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• W2015683693 abstract "Article20 August 2009free access A non-redundant role for MKP5 in limiting ROS production and preventing LPS-induced vascular injury Feng Qian Feng Qian Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Jing Deng Jing Deng Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Ni Cheng Ni Cheng Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Emily J Welch Emily J Welch Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Yongliang Zhang Yongliang Zhang Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Asrar B Malik Asrar B Malik Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Richard A Flavell Richard A Flavell Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Heaven, CT, USA Search for more papers by this author Chen Dong Chen Dong Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Richard D Ye Corresponding Author Richard D Ye Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Feng Qian Feng Qian Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Jing Deng Jing Deng Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Ni Cheng Ni Cheng Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Emily J Welch Emily J Welch Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Yongliang Zhang Yongliang Zhang Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Asrar B Malik Asrar B Malik Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Richard A Flavell Richard A Flavell Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Heaven, CT, USA Search for more papers by this author Chen Dong Chen Dong Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Richard D Ye Corresponding Author Richard D Ye Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA Search for more papers by this author Author Information Feng Qian1, Jing Deng1, Ni Cheng1, Emily J Welch1, Yongliang Zhang2, Asrar B Malik1, Richard A Flavell3, Chen Dong2 and Richard D Ye 1 1Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL, USA 2Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 3Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Heaven, CT, USA *Corresponding author. Department of Pharmacology, University of Illinois at Chicago, 835 South Wolcott Avenue, M/C 868, Chicago, IL 60612, USA. Tel.: +1 312 996 5087; Fax: +1 312 996 7857; E-mail: [email protected] The EMBO Journal (2009)28:2896-2907https://doi.org/10.1038/emboj.2009.234 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info There are at least 11 mitogen-activated protein kinase (MAPK) phosphatases (MKPs) and only 3 major groups of MAPKs, raising the question of whether these phosphatases have non-redundant functions in vivo. Using a modified mouse model of local Shwartzman reaction, we found that deletion of the MKP5 gene, but not the MKP1 gene, led to robust and accelerated vascular inflammatory responses to a single dose of LPS injection. Depletion of neutrophils significantly reduced the vascular injury in Mkp5−/− mice, whereas adoptive transfer of Mkp5−/− neutrophils replicated the LPS-induced skin lesions in wild-type recipients. Neutrophils isolated from Mkp5−/− mice exhibited augmented p38 MAPK activation and increased superoxide generation on activation. The p38 MAPK inhibitor, SB203580, significantly reduced p47phox phosphorylation and diminished superoxide production in neutrophils. p38 MAPK phosphorylated mouse p47phox, and deletion of the p47phox gene ablated the LPS-induced vascular injury in Mkp5−/− mice. Collectively, these results show an earlier unrecognized and non-redundant function of MKP5 in restraining p38 MAPK-mediated neutrophil oxidant production, thereby preventing LPS-induced vascular injury. Introduction Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinases (ERK), p38 MAPK and c-Jun N-terminal kinases (JNK), are activated through dual phosphorylation of their tripeptide motifs Thr-Glu-Tyr (ERK), Thr-Gly-Tyr (p38) and Thr-Pro-Tyr (JNK) within the activation loop (reviewed in Chen et al, 2001; Morrison and Davis, 2003). Dual phosphorylation of the Thr and Tyr residues is mediated through a highly conserved protein kinase cascade. In mammalian cells, ERK is activated by the MAPK kinases MKK1 and MKK2, p38 MAPK is activated by MKK3, MKK4 and MKK6 and JNK is activated by MKK4 and MKK7. There are also upstream kinases that respond to growth factors, inflammatory cytokines and stress signals and activate these MAPK kinases (Dong et al, 2002). The activated MAPKs then translocate to the nucleus and serve important functions in transcriptional regulation. In immune cells such as T lymphocytes and macrophages, MAPKs can be activated by inflammatory cytokines including TNF-α and IL-1β. These cells also respond to bacterial products including LPS and formylated chemotactic peptides with potent activation of selected MAPKs. As a result of these signalling events, MAPKs regulate the development, differentiation and activation of T lymphocytes as well as cytokine production in macrophages. The physiological importance and complexity of the MAPK cascade is evidenced by the presence of multiple forms of the MAPKs within each group, and by the profound effects that MAPK inhibition or gene deletion produce in vitro and in vivo (Chen et al, 2001). Given the important functions of MAPKs in all mammalian cells, proper regulation of their activities is crucial. Protein kinase phosphatases that act on either the phosphorylated threonines (e.g. PP2A, PP2C) or the phosphoylated tyrosines (e.g. PTPN5, PTPN7 and PTPRR) in activated MAPKs are the functional phosphatases for MAPKs (Alessi et al, 1995; Keyse, 2000; Farooq and Zhou, 2004). Dual specificity phosphatases (DUSPs) are MAP kinase phosphatases (MKPs) that selectively dephosphorylate both the phosphorylated threonines and phosphorylated tyrosines in activated MAPKs, a reaction involving the active site Cys and Asp residues in a MKP (Sun et al, 1993; Camps et al, 2000; Bhalla et al, 2002; Farooq and Zhou, 2004; Lang et al, 2006; Jeffrey et al, 2007; Liu et al, 2007; Zhang and Dong, 2007). Since the discovery of MKP1 (Sun et al, 1993), a total of 11 DUSPs have been identified as MKPs (Lang et al, 2006; Jeffrey et al, 2007; Liu et al, 2007; Zhang and Dong, 2007). These MKPs are expressed broadly in tissues and localized to different subcellular compartments (Camps et al, 2000; Farooq and Zhou, 2004). The fact that the number of available MKPs exceeds the number of MAPKs suggests different regulatory mechanisms for MKPs, which may lie in their substrate specificity, induced expression and tissue distribution. In vitro studies have shown that MKP1 and MKP5 share similar substrate specificities (p38 MAPK≈JNK>ERK), although MKP5 has an extra fragment N-terminal to its catalytic domain and belongs to a different subfamily of DUSPs (Tanoue et al, 1999; Theodosiou et al, 1999; Farooq and Zhou, 2004). There are also reports suggesting that the in vitro substrate selectivity of an MKP may differ from its selectivity in vivo (Lang et al, 2006; Jeffrey et al, 2007; Liu et al, 2007; Zhang and Dong, 2007). Therefore, it is presently unclear whether these MKPs have unique spatial and temporal characteristics or are functionally redundant. Recent studies using genetically altered mice lacking MKP1 (Zhao et al, 2005, 2006; Salojin et al, 2006), MKP5 (Zhang et al, 2004) and PAC1 (Jeffrey et al, 2006) have shown that these MKPs have important functions in regulating the expression of pro-inflammatory and immunomodulatory cytokines. These regulatory functions of MKPs are mediated through their suppression of the transcription-stimulating activities of the MAPKs. However, it was unclear whether MKPs regulate the activities of MAPKs in cytosolic compartment. In this study, we used a modified mouse model of local Shwartzman reaction (LSR) to examine a role of MKP5 in regulating host responses to inflammatory stimuli. LSR is a delayed vascular response to bacterial LPS and the inflammatory cytokine TNF-α, and is induced by consecutive injections of LPS (or LPS followed by TNF-α) into the skin of rabbits and mice (Stetson and Good, 1951; Brozna, 1990). As MAPKs are critical downstream effectors of the LPS signalling cascade, the LSR is an appropriate model for investigating the potential functions of MKPs in regulating inflammatory cell activation. Our results show a non-redundant function of MKP5 in the protection against LPS-induced vascular injury resulting from excessive p38 MAPK activation and neutrophil superoxide production. Results LPS induces a robust and accelerated vascular response in mice lacking MKP5 We used a mouse model of LSR to examine the physiological roles of MKP5 in maintaining homeostasis of host response to bacterial toxins and protection against tissues injury. In the classic LSR (Stetson and Good, 1951; Brozna, 1990), two injections of LPS separated by 18–24 h, or an LPS injection followed by a TNF-α injection 18–24 h later (Figure 1A), were required to induce skin lesions resembling those of thrombohaemorrhagic vasculitis. Surprisingly, all Mkp5−/− mice examined (15/15) reacted strongly to the first injection of LPS, which did not induce macroscopic lesions in WT mice (Figure 1A). Haemorrhage and dermal tissue necrosis was visible at the injection site 24 h after a single LPS administration (Figure 1B, left panels), and the extent of haemorrhage was quantified (Figure 1C). In contrast, mice lacking MKP1, which exhibits substrate specificity similar to that of MKP5, did not display exaggerated vascular response to LPS (Figure 1B, right panels). The Mkp1−/− mice responded similarly to their WT littermates in the classic LSR after the second injection (data not shown). Histological examination of dermal tissues from the Mkp5−/− mice showed erythrocyte extravasation, microcapillary thrombus formation and neutrophil accumulation at the site of LPS injection (Figure 1D, lower right image). No vascular injury and skin lesions were observed at the site of PBS injection in the same animals. Figure 1.LPS-induced microvascular injury in Mkp5+/+ and Mkp5−/− mice. (A) Experimental scheme and results of a classical local Shwartzman reaction (LSR) induced by consecutive injections of LPS and then TNF-α. Dorsal skin of WT mice were first injected s.c. with LPS (80 μg, right side of each panel) or PBS control (left side). After 24 h, TNF-α (0.2 μg) or PBS in same volume was injected s.c. into the same site that received LPS. The mice were killed 24 h after the second injection, and the skin tissues were examined macroscopically. Representative sample images from one of the five experiments are shown. (B) Experimental scheme and results of a modified (one-injection) LSR showing macroscopic appearance of dorsal skin in Mkp5+/+ (WT) and Mkp5−/− (KO) mice (panels on the left), compared with that of the Mkp1+/+ (WT) and Mkp1−/− (KO) mice (panels on the right). The WT and KO littermates in each group received an injection of either LPS (80 μg; right to the dotted line) or PBS (left to the dotted line). A total of 11 WT and 15 Mkp5−/− mice, and 8 WT and 8 Mkp1−/− mice were examined 24 h after the LPS injection. Representative images are shown. (C) The degree of haemorrhage in the Mkp5+/+ and Mkp5−/− group above was estimated based on densitometry analysis of skin samples receiving either LPS or PBS injection. **P<0.01. (D) Grouped images showing representative photomicrographs of H&E-stained skin sections from WT (upper panels) and Mkp5−/− (lower panels) mice that were treated with LPS or PBS as marked. Erythrocyte extravasation, thrombus formation and neutrophil accumulation are evident in the sample from LPS-treated Mkp5−/− mice 24 h after LPS injection. Download figure Download PowerPoint Mkp5−/− neutrophils are necessary for the augmented vascular response to LPS Neutrophil infiltration at the LPS injection site was quantified based on neutrophil myeloperoxidase (MPO) activity. As shown in Figure 2A, increased MPO activity was detectable in Mkp5−/− mice 12 and 24 h after LPS injection. However, peripheral blood neutrophil counts in WT and Mkp5−/− (KO) mice were similar (Figure 2B), suggesting increased neutrophil infiltration but not expansion of the neutrophil pool in the Mkp5−/− mice. Using an air pouch model, we determined pro-inflammatory chemokine and cytokine expression. LPS induced significantly more secretion of TNF-α, keratinocyte-derived chemokine (KC) and IL-6 in the Mkp5−/− mice than in WT littermates (Figure 2C–E). Elevated KC level could contribute to neutrophil accumulation at the LPS injection site. Figure 2.LPS-induced tissue neutrophilia and cytokine production in Mkp5+/+ and Mkp5−/− mice. (A) Time-dependent neutrophil accumulation at the LPS injection sites in WT and Mkp5−/− (KO) mice, based on the activity of neutrophil myeloperoxidase (MPO) in tissue extract. The values of MPO activity represent the means±s.e.m. of measurements in four mice. The difference between the WT and KO mice at 12 and 24 h time points was statistically significant (*P<0.05). (B) Peripheral blood neutrophil count in WT and Mkp5−/− (KO) mice before (0 h) and after (24 h) receiving 80 μg LPS (n=5). (C–E) Change in the expression level of TNF-α, KC and IL-6 in WT and Mkp5−/− mice. Air pouch was established by injecting sterile air into the dorsal skin of WT and Mkp5−/− (KO) mice. LPS (80 μg) was then injected into the air pouch. After 2 h, lavage fluid was collected from the air pouch and the concentration of TNF-α, KC and IL-6 was determined. Values shown are means±s.e.m. of measurements using four mice. *P<0.05 and **P<0.01, compared with LPS-stimulated WT mice. Download figure Download PowerPoint To determine whether neutrophil accumulation is critical to the LPS-induced vascular inflammation, anti-Gr-1 antibody was administered intraperitoneal (i.p.) to deplete neutrophils. The antibody effectively reduced neutrophil count in peripheral blood by ∼85%, whereas an isotype-matched IgG control produced no significant change in 48 h (P<0.01; Figure 3A). Mkp5−/− mice that received anti-Gr-1 or an IgG control were then given either LPS or PBS. As shown in Figure 3B, mice receiving anti-Gr-1 displayed significantly reduced (P<0.05) loss of MPO activity at the site of LPS injection, suggesting reduced neutrophil infiltration. Mice receiving anti-Gr-1 also showed diminished skin lesions on LPS stimulation, with reduced haemorrhage (Figure 3C). These results indicate that local accumulation of neutrophils is necessary for the LPS-induced vascular inflammatory response in the Mkp5−/− mice. Figure 3.Role of neutrophils in LPS-induced microvascular injury in Mkp5−/− mice. (A) Peripheral blood neutrophil count showing anti-Gr-1-mediated depletion of neutrophils in Mkp5−/− mice, determined at 0 and 48 h after i.p. injection of the antibody (filled bars) and compared with mice receiving isotype-matched IgG (Iso-IgG, open bars). (B) Neutrophil accumulation at the site of LPS or PBS injection in Mkp5−/− mice receiving anti-Gr-1 (filled bars) or isotype-matched IgG (open bars). (C) Macroscopic appearance of dorsal skin in LPS-injected Mkp5−/− mice that received anti-Gr-1 or isotype-matched IgG (100 μg each) 24 h before LPS challenge. Representative images from five independent experiments are shown. The degree of haemorrhage was quantified by densitometry and shown on the right side. (D, E) WT mice were intravenously injected with bone marrow neutrophils (2 × 106 in D, and 1 × 107 in E) isolated from either Mkp5−/− mice (KO to WT) or WT mice (WT to WT). After 10 min, dorsal skin was injected s.c. with LPS (80 μg). Macroscopic appearance of the LPS injection site was imaged at 24 h (n=5 in each group), and representative images are shown. For (C), (D) and (E), the degree of haemorrhage was estimated by densitometry as described in Figure 1, and the results are shown as means±s.e.m. on the right side of the images. *P<0.05; **P<0.01, based on five mice in each group. Download figure Download PowerPoint Administration of the anti-Gr-1 antibody resulted in a small decrease in peripheral monocytes count, as the antibody used recognizes a subset of monocytes. The change in monocytes count, however, is not statistically significant (Supplementary Figure S1A). To determine whether neutrophils are the primarily contributor to the LPS-induced tissue damage in Mkp5−/− mice, we adoptively transferred Mkp5−/− neutrophils to recipient WT mice. Transfer of Mkp5−/− bone marrow-derived neutrophils, but not WT neutrophils, reconstitutes the thrombohaemorrhagic lesions in WT recipients on LPS stimulation. Moreover, the extent of LPS-induced haemorrphage was positively correlated with the number of neutrophils transferred (Figure 3D and E). As the bone marrow-derived neutrophils were approximately 78% Gr-1+, we further determined whether neutrophils or other cell populations were responsible for the observed skin lesions, using flow cytometry to enrich the neutrophil population to >99% purity (Supplementary Figure S1B and C). When adoptively transferred to the recipient mice, these neutrophils produced results similar to those in Figure 3D and E (Supplementary Figure S2). On the basis of these findings, we conclude that the Mkp5−/− neutrophils are both necessary and sufficient for the LPS-induced vascular injury in mice. Loss of Mkp5 leads to augmented neutrophil superoxide production The ability to release granule contents and produce superoxide by activated neutrophils is essential to host defence against invading microbial pathogens. These bactericidal functions, however, also contribute to vascular injury in many inflammatory diseases. In a classic LSR, vascular injury resulting from LPS and TNF-α injections depends on the release of neutrophil elastase and requires activation of a complement C3-triggered, Mac-1-dependent signalling pathway (Hirahashi et al, 2006). Although neutrophil-derived superoxide and other oxygen radicals are highly toxic to bacteria as well as mammalian cells (Nauseef, 2007), a role for oxygen radicals in LSR has not been established. To determine whether major bactericidal activities were altered in Mkp5−/− neutrophils, we conducted chemotaxis assay using the mouse chemokine KC. Both WT and Mkp5−/− neutrophils behaved similarly in chemotaxis towards KC (Supplementary Figure S3A). These neutrophils also responded to C5a, an anaphylatoxin with a demonstrated role in the LSR and septic responses (Shin et al, 1968; Rothstein et al, 1988), in chemotaxis and degranulation assays, with no significant difference between the WT and Mkp5−/− mice (Supplementary Figure S3B and C). Likewise, fMet-Leu-Phe (fMLF) induced similar chemotaxis and β-glucuronidase release in Mkp5−/− mice and WT littermates (Supplementary Figure S4A and B). In contrast, neutrophils from the Mkp5−/− mice produced significantly more superoxide than neutrophils from the WT littermates when stimulated with C5a (Figure 4A). Interestingly, neutrophils from the Mkp1−/− mice showed no difference from their WT littermates in C5a-induced superoxide production (Figure 4B). The Mkp5−/− neutrophils also exhibited enhanced superoxide production in response to TNF-α stimulation (Figure 4C), and adherence to fibrinogen-coated surface further increased and extended the superoxide production (Figure 4D). These changes were also observed in Mkp5−/− neutrophils stimulated with fMLF and the phorbol ester PMA (Supplementary Figure S4C and D), suggesting that MKP5 deficiency broadly altered neutrophil superoxide production induced by soluble activators of NADPH oxidase. Together, these results indicate that MKP5, but not MKP1, has a function in the negative regulation of neutrophil NADPH oxidase. Figure 4.Augmented superoxide production in Mkp5−/− neutrophils. Representative tracings showing the production of superoxide as counts per second (CPS) by neutrophils (5 × 105) from the Mkp5−/− mice (A), Mkp1−/− mice (B) and their WT littermates, stimulated in suspension with 100 nM C5a or PBS. In (C) neutrophils from the Mkp5−/− mice and their WT littermates were stimulated with 100 ng/ml TNF-α or PBS, and superoxide production was measured. In (D) neutrophils from Mkp5−/− mice and their WT littermates were let adherent to fibrinogen-coated surface and stimulated with 100 ng/ml TNF-α. Superoxide generation in these experiments was detected using isoluminol-ECL. Shown on the right to the tracings are quantifications of cumulative superoxide production as means±s.e.m., based on measurement of integrated chemiluminescence (CL) in the course of the assays as indicated (n=5). *P<0.05; **P<0.01. Download figure Download PowerPoint Augmented superoxide production and vascular injury in Mkp5−/− neutrophils require activation of p38 MAPK To delineate the mechanisms by which MKP5 protects vascular cells from LPS-induced damage, we sought to determine the effect of Mkp5 deletion on MAPK activation in neutrophils. Studies conducted in vitro suggest that MKP5 preferentially dephosphorylates activated MAPKs in the order of p38 MAPK≈JNK>ERK (Tanoue et al, 1999; Theodosiou et al, 1999). To determine the effect of Mkp5 deletion in MAPK activation in intact cells, neutrophils were purified from the Mkp5−/− and WT mice and stimulated with C5a (10 nM). Phosphorylation of MAPK was determined by western blotting using specific anti-phospho-MAPK antibodies. Under these experimental conditions, the Mkp5−/− neutrophils exhibited enhanced ERK phosphorylation with unaltered kinetics that promptly terminate after 2 min (Figure 5A). No significant changes in JNK phosphorylation beyond a slightly increased basal level were observed in Mkp5−/− neutrophils (Figure 5B). In contrast, C5a stimulated a sustained increase in p38 MAPK phosphorylation in the Mkp5−/− neutrophils compared with neutrophils from WT littermate (Figure 5C). The changes in the MAPK phosphorylation pattern suggest that MKP5 selectively regulates p38 MAPK activation and produces lesser effects on ERK and JNK activation in neutrophils. Figure 5.C5a-induced MAPK activation and superoxide production in WT and Mkp5−/− neutrophils. Phosphorylation of ERK1/2 (A), JNK (B) and p38 MAPK (C) in C5a (10 nM)-stimulated WT and Mkp5−/− neutrophils was determined by western blotting using anti-phospho-antibodies against the individual MAPKs. Total MAPKs in the samples were shown below the phospho-MAPK blots. Densitometry analysis was conducted to determine the relative level of induced p38 MAPK phosphorylation, and the results are shown in (C). (D) The effects of MAPK inhibitors on C5a-stimulated superoxide production in WT (open bars) and Mkp5−/− (KO, filled bars) neutrophils, shown as integrated chemiluminescence (CL) in a 15 min period after stimulation. The cells were treated for 15 min with either the p38 MAPK inhibitor SB203580 (SB, 3 μM), the JNK inhibitor SP600125 (SP, 10 μM) or the MEK inhibitor PD98059 (PD, 30 μM), before C5a stimulation. Data shown are means±s.e.m. from three independent experiments. **P<0.01. Download figure Download PowerPoint We postulated that C5a produced at the site of LPS injection could stimulate enhanced superoxide generation in the Mkp5−/− neutrophils through hyperactivation of p38 MAPK. This possibility was examined ex vivo using pharmacological inhibitors for MAPKs. As shown in Figure 5D, the enhanced superoxide production in Mkp5−/− neutrophils was ablated with the p38 MAPK inhibitor SB203580 (P<0.01). In contrast, SP600125 and PD98059, inhibitors for the JNK and ERK signalling pathways, respectively, produced no significant effect on C5a-induced superoxide generation in either WT or Mkp5−/− neutrophils (Figure 5D, middle and right panels). Taken together, these results show that p38 MAPK, but not JNK and ERK, is a primary target of MKP5 in regulating neutrophil superoxide production. We next determined whether inhibition of p38 MAPK could affect the LPS-induced skin lesions in vivo. Mkp5−/− mice were given LPS alone or LPS and SB203580 together. As shown in Figure 6A and B, co-administration of SB203580 and LPS to Mkp5−/− mice significantly reduced skin lesions compared with mice receiving LPS alone (P<0.01). Injection of SB203580 together with LPS markedly decreased the LPS-induced ATF-2 phosphorylation (Figure 6C and D), showing that pharmacological inhibition of p38 MAPK could effectively block the exacerbated LSR. Figure 6.In vivo effects of SB203580 on LPS-induced vascular injury and p38 MAPK activation. The p38 MAPK inhibitor SB203580 (100 μg) was co-administered s.c. with LPS (80 μg) to Mkp5−/− mice. Control mice received either PBS or LPS alone. The mice were killed 24 h after injection, and skin tissue at the injection site was examined (A). The degree of a haemorrhage was quantified by densitometry analysis of skin sample and shown in (B). (C) Skin tissues at the injection site were excised and ATF-2 phosphorylation in tissue homogenate was determined by western blotting, using an antibody recognizing phosphorylated ATF-2. The relative level of ATF-2 phosphorylation was shown in (D). **P<0.01 and *P<0.05, based on four experiments. Download figure Download PowerPoint Mkp5 deletion increases p38 MAPK-mediated phosphorylation of mouse p47phox Phosphorylation of p47phox leads to conformational changes necessary for its membrane translocation. Biochemical characterization of human p47phox has led to the identification of Ser345 phosphorylation by ERK and p38 MAPK, which represents convergent signalling of the MAPKs leading to neutrophil NADPH oxidase priming at inflammatory sites (Dang et al, 2006). The sequence surrounding this phosphorylation site in mouse p47phox is quite different (Figure 7A), and there are three potential phosphorylation sites for p38 MAPK (Xue et al, 2006). Through DNA mutagenesis, we individually replaced these sites with alanine, and expressed the resulting mouse p47phox as GST fusion proteins for in vitro kinase assay. Ala substitution of Thr356, but not Ser346 and Ser355, resulted in a significant reduction in p38 MAPK-catalysed substrate phosphorylation (Figure 7B). This result suggests that The356 is a site for p38 MAPK phosphorylation. Figure 7.Identification of Thr356 in mouse p47phox as a p38 MAPK phosphorylation site. (A) Alignment of sequence of human and mouse p47phox proteins surrounding the potential p38 MAPK phosphorylation site. (B) Autoradiograph of in vitro kinase assay using full-length WT or mutated mp47phox fused to GST as substrates and an activated GST–p38α, as detailed in the section ‘Materials and methods’. GST without mp47phox was used as a negative control. Two phosphorylated bands were identified in the autoradiograph: a phosphorylated GST–mp47phox (apparent molecular weight 72 kDa), and an autophosphorylated GST–p38 (apparent molecular weight 68 kDa). (C) Coomassie blue staining showing the positions and levels of the proteins in the gel. (D) The extent of substrate phosphorylation was quantified by densitometry, and fold change relative to the phosphorylated WT GST–mp47phox was shown. Two independent kinase assays were performed, and similar results were obtained. Download figure Download PowerPoint We then compared C5a-induced p47phox phosphorylation in WT and Mkp5−/− neutrophils. After treatment with SB203580 or vehicle control (DMSO) for 15 min, neutrophils were stimulated with C5a for 1 min, and cell lysate was prepared. Phosphorylated proteins were collected using affinity chromatography. The phosphoproteins were separated on SDS–PAGE, transferred to membrane and blotted with an anti-p47phox antibody. As shown in Figure 8, C5a stimulated p47phox phosphorylation in both WT and Mkp5−/− neutrophils, with the latter showing a significant increase in p47phox phosphorylation (P<0.05). Treatment of neutrophils with SB203580 reduced p47phox phosphorylation by approximately 50%. The partial inhibition of C5a-induced p47phox phosphorylation by SB203580 is expected because other protein kinases, such as PKC, are also activated and can contribute to p47phox phosphorylation (el Benna et al, 1994). Figure 8.Posphorylation of p47phox in WT and Mkp5−/− neutrophils on C5a stimu" @default.
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• W2015683693 title "A non-redundant role for MKP5 in limiting ROS production and preventing LPS-induced vascular injury" @default.
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