CREB Pathway Links PGE2 Signaling with Macrophage Polarization

CREB pathway links PGE2 signaling with macrophage polarization

Bing Luana,b,1, Young-Sil Yoona, John Le Layc, Klaus H. Kaestnerc, Susan Hedricka, and Marc Montminya,1

aPeptide Biology Laboratories, The Salk Institute, La Jolla, CA 92037-1002; bDepartment of Endocrinology, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai 200092, China; and cDepartment of Genetics, Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145

Contributed by Marc Montminy, October 8, 2015 (sent for review September 3, 2015; reviewed by Seung-Hoi Koo and Ling Qi) Obesity is thought to promote insulin resistance in part via activation required for M2 macrophage polarization. As disruption of this of the innate immune system. Increases in proinflammatory cytokine pathway increases insulin resistance in the setting of dietary production by M1 macrophages inhibit insulin signaling in white obesity, our results suggest that small molecules that enhance adipose tissue. In contrast, M2 macrophages have been found to its activity in macrophages may provide therapeutic benefit to enhance insulin sensitivity in part by reducing adipose tissue diabetic individuals. inflammation. The paracrine hormone prostaglandin E2 (PGE2) enhances M2 polarization in part through activation of the cAMP Results pathway, although the underlying mechanism is unclear. Here we We evaluated the effect of cAMP signaling on the expression of show that PGE2 stimulates M2 polarization via the cyclic AMP- M2 macrophage marker genes, including arginase-1 (Arg1), the responsive element binding (CREB)-mediated induction of Krupple- mannose receptor (Mrc1), resistin-like α (Fizz1, Retnla), and like factor 4 (KLF4). Targeted disruption of CREB or the cAMP-regulated chitinase 3-like 3 (Ym1, Chi3l3) in cultured bone marrow mac- transcriptional coactivators 2 and 3 (CRTC2/3) in macrophages down- rophages (BMMs) (12, 13). Exposure to PGE2, potentiated IL- regulated M2 marker gene expression and promoted insulin re- 4–induced increases in M2 marker gene expression (Fig. 1 A and sistance in the context of high-fat diet feeding. As re-expression B). We observed similar effects using other cAMP agonists, in- of KLF4 rescued M2 marker gene expression in CREB-depleted cells, cluding the β2 adrenergic receptor agonist, isoproterenol, as well our results demonstrate the importance of the CREB/CRTC pathway as a number of bacterial toxins (Fig. S1A). CELL BIOLOGY in maintaining insulin sensitivity in white adipose tissue via its Overnutrition triggers leptin-mediated increases in catechol- effects on the innate immune system. amine signaling to white and brown adipose tissues (WAT and BAT) that promote fat burning. Indeed, leptin injection into ob/ob cAMP | CREB/CRTC | M2 macrophage | insulin resistance mice up-regulated circulating concentrations of norepinephrine, leading to elevations in intracellular cAMP (14, 15). In keeping nder obese conditions, macrophage infiltration and activa- with these effects, leptin administration augmented M2 marker Ution in adipose tissue leads to a chronic inflammatory state expression in WAT from leptin-sensitive ob/ob mice; these ef- with increased secretion of proinflammatory cytokines (1). The fects were blocked by pretreatment with β-adrenergic receptor activation of IkB and Jun N-terminal kinases impairs insulin antagonist propranolol (Fig. 1 C and D). signaling in metabolic tissues and thereby contributes to insulin In contrast with acute effects of overnutrition, chronic high-fat resistance (2–4). Classically activated M1 macrophages secrete diet (HFD) feeding causes leptin resistance and attenuates proinflammatory cytokines, such as TNF-α and IL-12, which sympathetic catecholamine signaling to WAT (14). As a result, promote insulin resistance. Alternatively activated M2 macro- M2 marker gene expression was down-regulated in WAT from phages are thought to protect adipocytes from the development HFD mice; these effects were reversed by administering the of insulin resistance in response to IL-4 signaling (5, 6). In- phospho-diesterase 4 (PDE4) inhibitor Rolipram (5 mg/kg) (Fig. creases in STAT6 activity stimulate the expression of Krupple- 1 E and F) and increasing intracellular concentrations of cAMP. like factor 4 (KLF4), which in turn promotes expression of the Taken together, these results show that increases in cAMP signaling M2 program. Obesity causes an M2-to-M1 shift in adipose promote M2 macrophage polarization. tissue that leads to insulin resistance (7). The eicosanoid prostaglandin E2 (PGE2) has been found to Significance promote M2 macrophage polarization in part via induction of the cAMP pathway. Indeed, circulating catecholamines also ex- The second messenger cAMP exerts potent immunosuppres- ert potent anti-inflammatory effects on macrophage function via sive effects on the innate immune system, in part by activating cAMP signaling (8). In this regard, a number of bacteria appear class IIa histone deacetylases, and thereby inhibiting NF-κB– to evade the innate immune system by producing toxins that dependent transcription. We found that cAMP also promotes enhance cAMP production. cAMP stimulates the expression of M2 macrophage polarization by stimulating the cyclic AMP- cellular genes in part via the phosphorylation of CREB at Ser133 responsive element binding (CREB)/cAMP-regulated transcriptional and via the dephosphorylation of the cAMP regulated tran- coactivators TORC1 pathway. In the setting of acute overnutrition, scriptional coactivators (CRTC) family of coactivators (9). Fol- macrophage CREB inhibits the production of inflammatory medi- lowing its activation, the cyclic AMP-responsive element binding ators and contributes to the maintenance of insulin sensitivity. (CREB) pathway appears to block M1 macrophage function in Author contributions: B.L. and M.M. designed research; B.L., Y.-S.Y., J.L.L., K.H.K., and S.H. part via the induction of the anti-inflammatory cytokine IL-10 performed research; B.L., Y.-S.Y., J.L.L., K.H.K., S.H., and M.M. analyzed data; and B.L. (10, 11). Superimposed on these effects, cAMP also inhibits the wrote the paper. expression of proinflammatory cytokines via the induction of Reviewers: S.-H.K., Korea University; and L.Q., Cornell University. class IIa histone deacetylases (HDACs) and subsequent de- The authors declare no conflict of interest. κ acetylation of NF- B. 1To whom correspondence may be addressed. Email: [email protected] or montminy@ Here we explore the potential roles of the class IIa HDAC and salk.edu. CREB/CRTC pathways in M2 macrophages. We found that, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. although PGE2 stimulates both pathways, only one of these is 1073/pnas.1519644112/-/DCSupplemental.

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Fig. 1. cAMP promotes M2 macrophage polarization. (A and B) Effect of PGE2 exposure on M2 macrophage marker mRNA amounts (A)(Arg1, Mrc1, Fizz1, Ym1) and Arg1 protein level (B) in BMMs treated with IL-4. (C and D) Effects of leptin on M2 macrophage marker mRNA (C) and Arg1 protein amounts (D)in fat pads of ob/ob mice. Effect of β-adrenergic antagonist (Propanolol; pro) shown. Body weights indicated (n = 4 per group). (E and F) Effect of normal chow (NC) or HFD feeding on M2 macrophage marker mRNA amounts (E) and on Arg1 protein levels (F) in WAT. Coinjection of phospho-diesterase inhibitor Rolipram (5 mg/kg) in HFD mice indicated. (n = 4 per group). (G) Effect of PGE2 exposure on amounts of phosphorylated CREB, CRTC2/3, or HDAC4 in wild- type BMMs. *P < 0.05 and **P < 0.01.

cAMP triggers the activation of the CREB/CRTC and class IIa disrupts the DNA binding activity of all CREB family members HDAC pathways (9, 16, 17) (Fig. S1B). Of the three members of the (CREB1, CREM, and ATF1). Similar to CREB1 knockout cells, class IIa HDAC family (HDAC4, -5, and -7), HDAC4 is expressed at ACREB expression in BMMs disrupted M2 marker gene ex- highest levels in macrophages, prompting us to use cells from mice pression following exposure to IL-4 plus PGE2 (Fig. S4 A and B). with a macrophage-specific knockout of HDAC4 (HDAC4 MKO) CRTC proteins are potent coactivators of CREB. Sequestered to evaluate the importance of this pathway for M2 polarization. in the cytoplasm under basal conditions through phosphorylation- Although exposure of BMMs to PGE2 plus IL-4 promoted HDAC4 dependent interactions with 14-3-3 proteins, CRTCs shuttle to the dephosphorylation and activation (Fig. 1G), M2 marker gene nucleus following their dephosphorylation in response to cAMP, expression was up-regulated comparably between wild-type and where they bind to CREB over relevant promoters (9). Of the three HDAC4 MKO BMMs (Figs. S2A and S3 A and B), arguing against family members, CRTC2 and CRTC3 proteins, but not CRTC1, a significant role for the HDAC4 pathway in this setting. were detected in cultured BMMs by immunoblot assay (Fig. S2C). Next we tested the importance of the CREB pathway for M2 Based on their overlapping effects on CREB target gene polarization using homozygous CREB floxed (CREB fl/fl) mice expression, we evaluated the importance of both CRTC2 and expressing a macrophage-specific LysM-cre transgene. Exposure CRTC3 (CRTC2/3 DKO) for macrophage polarization. M2 of wild-type BMMs to PGE2 increased CREB Ser133 phos- marker expression was reduced in CRTC2/3 DKO BMMs exposed phorylation and augmented CRTC2 and CRTC3 dephosphorylation to IL-4 plus PGE2 (Fig. 2 C and D,andFig. S2C). Taken together, (Fig. 1G) (14). Exposure to IL-4 and PGE2 increased M2 marker these results demonstrate that the CREB/CRTC pathway is re- gene expression cooperatively in wild-type cells, and these effects quired for cAMP induction of M2 marker genes (Fig. 2D). were potently disrupted in CREB mutant BMMs (Fig. 2 A and B, Although CREB and CRTC2/3 appeared important for induc- and Fig. S2B). tion of M2 marker genes by PGE2, the absence of conserved We further evaluated the role of CREB in M2 polarization by CREB binding sites on these promoters pointed to the involvement expressing the dominant-negative CREB inhibitor ACREB, a of additional transcriptional activators in mediating these effects. synthetic polypeptide that selectively heterodimerizes with and In this regard, KLF4 has been shown to cooperate with STAT6 in

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Relative mRNA levels Relative mRNA 100 2 0 0 WT Creb MKO WT Creb MKO 0 0 WT Crtc2/3 DKO WT Crtc2/3 DKO ** 1000 * 1400 9000 Fizz1 Ym1 10000 Fizz1 ** Ym1 ** 900 9000 8000 UT 1200 800 UT 7000 8000 700 IL4 1000 IL4 6000 7000 600 IL4 6000 5000 800 IL4 500 +PGE2 5000 +PGE2 4000 600 400 4000 3000 300 3000 400 2000 200 2000

Relative mRNA levels Relative mRNA levels Relative mRNA 200 1000 100 1000 0 0 0 0 WT Creb MKO WT Creb MKO WT Crtc2/3 DKO WT Crtc2/3 DKO

B Creb D Crtc2/3 WT MKO WT DKO CELL BIOLOGY IL4 - + + - + + IL4 - + + - + + PGE2 - - + - - + PGE2 - - + - - +

Arg1 37kd Arg1 37kd α αTubulin 50kd Tubulin 50kd

Fig. 2. PGE2 stimulates M2 polarization via the CREB pathway. (A and B) Effect of IL-4 and PGE2 on M2 macrophage marker mRNA (A) and Arg1 protein amounts (B) in in BMMs from mice with a deletion of the CREB gene in macrophages (CREB MKO) or control littermates. (C and D) Effect of IL-4 and PGE2 on M2 macrophage marker mRNA (C) and Arg1 protein amounts (D) in wild-type and CRTC2/3 DKO BMMs. *P < 0.05 and **P < 0.01.

promoting M2 macrophage polarization; deletion of the KLF4 may protect against the development of insulin resistance in gene in macrophages disrupts M2 function and increases proin- this setting. To test this notion, we evaluated effects of HFD flammatory gene expression (13). Moreover, the mouse KLF4 feeding on insulin sensitivity in CREB MKO versus wild-type promoter contains CREB binding sites at −269 and −232 relative littermates. Although body weights, adiposity, and leptin levels to the transcription start site. Correspondingly, exposure to FSK were comparable between the two groups (Fig. 4 A and B and up-regulated KLF4-luciferase reporter activity in HEK293T Fig. S5A), CREB MKO mice exhibited higher fasting glucose cells. CRTC2 overexpression further enhanced KLF4 promoter and insulin concentrations in the context of HFD feeding activity, whereas A-CREB blocked it (Fig. 3A). Consistent with a compared with wild-type (Fig. 4C and Fig. S5B); they were also direct effect on KLF4 expression, exposure to PGE2 increased more glucose intolerant and had reduced glucose clearance rela- phospho-CREB and CRTC2 occupancy over the KLF4 promoter tive to controls by glucose and Insulin tolerance testing (Fig. 4D in BMMs (Fig. 3B). Exposure of wild-type BMMs to PGE2 in- and Fig. S5C). Indeed, macrophage infiltrates in WAT were more creased endogenous KLF4 mRNA amounts maximally after 4 h; pronounced in CREB MKO mice compared with wild-type (Fig. KLF4 expression was substantially down-regulated in CREB MKO 4E). M1 macrophage markers were significantly elevated in CREB as well as CRTC2/3 DKO BMMs (Fig. 3C). mutants, whereas M2 markers were reduced (Fig. 4F). Taken We tested whether the induction of KLF4 is required for the up- together, these results demonstrate that the macrophage CREB/ regulation of M2 marker genes in response to PGE2. Supporting CRTC pathway promotes M2 polarization that protects adipose this idea, IL-4/PGE2-dependent induction of M2 marker genes tissue from insulin resistance in the setting of obesity. were down-regulated in CREB and CRTC2/3 mutant cells relative to control; these effects were rescued following lentiviral expression Discussion of KLF4 (Fig. 3D). We detected similar changes in Arg1 protein In addition to its role as a starvation state signal, the second expression following KLF4 expression (Fig. 3E). Taken to- messenger cAMP exerts potent anti-inflammatory effects gether, these results indicate that the CREB/CRTC pathway pro- through the inhibition of macrophage function. cAMP attenuates motes M2 polarization via the up-regulation of KLF4 in response proinflammatory M1 macrophage gene expression, for example, via to IL-4/PGE2 signaling. induction of the anti-inflammatory cytokine IL-10 (10, 11) and via M2 macrophages have been shown to maintain insulin the induction of HDAC4. Superimposed on these effects, we sensitivity in adipose tissue by reducing proinflammatory cy- found that cAMP promotes anti-inflammatory M2 macrophage tokine expression (18). The loss of M2 macrophages leads to gene expression through up-regulation of the CREB/CRTC reciprocal increases in M1 polarized macrophages in adipose pathway (Fig. 4G). Both CREB and class IIa HDAC pathways tissue that promote insulin resistance. Based on its effects on inhibit the production of inflammatory mediators and contribute to M2 marker genes, we speculated that the CREB/CRTC pathway the maintenance of insulin sensitivity in the setting of overnutrition.

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ChIP signal ChIP 4 Klf4 promoter) 2

0.02 levels Relative Klf4 mRNA ** 1 2 * * ** 0 * ** * Relative Luciferase reporter activity ( 0 0 Con ACREB CRTC2 IgG pCREB CRTC2 PGE2(hr) 0 2 4 8 16 24

** ** D 1200 8 E Arg1 Mrc1 7 1000 6 Creb MKO 800 5 WT Creb MKO +Klf4 600 4 IL4 - + + - + + - + + 3 PGE2 - - + - - + - - + 400 2 Arg1 37kd 200 1 αTubulin 50kd 0 0 WT Creb MKO Creb MKO WT Creb MKO Creb MKO +Klf4 +Klf4 ** 9000 ** ** 900 8000 Fizz1 800 Ym1 7000 700 Relative mRNA levels Relative mRNA 6000 600 5000 500 4000 400 3000 300 2000 200 1000 100 0 0 WT Creb MKO Creb MKO WT Creb MKO Creb MKO +Klf4 +Klf4

Fig. 3. The CREB/CRTC pathway promotes M2 function via induction of KLF4. (A) Transient assay of HEK293T cells showing effects of FSK on KLF4 reporter activity. Cotransfection with ACREB or CRTC2 indicated. (B) ChIP (26) assay of pCREB and CRTC2 recruitment to the KLF4 promoter in BMMs exposed to PGE2. (C) Effect of PGE2 exposure for various times on KLF4 mRNA amounts in BMMs from wild-type, CREB MKO, and CRTC2/3 DKO mice. (D and E) Effect of lentiviral KLF4 expression on M2 macrophage marker mRNA amounts (D) and Arg1 protein level (E) in wild-type and CREB MKO BMMs exposed to IL-4 and PGE2. *P < 0.05 and **P < 0.01.

In addition to KLF4, a number of other CREB target genes, obtain by cross of CRTC2 fl/fl with CRTC3 fl/fl. In studies with CRTC2/3 DKO most notably CEBPβ and SOCS3, have been shown to promote macrophages, BMMs from floxed CRTC2/3 mice were infected with cre- macrophage polarization (19, 20). Future studies should provide expressing or control lacz-expressing lentivirus. For studies with KO mice, further insight into these and other mechanisms by which cAMP age-matched wild-type littermates were used as controls. For HFD studies, signaling modulates innate immunity. 6-wk-old mice were transferred to a 60% HFD (Research Diets, D12492) for 12 wk. MRI scans for fat and lean mass were performed using an Echo MRI- Experimental Procedures 100 instrument according to the manufacturer’s instructions. All mice were housed in colony cages with a 12-h light/dark cycle in a temperature-controlled Cells. HEK293T cells were maintained in DMEM and exposed to FSK (10 μM) environment. Animal studies were approved by the Institutional Animal Care and for transient transfection assays. BMMs were prepared as described pre- Use Committee at the Salk Institute. viously (21). Cells were treated with IL-4 (10 ng/mL) and PGE2 (10 nm), isoproterenol (10 μm), pertusis toxin (1 μg/mL), cholera toxin (1 μg/mL), edema factor (0.1 μg/mL) + protective antigen (0.3 μg/mL) for 16 h, and Glucose Tolerance Testing and Insulin Tolerance Testing. For glucose tolerance mRNA or protein was extracted for examination. testing, mice were fasted for 16 h and then injected intraperitoneally with glucose (1.5 mg/kg). For insulin tolerance testing, mice were fasted 2 h and Mice. C57BL/6J, ob/ob, and LysMcre mice were purchased from The Jackson injected intraperitoneally with insulin (Humulin; 1 U/kg). Blood was collected Laboratory. CREB1 fl/fl mice were a generous gift of E. Nestler (Mount Sinai from the tail vein at indicated times and glucose levels were measured with a Hospital, New York, NY). Macrophage-specific knockout of CREB was One Touch Ultra Glucometer (Johnson & Johnson). obtained by a two-step cross of CREB1 fl/fl mice with LysMcre mice according to The Jackson Laboratory protocol. CRTC2 fl/fl mice were as described ChIP and Quantitative PCR. BMMs were plated in 150-mm plates and exposed previously (22). CRTC3 fl/fl mice were generated with loxP sites flanking to PGE2 (100 nM) for 1 h. ChIP assays were performed as described previously exon 4 (ES cell clone obtained from KOMP). Floxed CRTC2/3 mice were (23). BMMs were stimulated for indicated times. RNA was isolated by RNeasy

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Body Weight (g ) Body Weight 10 10 10 leptin (ng/ml )

Body mass (g ) 5 0 0 0 Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Fat mass Lean mass CD 600 WT 14 250 WT 250 * 500 ** Creb MKO 12 * Creb MKO 200 200 ** * 10 400 150 * 150 8 300 * 6 100 100 200 4 50 Insulin (ng/ml ) 100 50 2 Blood glucose (mg/dl ) 0 0 0 0 Blood glucose (mg/dl ) ad lib fast Mins 0 15 30 60 90 120 0 15 30 60 90 120

EF3 M1 Markers * 2.5 F4/80 WT ** Creb MKO 2 * 4 Mφ Markers 1.5 WT 3.5 ** * 3 1 2.5 0.5

2 levels Relative mRNA 0 1.5 Mcp1 Rantes Nos2 Tnfa 1 CELL BIOLOGY Creb 0.5 WT

MKO levels Relative mRNA 0 1.4 M2 Markers Creb MKO Cd68 F4/80 1.2 1 0.8 G cAMP IL4 ** * 0.6 * * 0.4 0.2 0 Relative mRNA levels Relative mRNA Mrc1 Fizz1 Ym1 Arg1

CRTC Klf4 Creb +

M2 Macrophage

Fig. 4. Loss of M2 macrophages and insulin resistance in CREB MKO mice. (A and B) Relative weight gain (A)(n = 8 per group) as well as fat mass (n = 4 per group) and circulating leptin levels (B)(n = 8 per group) in CREB MKO and control littermates under HFD. (C) Circulating glucose and insulin concentrations in CREB MKO compared with control littermates maintained on a HFD for 12 wk (n = 8 per group). (D) Glucose tolerance (Left) and insulin tolerance (Right) testing of CREB MKO and control littermates maintained on HFD (n = 12 per group). (E) Relative macrophage infiltration in WAT by immunohistochemical (Left) and quantitative PCR (Right)analyses. (Scale bar, 50 μm.) (F) Expression of M1 and M2 marker genes in WAT by quantitative PCR analyses. (G) Schematic of proposed mechanism. *P < 0.05 and **P < 0.01.

kit (Qiagen). Quantitative PCR was carried out with SYBR Green, as described Statistical Analyses. All studies were performed on at least three independent previously (23). occasions. Results are reported as mean ± SEM. The comparison of different groups was carried out using two-tailed unpaired Student’s t test or two- Blotting and Immunostaining. Immunoblot and immunostaining assays were per- way ANOVA test. Differences were considered statistically significant at *P < formed as described previously (24). Quantitated results for Arg1 protein level are 0.05 and **P < 0.001. shown in Fig. S6. Mouse adipose tissues were fixed and paraffin-embedded. Sec- μ tions (5 m) were used for immunostaining with F4/80 antibody. ACKNOWLEDGMENTS. This work was supported by National Institutes of Health Grants R37DK083834 (to M.M.) and P01-DK049210 (to K.H.K.); the Luciferase Reporter Assay. HEK293T cells were transfected with KLF4-Luc Clayton Foundation for Medical Research; the Kieckhefer Foundation; and reporter, RSV-βgal, and indicated plasmids for 48 h, and luciferase assays the Leona M. and Harry B. Helmsley Charitable Trust Grant #2012-PG- were performed as described previously (25). MED002 (to M.M.).

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