophages. Additional support of this model is provided by the observation that pharmaceutical inhibition of p38 MAPK, but not JNK, can completely block the increased IL-10 synthesis in MKP-1–/– macrophages [28
]. Finally, ex vivo studies using MKP-1–/– macrophages demonstrated unanimously that MKP-1 is not involved in ERK1/2 dephosphorylation, confirming the substrate specificity of MKP-1 with its preference for p38 MAPK and JNK [7
, 21
, 40
].
Our KO studies also indicate that MKP-1 expression is important to prevent development of autoimmunity, in particular arthritis [29
]. After immunization with collagen, MKP-1–/– mice responded with a dramatically exacerbated arthritic response compared with wild-type controls, which manifested in the increased incidence and severity of the autoimmune response, including joint-swelling and clinical signs of inflammation in the ankle and wrist joints. Moreover, collagen-immunized MKP-1–/– mice displayed significantly increased serum levels of TNF- and IL-6, which were sustained throughout the entire study period. The concept that innate immune responses and local release of proinflammatory cytokines produced by infiltrating cells play a critical role in the development of arthritis is supported by several reports. TLR4-deficient mice treated with anticollagen antibody and LPS exhibit delayed arthritis progression and decreased production of the proinflammatory mediators TNF- and COX-2 in synovial tissue [69
, 70
]. Furthermore, deletion of MyD88 prevents the development of joint inflammation and accumulation of proinflammatory cytokines and chemokines in synovial tissue of mice injected with the streptococcal cell wall [71
]. Animal studies also demonstrated the role of immune responses induced by bacterial DNA and CpG in the onset of a TH1 cell-dependent and IL-1/IFN--mediated, joint-specific inflammation in Lewis (LEW) and LEW.1AV1 rats [72
]. Finally, p38 MAPK has been implicated directly in the pathogenesis of synovial inflammation and the progression of IL-1- and TNF-induced bone resorption and joint destruction in experimental arthritis and in patients with rheumatoid arthritis (RA) [11
, 73
74
75
76
]. Recently, p38 MAPK inhibitors were shown to reduce the incidence and progression in the rat streptococcal cell wall arthritis model [76
]. In addition, a recent report showed that targeted disruption of MKK3 leads to deficient expression of IL-1ß and IL-6 in synoviocytes [11
]. MKK3 is also required for p38 MAPK- and NF-B-mediated IL-6 production induced by TNF-. As expected, MKK3–/– mice exhibit significantly reduced inflammatory cell infiltration and joint destruction compared with control cohorts when challenged in the K/BxN antiglucose-6-phosphate isomerase (anti-GPI) antibody transfer model of RA. This model is mediated by mast cells and neutrophils, which are activated through C5a and FcRs after binding complement factors and anti-GPI/GPI immune complexes formed on the cartilage and synovial surfaces.
In summary, studies performed with KO mice and cells demonstrated unequivocally that MKP-1 has a pivotal and nonredundant role in controlling cytokine release and serves as a key negative regulator of TLR-mediated proinflammatory cytokine production and innate immunity mediated by primary macrophages.
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MKP-5 (DUSP10)
|
|---|
MKP-5 is a ubiquitously expressed, 52.6-kD phosphatase, which displays substrate specificity for JNK and to a lesser extent, for p38 MAPK [44
]. MKP-5 mRNA message is up-regulated markedly by stress stimuli such as anisomysin and osmotic stress in cultured cells [44
] and in human leukocytes activated with various stimuli, including TLR ligands (our unpublished data). The induction of MKP-5 was observed in the myeloid cell line RAW, stimulated with LPS [7
]. In contrast, naïve CD4+ T cells express MKP-5 constitutively, whereas activation of T cells leads to a significant decrease in MKP-5 message levels.
Targeted deletion of the mouse MKP-5 gene had no apparent effect on lymphoid and myeloid cell development, but it led to markedly increased IFN- and IL-4 production by activated TH1 and TH2 lymphocytes in comparison with the wild-type response [7
]. This result is consistent with a role of MKP-5 in negative regulation of effector T cell function. However, MKP-5–/– CD4+ T cells stimulated with a combination of CD3 and CD28 antibodies exhibited deficient proliferative responses [7
]. This diminished response may account for the reduced antigen-specific expansion of MKP-5–/– versus MKP-5–+/+ T cells of mice immunized with keyhole limpet hemocyanin [7
]. Correspondingly, MKP-5–/– mice also showed resistance to EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide [7
]. These observations indicate that MKP-5 plays an important role in adaptive immunity in the context of the initial expansion of naïve T cells in response to self and nonself peptide antigens.
Further studies suggest that MKP-5 plays a more complex, dual role in the immune system, connecting adaptive and innate immunity. LPS-activated APC of MKP-5–/– mice display a noticeably increased ability to provide costimulation and prime T cells to produce greater amounts of IL-2 and proliferate in response to antigen challenge. In addition, MKP-5–/– peritoneal macrophages exhibit significantly increased release of TNF- and IL-6 proinflammatory cytokines ex vivo in response to LPS challenge. MKP-5–/– macrophages were also hyper-responsive to a broad range of TLR signals and produced markedly elevated levels of TNF- and IL-6 after ligation of TLR2 and TLR3 with peptidoglycan and poly(I:C), respectively, and after infection with Listeria monocytogenes [7
]. The uncontrolled up-regulation of proinflammatory cytokine release and dramatically elevated levels of effector cytokines TNF- and IFN- produced by MKP-5-deficient T cells may explain the exacerbated, inflammatory immune responses and increased mortality of MKP-5–/– mice upon secondary infection with LCMV [7
].
Stimulation of MKP-5-deficient mouse TH1 and TH2 cells with CD3 antibody led to increased JNK activity. In contrast, the absence of MKP-5 had no effect on p38 MAPK and NF-B activity in TH1 and TH2 cells, despite the ability, demonstrated previously, of MKP-5 to dephosphorylate JNK and p38 MAPK in vitro [21
, 22
]. Thus, results obtained with KO cells are more consistent with the notion that DUSP other than MKP-5, primarily MKP-1, play a more significant, regulatory role in p38 MAPK and NF-B activation in vivo.
|
PAC-1 (DUSP2)
|
|---|
PAC-1 is a 32-kDa protein originally cloned from human T cells as an immediate-early gene [33
]. It preferentially dephosphorylates ERK and p38 but not JNK in vitro [33
]. In mice, PAC-1 is expressed predominantly in murine hematopoietic tissues, especially tissues with high T cell content (thymus, spleen, lymph nodes, and peripheral blood), but significant expression can also be detected in activated B cells, as well as circulating neutrophils and monocytes (Table 1)
. Its expression is induced rapidly in mitogen-stimulated T and B cells after stimulation [33
, 77
]. In addition, Jeffrey et al. [31
] reported high expression and rapid activation-induced up-regulation of PAC-1 in human mast cells, eosinophils, neutrophils, and macrophages as well as in mouse mast cells and activated macrophages. Transcriptional induction of PAC-1 by MAPKs has been proposed as a feedback mechanism that negatively regulates MAPK signaling in activated lymphocytes [77
]. It is notable that a comprehensive analysis of transcript regulation of known DUSP in primary human leukocytes identified PAC-1, hVH3 (DUSP5), and MKP-2 (DUSP4) as the most highly induced DUSP in T cells, whereas the expression levels of the other DUSP, including MKP-5 and MKP-1, were markedly lower in comparison [31
].
Thus far, the function of PAC-1 has been described generally in the context of negative regulation of MAPK signaling, similarly to other members of the DUSP family. However, a recent report by Jeffrey et al. [31
] revealed a positive regulation of innate and inflammatory cell signaling by PAC-1. In striking contrast to previous ex vivo findings, targeted disruption of the PAC-1 gene in mice led to weakened effector immune cell function and altered phosphorylation and kinase activity of ERK and p38 MAPK. The gene deletion also affected the activity of effectors downstream of MAPKs, including the transcription factors Elk-1, NFAT, and AP-1, in mast cells and macrophages, which were activated through the Rc
RI and TLR4, respectively. This impairment in the activity of MAPKs, previously thought to be dephosphorylated by PAC-1, was accompanied by enhanced kinase activity of JNK.
Although PAC-1–/– mice showed no apparent abnormalities in lymphoid and myeloid development, LPS-stimulated PAC-1–/– macrophages were deficient in their ability to produce proinflammatory cytokines (such as TNF-, IL-6, and IL-12), chemokines, and mediators of inflammation such as PGE2 and NO. Moreover, PAC-1-deficient macrophages displayed reduced expression of various inflammation-related genes after LPS activation, including complement/chemoattractant receptors (C5aR and C3aR), matrix metalloproteinases, adhesion molecules, and apoptosis-related genes. Finally, PAC-1 deficiency led to reduced mast cell survival in IL-3-dependent cultures, consistent with the proposed role of ERK in antiapoptotic signaling, which is normally counterbalanced by the proapoptotic function of JNK. In agreement with the impaired effector function of cells involved in innate immunity and inflammation, including macrophages, neutrophils, and mast cells, PAC-1–/– mice exhibited markedly reduced inflammatory responses in the passive K/BxN arthritis model.
In conclusion, in vivo studies using PAC-1–/– mice speak against a negative role of PAC-1 in regulation of MAPK signaling. Instead, PAC-1 has emerged as a crucial, positive mediator of inflammatory cell signaling, innate immunity, and effector immune mechanisms, controlled by signaling cascades downstream of ERK, p38 MAPK, and JNK.
|
MKP-6 (DUSP14)
|
|---|
MKP-6, also known as MKP-L, is a ubiquitously expressed, 25-kDa phosphatase identified by yeast-hybrid technology as a CD28 cytoplasmic tail-interacting protein [78
]. In the immune system, MKP-6 transcript can be detected in the thymus, lymph nodes, and spleen, and it is induced rapidly after activation of T and B cells, neutrophils, and monocytes (ref. [78
] and our unpublished data). In vitro, MKP-6 exhibits substrate specificity for ERK and JNK. Transfer of a dominant-negative form of MKP-6 (C111S) into human peripheral blood T cells enhanced CD28-induced secretion of IL-2 specifically and resulted in hyperphosphorylation of ERK and JNK but not p38 MAPK [78
]. Consistent with an important role of MKP-6 in CD28 costimulatory signaling, MKP-6 expression is enhanced strongly in T cells stimulated through CD28. Furthermore, the interaction of MKP-6 with the cytoplasmic region of CD28 is necessary for the negative-feedback control of CD28-induced IL-2 production and dephosphorylation of ERK and JNK by MKP-6. To date, no MKP-6 KO models have been published, but our preliminary results indicate that deletion of the MKP-6 gene did not result in significant developmental changes in the immune system. MKP-6-deficient mice also displayed normal innate immune responses and systemic cytokine production in response to LPS challenge. Moreover, MKP-6–/– mice immunized with OVA in CFA showed normal antigen-specific IgG responses, suggesting a somewhat redundant role played by MKP-6 in adaptive immunity. Further challenge studies and in vitro assays on purified MKP-6-deficient T cells, B cells, and macrophages will be required to determine the precise role of MKP-6 in innate and adaptive immune responses and inflammation.
|
ROLE OF DUSP IN APOPTOSIS, STRESS-INDUCED RESPONSES, AND CELL MOTILITY
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|---|
MAPKs have been studied extensively in vitro in the context of cell cycle, DNA repair, and apoptosis. The role of p38 MAPK in lymphocyte apoptosis remains controversial, and two reports demonstrate activation-induced apoptosis of CD8+ T cells mediated by the Fas-p38 MAPK-Bcl-2 signaling cascade [79
, 80
], and some reports indicate that p38 MAPK inhibits cell death by up-regulating the expression of antiapoptotic proteins [81
, 82
]. p38 MAPK and JNK signaling pathways can trigger programmed cell death in various cell types, including Fas-induced T cell apoptosis, by phosphorylating antiapoptotic Bcl-2 family members [80
, 83
, 84
], whereas ERK can inhibit apoptosis by phosphorylating Bim and targeting it for proteosomal degradation [85
].
Despite the identification of various DUSP, which regulate p38 MAPK and JNK, their precise roles and the mechanisms, whereby they mediate their cytoprotective effects, are not fully understood. In vitro studies indicate that conditional expression of MKP-1 inhibits a variety of biochemical events associated with cell death, including UV-induced JNK and p38 MAPK activity, DNA fragmentation, activation of caspase-3, and the proteolytic cleavage of the caspase-3 substrate poly(adenosine 5'-diphosphate ribose) polymerase [86
]. Cytoprotection against UV-induced apoptosis appears to be specific to MKP-1 and its substrates, whereas the DUSP, PAC-1, failed to promote cell survival. Consistent with this notion is the observation that upon DNA damage, down-regulation of MKP-1 leads to activation of the JNK pathway [87
]. Moreover, Wu and Bennett [50
] demonstrated that MKP-1-deficient fibroblasts display reduced cell growth as a result of enhanced cell death. MKP-1 deficiency also results in increased sensitivity of fibroblasts to anisomycin-induced apoptosis mediated by p38 MAPK. Another interesting mechanism of regulation of cell survival by two members of the phosphatase family, PP2A and MKP-3, was demonstrated in macrophages exposed to hyperoxia [18
]. These cells survive in the lung for prolonged periods of time as a result of sustained activation of ERK as a result of PP2A and MKP-3 down-regulation. Finally, resistance of human DU145 prostate cancer cells to Fas ligand-induced cell death has been linked to MKP-1 overexpression, suggesting that MKP-1 may play an important role in apoptosis of cancer cells [12
, 13
]. Thus, DUSP and other phosphatases may promote cell survival and attenuate stress-induced apoptosis by down-regulating activation of p38 MAPK and JNK. It remains to be established, however, through the use of appropriate KO models, whether the prosurvival and cytoprotective effects of MKP-1 observed in vitro have a similar impact on cell death of lymphocytes in vivo.
The critical role played by DUSP in leukocyte function also needs to be re-examined in the context of the studies, suggesting that MAPKs regulate chemoattraction of leukocytes to areas of inflammation and necrosis, cytoskeletal rearrangement, and cell migration/homing. Activation of JNK is associated with cytoskeletal rearrangements and cell motility mediated by various cytoskeletal proteins, including paxillin, microtubule-associated proteins, X-linked doublecortin, the stathmin family member SCG10, and the intermediate filament protein keratin 8 [88
89
90
]. p38 MAPK controls cell migration by phosphorylating MAPKAP 2/3 [88
]. Finally, ERK has been implicated in regulation of cell adhesion and motility mediated by myosin light chain kinase, calpain, or focal adhesion kinase [88
, 89
]. DUSP may control cell motility and adhesion by inhibiting activation of JNK, p38 MAPK, or ERK. Exposure of monocytes and macrophages to hypoxia or to the inflammatory cytokine TNF- results in a rapid increase of MKP-1 mRNA levels and dephosphorylation of MAPK, leading to inhibition of the MAPK-mediated chemoattractant signaling cascade [91
]. Another study by Jeong et al. demonstrated that stem cell factor (SCF)-induced migration of mast cells depends on p38 MAPK activation and that MKP-1 overexpression in dexamethasone-treated mast cells leads to down-regulation of p38 MAPK activity and inhibition of SCF-induced migration and production of proinflammatory cytokines by mast cells [92
]. Further work with KO models will be required to elucidate the precise mechanism by which DUSP control various aspects of cell motility and adhesion.
|
CONCLUSIONS
|
|---|
Recent studies using KO mice provide in vivo genetic evidence of a critical and nonredundant role played by the DUSP family of phosphatases in the regulation of innate and adaptive immunity mediated by MAPKs downstream of TLR. These studies underscore the essential role played by gene KO technology in contemporary biology, specifically, in dissecting the precise physiological roles of various genes in innate immunity and immune regulation. The KO approach provides a gold standard for examining gene function and facilitates target validation and drug discovery, which require the use of animal models of human diseases for the preclinical evaluation of functional inhibition of various signaling molecules.
Much of our current understanding of the role played by DUSP in the regulation of leukocyte activation is based on studies performed in vitro. In this regard, demonstration of increased and sustained JNK kinase activity in PAC-1-deficent macrophages and mast cells, as well as normal p38 MAPK and NF-B activities in MKP-5-deficient T cells, highlights the importance of the gene KO approach in defining the physiological use of substrates by various DUSP in vivo.
The DUSP family of phosphatases has emerged as a vital component of the protective immune mechanisms that control the threshold for and magnitude of innate inflammatory responses to bacterial and viral pathogens, as well as to self-antigens, during the course of autoimmune disease development. Even subtle genetic alterations in the DUSP-controlled signaling pathways may result in failure to down-regulate cytokine production and contribute to pathologically enhanced, inflammatory immune responses and the development of autoimmunity. Targeting DUSP activity may offer attractive, new opportunities for the development of therapeutics for a variety of inflammatory disorders and autoimmune diseases.
|
ACKNOWLEDGEMENTS
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|---|
We thank the phenotypic analysis groups at Lexicon Genetics Inc. for carrying out the comprehensive clinical diagnostic tests on the mutant mouse lines and C. A. Turner for critical reading of the manuscript.
Received October 18, 2006;
revised January 23, 2007;
accepted January 24, 2007.
|
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ABSTRACT
INNATE IMMUNITY AND MAPK...
