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p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Perdiguero E, Sousa-Victor P, Ruiz-Bonilla V, Jardí M, Caelles C, Serrano AL, Muñoz-Cánoves P - J. Cell Biol. (2011)

Bottom Line: Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood.Conversely, miR-21-AKT interference altered homeostasis during tissue repair.This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University, 08003 Barcelona, Spain.

ABSTRACT
Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

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Increased AKT activity in MKP-1−/− macrophages is controlled by p38-induced miR-21 expression. (A) Phospho-AKT (P-AKT)–positive macrophages were counted from immunostained serial cryosections of gastrocnemius muscles obtained from WT and MKP-1−/− mice postinjury (P.I.; see pictures in Fig. S5 D). (B) Gene expression analysis by RT-PCR in FACS-isolated macrophage populations at 6 d after injury from mice treated with wortmannin or vehicle for the preceding 3 d. (C) WT and MKP-1−/− BM macrophages were stimulated with LPS at the indicated times. Western blotting analysis using phospho-AKT and AKT antibodies. (D) Expression of miR-21 was analyzed by qPCR in isolated macrophage populations at 3 and 6 d after injury from WT and MP-1−/− mice or WT mice treated with SB203580 (SB). (E) Total number of macrophages per milligram of muscle at 6 d after injury. Muscles were injected with scrambled or mimic miR-21 oligonucleotides at 3 d after injury. (F) Primary macrophages were transfected with scrambled or ant–miR-21 oligonucleotides and stimulated with LPS for the indicated times; IL-1β or IL-10 expression analysis. (G) As in F, primary macrophages were transfected with scrambled (Scr) or miR-21 mimic oligonucleotides. (H) Primary macrophages infected with a retrovirus expressing MKK6E or an empty retrovirus (C, control), in the absence or presence of SB203580, were analyzed for MKK6, phospho-p38 (P-p38), phospho-ATF2 (P-ATF2), and p38 expression by Western blotting (left) or for miR-21 mRNA expression by qPCR analysis (right). Means ± SEM of at least three experiments. # or ***, P < 0.001; **, P < 0.01; *, P < 0.05.
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fig6: Increased AKT activity in MKP-1−/− macrophages is controlled by p38-induced miR-21 expression. (A) Phospho-AKT (P-AKT)–positive macrophages were counted from immunostained serial cryosections of gastrocnemius muscles obtained from WT and MKP-1−/− mice postinjury (P.I.; see pictures in Fig. S5 D). (B) Gene expression analysis by RT-PCR in FACS-isolated macrophage populations at 6 d after injury from mice treated with wortmannin or vehicle for the preceding 3 d. (C) WT and MKP-1−/− BM macrophages were stimulated with LPS at the indicated times. Western blotting analysis using phospho-AKT and AKT antibodies. (D) Expression of miR-21 was analyzed by qPCR in isolated macrophage populations at 3 and 6 d after injury from WT and MP-1−/− mice or WT mice treated with SB203580 (SB). (E) Total number of macrophages per milligram of muscle at 6 d after injury. Muscles were injected with scrambled or mimic miR-21 oligonucleotides at 3 d after injury. (F) Primary macrophages were transfected with scrambled or ant–miR-21 oligonucleotides and stimulated with LPS for the indicated times; IL-1β or IL-10 expression analysis. (G) As in F, primary macrophages were transfected with scrambled (Scr) or miR-21 mimic oligonucleotides. (H) Primary macrophages infected with a retrovirus expressing MKK6E or an empty retrovirus (C, control), in the absence or presence of SB203580, were analyzed for MKK6, phospho-p38 (P-p38), phospho-ATF2 (P-ATF2), and p38 expression by Western blotting (left) or for miR-21 mRNA expression by qPCR analysis (right). Means ± SEM of at least three experiments. # or ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Mentions: To further understand mechanistically how p38–MKP-1 regulate these macrophage inflammatory transitions, we checked the AKT activation status of WT and MKP-1−/− macrophages in damaged muscle because an antiinflammatory role for AKT in response to LPS has been previously suggested (Guha and Mackman, 2002; Luyendyk et al., 2008). AKT activity was reduced in macrophages at early phases of tissue regeneration, coinciding with the mixed pro- and antiinflammatory profile, and in more advanced stages of the repair process, AKT activity remained higher in MKP1−/− macrophages (Fig. 6 A and Fig. S4 D). This higher activity correlated with loss of pro- and antiinflammatory cytokine gene expression in the absence of MKP-1 and suggested the likely requirement of AKT inhibition for pro- to antiinflammatory cytokine switching and subsequent cytokine silencing in WT macrophage at advanced stages of tissue repair.


p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair.

Perdiguero E, Sousa-Victor P, Ruiz-Bonilla V, Jardí M, Caelles C, Serrano AL, Muñoz-Cánoves P - J. Cell Biol. (2011)

Increased AKT activity in MKP-1−/− macrophages is controlled by p38-induced miR-21 expression. (A) Phospho-AKT (P-AKT)–positive macrophages were counted from immunostained serial cryosections of gastrocnemius muscles obtained from WT and MKP-1−/− mice postinjury (P.I.; see pictures in Fig. S5 D). (B) Gene expression analysis by RT-PCR in FACS-isolated macrophage populations at 6 d after injury from mice treated with wortmannin or vehicle for the preceding 3 d. (C) WT and MKP-1−/− BM macrophages were stimulated with LPS at the indicated times. Western blotting analysis using phospho-AKT and AKT antibodies. (D) Expression of miR-21 was analyzed by qPCR in isolated macrophage populations at 3 and 6 d after injury from WT and MP-1−/− mice or WT mice treated with SB203580 (SB). (E) Total number of macrophages per milligram of muscle at 6 d after injury. Muscles were injected with scrambled or mimic miR-21 oligonucleotides at 3 d after injury. (F) Primary macrophages were transfected with scrambled or ant–miR-21 oligonucleotides and stimulated with LPS for the indicated times; IL-1β or IL-10 expression analysis. (G) As in F, primary macrophages were transfected with scrambled (Scr) or miR-21 mimic oligonucleotides. (H) Primary macrophages infected with a retrovirus expressing MKK6E or an empty retrovirus (C, control), in the absence or presence of SB203580, were analyzed for MKK6, phospho-p38 (P-p38), phospho-ATF2 (P-ATF2), and p38 expression by Western blotting (left) or for miR-21 mRNA expression by qPCR analysis (right). Means ± SEM of at least three experiments. # or ***, P < 0.001; **, P < 0.01; *, P < 0.05.
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fig6: Increased AKT activity in MKP-1−/− macrophages is controlled by p38-induced miR-21 expression. (A) Phospho-AKT (P-AKT)–positive macrophages were counted from immunostained serial cryosections of gastrocnemius muscles obtained from WT and MKP-1−/− mice postinjury (P.I.; see pictures in Fig. S5 D). (B) Gene expression analysis by RT-PCR in FACS-isolated macrophage populations at 6 d after injury from mice treated with wortmannin or vehicle for the preceding 3 d. (C) WT and MKP-1−/− BM macrophages were stimulated with LPS at the indicated times. Western blotting analysis using phospho-AKT and AKT antibodies. (D) Expression of miR-21 was analyzed by qPCR in isolated macrophage populations at 3 and 6 d after injury from WT and MP-1−/− mice or WT mice treated with SB203580 (SB). (E) Total number of macrophages per milligram of muscle at 6 d after injury. Muscles were injected with scrambled or mimic miR-21 oligonucleotides at 3 d after injury. (F) Primary macrophages were transfected with scrambled or ant–miR-21 oligonucleotides and stimulated with LPS for the indicated times; IL-1β or IL-10 expression analysis. (G) As in F, primary macrophages were transfected with scrambled (Scr) or miR-21 mimic oligonucleotides. (H) Primary macrophages infected with a retrovirus expressing MKK6E or an empty retrovirus (C, control), in the absence or presence of SB203580, were analyzed for MKK6, phospho-p38 (P-p38), phospho-ATF2 (P-ATF2), and p38 expression by Western blotting (left) or for miR-21 mRNA expression by qPCR analysis (right). Means ± SEM of at least three experiments. # or ***, P < 0.001; **, P < 0.01; *, P < 0.05.
Mentions: To further understand mechanistically how p38–MKP-1 regulate these macrophage inflammatory transitions, we checked the AKT activation status of WT and MKP-1−/− macrophages in damaged muscle because an antiinflammatory role for AKT in response to LPS has been previously suggested (Guha and Mackman, 2002; Luyendyk et al., 2008). AKT activity was reduced in macrophages at early phases of tissue regeneration, coinciding with the mixed pro- and antiinflammatory profile, and in more advanced stages of the repair process, AKT activity remained higher in MKP1−/− macrophages (Fig. 6 A and Fig. S4 D). This higher activity correlated with loss of pro- and antiinflammatory cytokine gene expression in the absence of MKP-1 and suggested the likely requirement of AKT inhibition for pro- to antiinflammatory cytokine switching and subsequent cytokine silencing in WT macrophage at advanced stages of tissue repair.

Bottom Line: Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood.Conversely, miR-21-AKT interference altered homeostasis during tissue repair.This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Biology Group, Department of Experimental and Health Sciences, Pompeu Fabra University, 08003 Barcelona, Spain.

ABSTRACT
Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

Show MeSH
Related in: MedlinePlus