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巨噬细胞吞噬抑制因子

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巨噬细胞吞噬抑制因子 LETTERS Macrophage migration inhibitory factor stimulates AMP-activated protein kinase in the ischaemic heart Edward J. Miller1*, Ji Li1*{, Lin Leng2, Courtney McDonald2, Toshiya Atsumi5, Richard Bucala2,3* & Lawrence H. Young1,4* Understanding cellular response ...
巨噬细胞吞噬抑制因子
LETTERS Macrophage migration inhibitory factor stimulates AMP-activated protein kinase in the ischaemic heart Edward J. Miller1*, Ji Li1*{, Lin Leng2, Courtney McDonald2, Toshiya Atsumi5, Richard Bucala2,3* & Lawrence H. Young1,4* Understanding cellular response to environmental stress has broad implications for human disease. AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and -consuming pathways, and protects the heart against isch- aemic injury and apoptosis1. A role for circulating hormones such as adiponectin2 and leptin3 in the activation of AMPK has received recent attention. Whether local autocrine and paracrine factors within target organs such as the heart modulate AMPK is unknown. Here we show that macrophage migration inhibitory factor (MIF), an upstream regulator of inflammation4, is released in the ischaemic heart, where it stimulates AMPK activation through CD74, promotes glucose uptake and protects the heart during ischaemia-reperfusion injury. Germline deletion of theMif gene impairs ischaemic AMPK signalling in the mouse heart. Human fibroblasts with a low-activityMIF promoter polymorph- ism5 have diminished MIF release and AMPK activation during hypoxia. Thus, MIF modulates the activation of the cardioprotec- tive AMPK pathway during ischaemia, functionally linking inflammation and metabolism in the heart. We anticipate that genetic variation in MIF expression may impact on the response of the human heart to ischaemia by the AMPK pathway, and that diagnostic MIF genotyping might predict risk in patients with coronary artery disease. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls the inflammatory ‘set point’ by regulating the release of other pro-inflammatory cytokines6. MIF is expressed in several cell types, including monocytes/macrophages7, vascular smooth muscle8 and cardiomyocytes9, and is released on stimulation from pre-formed storage pools. MIF is involved in the pathogenesis of inflammatory diseases, such as atherosclerosis8,10, rheumatoid arthritis5, sepsis4, asthma11 and acute respiratory distress syndrome12. HumanMIF gene expression is determined by promoter polymorph- isms, including a tetra-nucleotide CATT repeat at position –794 (ref. 5). MIF signalling is known to activate ERK1/2 MAPK (ref. 13) through a receptor complex comprising CD74 (ref. 14) and CD44 (ref. 15). In contrast, the chemokine receptors CXCR2 and CXCR4 participate in MIF-mediated migratory function10. MIF also stimulates glycolysis during sepsis, increasing the syn- thesis of fructose 2,6-bisphosphate and cellular glucose uptake16. The signalling pathways by which MIF exerts its metabolic effects are unknown, but one candidate is the AMP-activated protein kinase (AMPK)—an important regulator of both glycolysis and glucose uptake during cellular stress1. AMPK senses the cellular energy state and affects diverse pathways to increase cellular ATP production and limit energy consumption. AMPK activity is regulated by AMP binding to its regulatory c-subunit17 and by phosphorylation of the catalytic a-subunit by upstream kinases, including LKB1 (ref. 18) and CaMKKb (ref. 19). In the heart, AMPK stimulates 6-phosphofructo- 2-kinase activity and glycolysis20, induces glucose transporter-4 (GLUT4, encoded by the SLC2A4 gene) translocation21, increases ischaemic glucose uptake1,22 and limits myocardial injury and apoptosis1. AMPK phosphorylation is also modulated by the adipocyte- derived circulating hormones leptin3 and adiponectin23, raising the possibility that cytokines might also activate AMPK. We hypothe- sized that AMPK might be activated in an autocrine/paracrine fash- ion by MIF in the heart during ischaemia, linking the regulatory control of inflammation and metabolism. Initial experiments examined whetherMIF has a role in the stimu- lation of the AMPK pathway during hypoxia in rat heart muscles. Hypoxic activation of AMPK (Fig. 1a) was associated with a twofold increase in muscle MIF release (Fig. 1b), the latter consistent with previous results in cardiomyocytes24. Pre-treatment with anti-MIF antibody reduced hypoxic AMPK activation by 67% (Fig. 1c). One of the important AMPK actions during hypoxia and ischaemia is to increase glucose transport1,22. Hypoxic glucose transport was inhi- bited 38% by anti-MIF antibody (Fig. 1d), indicating that secreted extracellular MIF modulates downstream AMPK action. To investigate whether MIF modulates AMPK, we added MIF to normoxic heart muscles. MIF caused time- and dose-dependent increases in AMPK phosphorylation (Fig. 1e and f), and increased heart muscle glucose uptake (Fig. 1g). Hypoxia and insulin- stimulated glucose uptake in the heart are mediated by translocation of the glucose transporter GLUT4 to the cell surface where it is physiologically active21. We used a cell-membrane impermeant photolabel compound and found significant translocation of GLUT4 to the cell surface (Fig. 1h), elucidating the mechanism through which MIF increases glucose uptake. We next examined whether MIF modulates AMPK signalling in the ischaemic heart.MIF is expressed by cardiomyocytes9,24, endothe- lial cells, monocytes and macrophages7. We used the isolated mouse heart perfused with crystalloid buffer, eliminating the potential contribution of MIF from circulating cells. MIF was highly expressed in cardiomyocytes, according to immunohistochemical data (Fig. 2a). Ischaemia triggered cardiac MIF release into the coronary venous effluent and decreased heart MIF content after ischaemia- reperfusion (Fig. 2b). To determine whetherMIF plays a part in ischaemic AMPK activa- tion, we used hearts fromMif2/2mice25 and compared them to wild- type controls. Mif2/2 mice demonstrated a normal baseline cardiac phenotype with respect to left ventricular size and function, histology and the expression of AMPK and glucose transporter proteins *These authors contributed equally to this work. 1Cardiovascular Medicine Section of the Department of Internal Medicine, 2Rheumatology Section of the Department of Internal Medicine, 3Department of Pathology, and 4Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. 5Department of Medicine II, Hokkaido University, Sapporo, 060-8638, Japan. {Present address: University of Wyoming School of Pharmacy, Laramie, Wyoming 82072, USA. Vol 451 | 31 January 2008 |doi:10.1038/nature06504 578 Nature Publishing Group©2008 (Supplementary Fig. 1). When perfused with mixed-substrate buffer and subjected to 15min of global ischaemia, AMPK activation was significantly blunted in the Mif2/2 hearts owing to decreased phos- phorylation of the activating Thr 172 site (Fig. 3a). The tumour- suppressor kinase LKB1 (also known as SKT11) has an important role in mediating AMPK phosphorylation during ischaemia18. However, we observed no change in the expression of LKB1, or CaMKKb (also known as Camkk2), another potential upstream kinase (Supplementary Fig. 1). Because AMPK mediates glucose uptake during ischaemia1, we examined whether the defect in AMPK signalling in the Mif2/2 hearts also diminished downstream glucose uptake. Although glu- cose uptake was normal inMif2/2 hearts during control perfusions, the stimulation of glucose uptake during ischaemia-reperfusion was significantly blunted compared towild-type hearts (Fig. 3b). This was associated with impaired glycogen synthesis inMif2/2 hearts during post-ischaemic reperfusion, despite a comparable amount of gly- cogen breakdown during ischaemia (Supplementary Fig. 2). Consistent with prior observations that AMPK deficiency is func- tionally deleterious to the heart during ischaemia-reperfusion1, Mif2/2 hearts also demonstrated impaired ischaemic tolerance (Fig. 3c).Mif2/2 hearts subjected to ex vivo ischaemia had decreased post-ischaemic left ventricular function (Fig. 3c) as well as increased ischaemic diastolic pressure and reduced contractility during reper- fusion (Supplementary Fig. 3).Mif2/2 hearts subjected to in vivo left coronary occlusion/reperfusion showed 2.3-fold greater infarct size compared to wild-type controls (Fig. 3d). These results indicate that MIF promotes early adaptive responses in the heart during ischaemia-reperfusion. Human gene mutations influence AMPK signalling and MIF expression. Individuals with rare mutations in the AMPK c2 subunit -pAMPK (Thr 172) -AMPKα -pAMPK (Thr 172) -AMPKα Hypoxia * * † † † * * * * * -pAMPK (Thr 172) -AMPKα g -AMPKα * * MIF (ng ml–1) MIF (400 ng ml–1) * * * * * MIF (ng ml–1) 0.0 2.5 5.0 7.5 10.0 α1 α2 α1 Control Hypoxia Control 0 1 2 Control Hypoxiaα2 Hypoxia A M P K a ct iv ity (p m ol m in –1 g –1 ) M IF p ro d uc tio n (n g p er m in p er m g of m us cl e) p A M P K (r el at iv e un its ) p A M P K (r el at iv e un its ) p A M P K (r el at iv e un its ) G lu co se u p ta ke (n m ol g –1 m in –1 ) G lu co se u p ta ke (n m ol g –1 m in –1 ) C el l- su rf ac e G LU T4 (fo ld in cr ea se ) 0 20 40 Control 100 200 100 200 400 MIF (400 ng ml–1) 30 60 120 -pAMPK (Thr 172) (min) 0 1 2 Control 30 60400 (min)120 0 1 2 3 4 Anti-MIF-mAb– + – IgG– – + Hypoxia Anti-MIF-mAb– + – – – + 0 20 40 60 80 0 20 40 Control 200 400 800 IgG h -t-GLUT4 -s-GLUT4 Control MIF 0.0 0.5 1.0 1.5 * Control MIF (400 ng ml–1) dcba f MIF (ng ml–1) e Figure 1 | Role of MIF in heart muscle AMPK signalling during hypoxia. a, Immunoblots show phosphorylated and total AMPK, bars show a2 or a1 AMPK activity. *P5 0.001, versus control; {P5 0.012, a1 versus a2. b, Muscle MIF release. *P5 0.03, versus control. c, d, Inhibition of AMPK activation and downstream glucose transport by anti-MIF antibody (100mgml21). *P5 0.02, versus control; {P5 0.04, versus hypoxia alone. e, AMPK activation by recombinantMIF (60min). *P, 0.02, versus control. f, AMPK activation by recombinant MIF (400ngml21). *P, 0.05, versus control. g, Glucose uptake during incubation with recombinant MIF. *P, 0.05, versus control. h, Immunoblots of cell-surface (s-GLUT4) and total (t-GLUT4)GLUT4 inmuscles incubatedwithout orwith 400 ngml21 rat MIF.*P, 0.001versus control,n5 3–6per group.Values aremeans6 s.e.m. a b Anti-MIFIgG -MIF 4 M IF r el ea se (n g p er m in p er g o f w ild -t yp e he ar t) M IF d en si to m et ry (r el at iv e un its ) 3 2 1 0 0 1 -MIF -AMPKα WT Baseline Reperfusion Baseline Reperfusion Baseline Reperfusion Mif–/– Figure 2 | Heart MIF expression and release triggered by ischaemia. a, Immunohistochemistry of wild-type (WT) mouse hearts with MIF antibody or non-immune immunoglobulin G (IgG). Immunoblots of heart lysates confirm the lack of immunoreactivity of the MIF antibody inMif2/2 hearts. Total AMPK is shown for loading comparison. b, Coronary effluent MIF production from wild-type hearts during baseline normal perfusion or during reperfusion after 10min of ischaemia. MIF concentration was multiplied by the coronary flow to calculate the production rate. *P5 0.01, versus baseline by unpaired t-test comparingmeans ofMIF concentration at five baseline and five reperfusion time points. MIF immunoblots of heart homogenates quantified by densitometry. *P5 0.003 versus control perfusions, n5 2–3 hearts each. Values are means6 s.e.m. NATURE |Vol 451 | 31 January 2008 LETTERS 579 Nature Publishing Group©2008 (PRKAG2, GeneID 51422) develop glycogen overload cardiomy- opathy and Wolf–Parkinson–White syndrome26. A common poly- morphism in the humanMIF promoter, containing 5, 6, 7 or 8 CATT tetra-nucleotide repeat units (–794 CATT5–8), also has functional consequences on MIF expression5. The CATT5 allele demonstrates low MIF promoter activity compared to the others5 and has been associated with less severe clinical manifestations of inflammatory diseases such as asthma11, cystic fibrosis27 and rheumatoid arthritis5, presumably owing to decreased MIF signalling. The MIF promoter genotype varies in the population according to ethnicity, but the low expression genotype is relatively commonwith 6%of Caucasians and 14.5% of African-Americans homozygous for the –794 CATT5 allele28. Despite demonstrable changes in MIF promoter activity, there are few data demonstrating the influence of the low expression genotype on the level of cellular MIF release. Thus, we examined whether polymorphisms in the human MIF promoter might lead to functional differences in MIF secretion and cellular AMPK activation, using early passage human dermal fibro- blasts. Cells from three of seven subjects were homozygous for the low expression 2794 CATT5 allele (‘5/5’ genotype) and the remain- der had at least one high expression 6-, 7- or 8-CATT repeat allele (‘non-5/5’ genotype). The 5/5 cells had significantly less MIF release into the culturemedia, during both normal and hypoxic incubations, when compared to non-5/5 cells (Fig. 4a). ReducedMIF release from the 5/5 cells was associated with less AMPK phosphorylation during hypoxic stress (Fig. 4b). To determine whether the relative MIF defi- ciency in the 5/5 cells was responsible for the impaired AMPK activa- tion during hypoxic stress, MIF (10 ngml21) was added to the media during hypoxic incubation. Exogenous MIF restored hypoxic AMPK activation in the 5/5 cells to levels that were equivalent to the non-5/5 cells (Fig. 4b). In contrast, MIF did not augment AMPK activation in hypoxic non-5/5 fibroblasts (Fig. 4b). Similarly, the addition of exo- genous MIF to hypoxic rat heart muscles did not augment AMPK activation (Supplementary Fig. 4). These data indicate that endogen- ous MIF release maximally modulates AMPK phosphorylation dur- ing hypoxia in normal heart tissue and cells. However, in relatively MIF-deficient cells (that is the 5/5 MIF promoter genotype), which have diminished MIF secretion during hypoxia, exogenous MIF augmented AMPK activation. The results indicate that recombinant MIF (or MIF agonists) might have a therapeutic effect by increasing AMPK activation during ischaemia or hypoxia in selected individuals with the low-expression 5/5 MIF promoter genotype. Thus, these experiments demonstrate that a common polymorphism in the MIF promoter leads to differential MIF release, which has conse- quences in cellular stress signalling in human cells. They also imply that exogenous MIF might have a beneficial effect in hypoxic tissues, specifically in patients with the 5/5 genotype. Taken together with the results implicating MIF in the activation of AMPK in the ischaemic heart, these data raise the possibility that a common polymorphism in theMIF promoter influences the suscep- tibility of patients with coronary artery disease to ischaemic injury. AMPK is under current investigation as a potential target molecule for the treatment of type 2 diabetes, because of its metabolic actions that increase skeletal muscle glucose uptake and suppress hepatic glucose production. AMPK is also a potential target in ischaemic heart disease, because of its cardioprotective effects1 and potential role in ischaemic preconditioning29. Treatment with MIF or MIF agonists warrants further study as an adjunctive therapy targeted at a 0.0 0.1 0.2 0.3 0.4 0.5 0.6 G lu co se u p ta ke (µ m ol m in –1 g –1 ) 14 4 3 2 1 0 p A M P K (r el at iv e un its ) A M P K α 2 ac tiv ity (p m ol m in –1 m g– 1 ) 12 10 8 6 4 2 0 WT Mif–/– WT Mif–/– WT Mif–/– WT Mif–/– WT pAMPK (Thr 172) AMPKα Mif–/– *† *† *† † † b c d 0 30 45 75 0 10,000 20,000 30,000 40,000 50,000 m m H g m in –1 * Time (min) Baseline Ischaemia Reperfusion -Coronary flow4 ml min–1 4 ml min–1 0 10 20 30 In fa rc t si ze (p er c en t of r is k ar ea ) * Co nt ro l Isc ha em ia Co nt ro l Isc ha em ia Co nt ro l Co nt ro l Isc ha em ia Isc ha em ia Co nt ro l Co nt ro l Isc ha em ia Isc ha em ia Co nt ro l Co nt ro l Isc ha em ia Isc ha em ia Figure 3 | Genetic MIF deletion impairs ischaemic heart AMPK activation and glucose uptake, and exacerbates post-ischaemic cardiac dysfunction and injury. a, AMPKphosphorylation and activity after ischaemic or control perfusions. *P, 0.05, versusMif2/2 ischaemic hearts; {P, 0.05, ischaemic versus control, n5 3–4 hearts for each genotype. b, Glucose uptake during control perfusion and during reperfusion after ischaemia (n5 5 for each genotype). *P5 0.01, versus wild-type baseline, {P5 0.04, versus Mif2/2 reperfusion. c, Heart-rate–left-ventricular-developed pressure product during control perfusion and post-ischaemic reperfusion. n5 6–7 hearts for each genotype. *P5 0.03, by repeated measures ANOVA during reperfusion. d, Myocardial infarction induced by 15min of left coronary occlusion in vivo followed by 4 h of reperfusion. Viable myocardium stained red with TTC; infarcted tissue, white; and normal non-ischaemic tissue, blue. The infarct area was quantified and expressed as a per cent of the ischaemic area at risk. *P5 0.04 versus wild type. n5 5–6 hearts per genotype. Values are means6 s.e.m. LETTERS NATURE |Vol 451 | 31 January 2008 580 Nature Publishing Group©2008 AMPK activation during acute myocardial ischaemia or infarction. To the extent that MIF is released from the heart during ischaemic preconditioning, MIF agonists might also augment preconditioning by increasing AMPK activation during ischaemia. Therapy directed at AMPKmight prove most effective in patients with low-expression MIF promoter polymorphisms. These hypotheses deserve further investigation and might also be addressed by analysis of gene banks from large cardiovascular clinical trials. To define the proximal mechanisms linking MIF and AMPK activation better, we next examined whether components of MIF cell-surface receptor complex, which is comprised of the ligand- binding component, CD74 (ref. 14), and the signal-transducing component, CD44 (ref. 15), is involved in AMPK activation during hypoxia. Treatment of human fibroblasts with a CD74-specific short interfering RNA (siRNA) decreased MIF receptor CD74 protein expression and blunted hypoxia-stimulated AMPK phosphorylation (Fig. 4c). We also studied MIF-induced AMPK phosphorylation in CD74null/CD44null COS-7/M6 cells that were stably transfected with either CD74 alone, CD44 alone, or CD74 together with CD44 (ref. 15). COS-7/M6 cells that expressedCD74 orCD44 alone showed no AMPK response to either hypoxia or exogenously added MIF. In contrast, COS-7/M6 cells expressing both transmembrane proteins showed significant AMPK phosphorylation responses (Supplemen- tary Fig. 5). These results support an important role for the two- component receptor complex, consisting of the MIF binding CD74 protein and the signal-transducing CD44 protein, in MIF-mediated AMPK signalling during cellular hypoxia in the heart. A CD74- dependent interaction between MIF and CXCR2 also has been reported and has a role in inflammatory cell recruitment10. Whether MIF activation of CXCR2 also has a role in the cellular response to hypoxic injury beyond its migratory function is worthy of additional investigation. In conclusion, these results define new models of both MIF action and AMPK activation, establishing a link between pathways central to inflammation and metabolism. MIF release leads to autocrine/ paracrine activation of the AMPK-signalling pathway in the isch- aemic heart. In other inflammatory disease states, high levels of MIF signalling, potentially activating additional pathways, might be deleterious. A common polymorphism in the human MIF pro- moter influences AMPK activation, andmight predispose susceptible individuals to ischaemic injury and provide a potential new risk marker for patients with coronary artery disease. METHODS SU
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