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. 2017 Oct 26;9(5):422–434. doi: 10.1093/jmcb/mjx046

Myeloid adrenergic signaling via CaMKII forms a feedforward loop of catecholamine biosynthesis

Yan Luo 1,2,†, Bilian Liu 1,†, Xin Yang 2, Xiaoxiao Ma 3, Xing Zhang 2,4, Denis E Bragin 5, Xuexian O Yang 4, Wendong Huang 3, Meilian Liu 1,2,✉
PMCID: PMC5907839  PMID: 29087480

Abstract

Type 2 immune response has been shown to facilitate cold-induced thermogenesis and browning of white fat. However, whether alternatively activated macrophages produce catecholamine and substantially promote adaptive thermogenesis in adipose tissue remains controversial. Here, we show that tyrosine hydroxylase (TyrH), a rate-limiting enzyme of catecholamine biosynthesis, was expressed and phosphorylated in adipose-resident macrophages. In addition, the plasma level of adrenaline was increased by cold stress in mice, and treatment of macrophages with adrenaline stimulated phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and TyrH. Genetic and pharmacological inhibition of CaMKII or PKA signaling diminished adrenaline-induced phosphorylation of TyrH in primary macrophages. Consistently, overexpression of constitutively active CaMKII upregulated basal TyrH phosphorylation, while suppressing the stimulatory effect of adrenaline on TyrH in macrophages. Myeloid-specific disruption of CaMKIIγ suppressed both the cold-induced production of norepinephrine and adipose UCP1 expression in vivo and the stimulatory effect of adrenaline on macrophage-dependent activation of brown adipocytes in vitro. Lack of CaMKII signaling attenuated catecholamine production mediated by cytokines IL-4 and IL-13, key inducers of type 2 immune response in primary macrophages. Taken together, these results suggest a feedforward mechanism of adrenaline in adipose-resident macrophages, and that myeloid CaMKII signaling plays an important role in catecholamine production and subsequent beige fat activation.

Keywords: CaMKII, adrenaline, tyrosine hydroxylase, catecholamine, UCP1

Introduction

An adipose tissue-specific type 2 immune response is characterized by the infiltration of eosinophils and group 2 innate lymphoid cells (ILC2s), alternative activation of M2 macrophages by IL-4 and/or IL-13, and the production of Th2 (T helper 2) cytokines. Type 2 immune response plays a critical role in regulating browning of white adipose tissue (WAT), energy expenditure and glucose homeostasis (Nguyen et al., 2011; Molofsky et al., 2013; Rao et al., 2014; Brestoff et al., 2015). Alternative activation of adipose-resident macrophages appears to produce and secrete catecholamines including dopamine, epinephrine, and norepinephrine which mediates type 2 inflammation-induced WAT browning (Nguyen et al., 2011; Molofsky et al., 2013; Rao et al., 2014; Brestoff et al., 2015). In support of this, macrophages have been shown to be an important source of catecholamine (Brown et al., 2003; Flierl et al., 2007; Nguyen et al., 2011). However, this was challenged by the recent study demonstrating that alternatively activated macrophages do not synthetize catecholamine or contribute to thermogenesis and energy homeostasis (Fischer et al., 2017). In addition, there is also a controversy regarding whether IL-4/IL-13 signaling plays an essential role in catecholamine production and thermogenesis (Molofsky et al., 2013; Rao et al., 2014; Brestoff et al., 2015; Fischer et al., 2017). Given the therapeutic potential of brown and beige fat, more studies are urgently needed to clarify whether IL-4 and IL-13-driven alternative activation of macrophages are required for adipose thermogenesis.

Tyrosine hydroxylase (TyrH) is the first and rate-limiting enzyme in the biosynthetic pathway to catecholamine production (Daubner et al., 2011). TyrH catalyzes the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is then converted to dopamine and is followed by the production of norepinephrine and epinephrine (Daubner et al., 2011). The central nervous system (CNS) is the main source of norepinephrine where TyrH is highly enriched (Fitzpatrick, 2000; Dunkley et al., 2004). The phosphorylation of TyrH including Ser8, 19, 31, and 40 residues in its R domain, drives its activation differentially in response to various stimuli or signaling molecules (Daubner et al., 2011). Several types of kinase such as cAMP-dependent protein kinase (PKA), protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and mitogen-activated protein kinases (MAPK) have been shown to stimulate the phosphorylation of TyrH to varying degrees in a number of different neuronal populations (Fitzpatrick, 2000; Dunkley et al., 2004). In addition to the CNS, macrophages are newly discovered sources of catecholamine and express TyrH both in primary and immortalized macrophage cell lines (Tsao et al., 1998; Brown et al., 2003). Moreover, Nguyen et al. (2011) first reported that alternatively activated macrophages sustain catecholamine production, which promotes adaptive thermogenesis in adipose tissue. Additional studies further demonstrated that M2 macrophage activation orchestrates catecholamine production and WAT browning (Molofsky et al., 2013; Brestoff et al., 2015). Furthermore, activation of the IL-4 receptor α (IL-4rα: a component of both IL-4 and IL-13 receptors) downstream pathways including signal transducer and activator of transcription 6 (STAT6) is critical for catecholamine production in macrophages (Molofsky et al., 2013; Brestoff et al., 2015). However, little is known about the role of intracellular signaling pathways PKA, CaMKII, and MAPK in catecholamine production in macrophages.

On the other hand, while norepinephrine has been shown to mediate cold-induced adaptive thermogenesis and WAT browning through β3-adreneceptor signaling, it is not completely understood whether adrenaline (also named as epinephrine), another catecholamine hormone secreted by the adrenal medulla, is involved in cold response. Accumulated evidence indicates that the plasma level of adrenaline is elevated in response to cold stress (Dronjak et al., 2004), and administration of adrenaline with lowest infusion rate (venous plasma concentration, 94 ± 32 pg/ml) significantly increases the metabolic rate in vivo (Staten et al., 1987). Along with this, lacking epinephrine by ablation of phenylethanolamine N-methyl transferase (Pnmt−/−), the enzyme that catalyzes the conversion of norepinephrine to epinephrine, impairs cold-induction of thermogenic gene ucp1 and pgc1α (Sharara-Chami et al., 2010). However, the mechanisms underlying the physiological role of adrenaline in cold-induced thermogenesis remain unclear.

In the present study, we show that acute cold stress upregulated the circulating levels of adrenaline, and treatment of macrophages with adrenaline stimulated phosphorylation of TyrH as well as activation of PKA and CaMKII. Moreover, we found that PKA/CaMKII signaling was required for adrenaline-stimulated activation of TyrH and subsequent catecholamine production in macrophages. Furthermore, myeloid-specific CaMKIIγ ablation attenuated cold-induced norepinephrine secretion and UCP1 expression in inguinal fat in vivo and in vitro. In addition, lack of CaMKII signaling diminished IL-4 and IL-13-induced catecholamine production in primary macrophages. Our data suggest that PKA/CaMKII signaling promotes the activation of TyrH and catecholamine production in macrophages, which provides a novel feedforward mechanism for adrenaline in adipose-resident macrophages.

Results

Phosphorylation of TyrH is enriched in primary macrophages

Given the controversy regarding the expression of TyrH in macrophages, we performed surgical denervation in inguinal fat of 3-month-old C57BL/6 mice. Denervation led to a robust downregulation of TyrH and UCP1, indicating that sympathetic tone plays a predominant role in regulating adipose norepinephrine (Figure 1A). In support of this, a non-specific band (around 50 kDa) recognized by TyrH antibody was little affected by denervation (Figure 1A). The expression levels of TyrH in brown and inguinal fat were greater than that in epidydimal fat, and were comparable to its levels in the hypothalamus and the brain stem, the major sources of norepinephrine (Figure 1B). In addition, the phosphorylation not the protein level of TyrH was enriched in peritoneal primary macrophages as compared with adipose tissue, liver, heart, hypothalamus, and brain stem (Figure 1B). In support of this, an immunoprecipitation assay showed that both phosphorylation and protein of TyrH were present in peritoneal macrophage as well as in brown adipose tissue, and cold stress stimulated the phosphorylation of TyrH in brown adipose tissue (Figure 1C and D). In addition, an immunofluorescence study indicated the stimulation of TyrH phosphorylation by cold stress in adipose-resident macrophages (Figure 1E), suggesting that macrophages may play a role in adipose norepinephrine.

Figure 1.

Figure 1

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Phosphorylation of TyrH is enriched in macrophages. (A) Surgical denervation markedly suppressed protein levels of TyrH in inguinal fat. Three 3-month-old male C57BL/6 mice were surgically denervated in inguinal fat and recovered for 2 weeks, and then euthanized after cold exposure for 1 day for western blot analysis of inguinal fat. S, sham; D, denervation. (B) The protein level of TyrH was low, while its phosphorylation was enriched in peritoneal macrophages compared to hypothalamus, brain stem, liver, heart, and adipose tissue of 3-month-old male C57BL/6 mice. Hypo, hypothalamus; BS, brain stem; BAT, brown adipose tissue; iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue; P-Mϕ, peritoneal macrophage; Non, non-specific band. (C) Cold stress stimulated the phosphorylation of TyrH in brown adipose tissue. TyrH in brown fat from mice exposed with or without cold was immunoprecipitated using anti-TyrH. For each immunoprecipitation reaction, 100 μg protein was used. Normal IgG was used as a negative control. Lysate from neuroblastoma N1E115, a catecholamine-producing clone, was used as a positive control for TyrH. (D) TyrH in peritoneal macrophages was immunoprecipitated for western blot analysis of TyrH protein and phosphorylation. Normal IgG was used as a negative control. Lys, lysate. (E) Immunofluorescence study of co-localization of macrophage marker F4/80 with P-TyrH in inguinal fat of mice under room temperature or cold stress condition. Scale bar, 50 μm. The data in E are the Representative images from at least three mice within each group. The data in B, C, and D are the representative from at least three individual experiments with similar results.

Circulating adrenaline is upregulated by cold stress and treatment of macrophages with adrenaline stimulates activation of TyrH, PKA, and CaMKII

Although norepinephrine has been shown to mediate cold-induced thermogenic program, whether adrenaline (epinephrine) plays a role in regulation of thermogenesis in adipose tissue remains undefined. We observed that 10 h cold stress (6°C) elevated the plasma level of adrenaline (Figure 2A), suggesting that adrenaline may be involved in cold adaption. Consistent with in vivo study (Sharara-Chami et al., 2010), adrenaline treatment at physiological doses (20 and 100 nM) as well as treatment of β3 adrenoceptor agonist CL 316243 (1 μM) induced UCP1 expression and PKA activation in brown adipocytes (Figure 2B and C). Given that adrenaline has the lower affinity with β3-adrenoceptor than norepinephrine (noradrenaline) (Liggett, 1992), it is possible that adrenaline targets other types of adipose-resident cell as well as adipocytes. To investigate if adrenaline modulates the function of macrophages, a possible source of catecholamine, RAW264.7 macrophage cells were treated with or without adrenaline (Brown et al., 2003; Flierl et al., 2007; Nguyen et al., 2011). We observed that acute adrenaline treatment of macrophages activated phosphorylation of TyrH at Ser40 as well as CaMKII at Thr286 in a dose and time-dependent manner (Figure 2D–G). Moreover, stimulated PKA and CaMKII phosphorylation showed up earlier than TyrH phosphorylation in adrenaline-treated macrophages (Figure 2E and G), implying that adrenaline might promote catecholamine production through regulation of PKA/CaMKII signaling in macrophages and subsequent thermogenesis. In line with this, treatment of 100 nM adrenaline stimulated the accumulation of intracellular Ca2+ in a time-dependent manner in macrophages (Figure 2H and I). These data suggest that adrenaline may regulate thermogenesis through targeting both macrophages and adipocytes.

Figure 2.

Figure 2

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Treatment of adrenaline stimulates the phosphorylation of TyrH and CaMKII in macrophages. (A) Circulating level of adrenaline was elevated by 10 h cold stress (6°C) in the 3-month-old male mice. ELISA analysis was used to measure the level of adrenaline. (B) Adrenaline treatment at 20 nM and 100 nM induced UCP1 expression and PKA activation in differentiated brown adipocytes. CL316,243 was used as the positive control. (C) Analysis and quantification of UCP1 western blot in B. (D−G) Treatment of adrenaline stimulated the phosphorylation of TyrH at Ser40 and CaMKII Thr286 in a dose (D) and time (E)-dependent manner in RAW264.7 macrophages. The dose (F) and time (G) course data for phosphorylation of TyrH and CaMKII in D and E were quantified using ScnImage software and normalized by the protein level of TyrH and CaMKII. * and # indicate the significance for P-CaMKII/CaMKII and P-TyrH/TyrH, respectively. (H) False color images showing the fluorescence ratio increased over time following exposure to adrenaline. (I) Averaged population data showing the time course of adrenaline-stimulated intracellular Ca2+ increase measured in RAW264.7 macrophages (mean ± SEM, n = 10 cells). Data were expressed as both a background-corrected fluorescence ratio (left y-axis, red for 340 nm and blue for 380 nm) and the estimated Ca2+ (right y-axis). The data in B, D, E, and H are the representative from at least three individual experiments with similar results. The data in A, C, F, G, and I are presented with mean ± SEM. *,#P < 0.05, **P < 0.01, ***P < 0.005 compared with control.

PKA signaling drives activation of CaMKII and TyrH in macrophages

To gain insight into whether the PKA pathway, downstream of β-adrenergic signaling, is critical in regulating TyrH activation and catecholamine production in macrophages, we treated RAW264.7 macrophage cells with dibutyryl cyclic AMP (dbcAMP), an exogenous cyclic AMP (cAMP). Similar to adrenaline, acute treatment of dbcAMP stimulated phosphorylation of TyrH as well as CaMKII in a time and dose-dependent manner in macrophages (Figure 3A and B). In addition to phosphorylation of CaMKII and TyrH, the expression levels of TyrH were induced by 22 h treatment of 100 μM dbcAMP as well (Figure 3C). Consistently, dbcAMP treatment markedly elevated intracellular Ca2+ levels in a time-dependent manner in macrophages (Figure 3D and E). To further determine whether CaMKII signaling is required for cAMP-induced activation of TyrH, RAW264.7 cells were pretreated with 10 μM KN93 for 1 h followed with treatment of dbcAMP for 20 min (Figure 3F and G). KN93 is a CaMKII-specific inhibitor that binds to the Ca2+/calmodulin binding site on the kinase and thus prevents kinase activation via competition with Ca2+/calmodulin, suppressed dbcAMP-induced phosphorylation of TyrH in neuroblastoma cells (Tokumitsu et al., 1990; Sumi et al., 1991). We found that inhibiting CaMKII significantly suppressed dbcAMP-stimulated phosphorylation of TyrH (Figure 3F and G), suggesting that CaMKII is the downstream kinase of PKA, and plays an important role in adrenaline-induced catecholamine production in macrophages. Similar to KN93, treatment of 10 μM H89, a PKA inhibitor, significantly suppressed the phosphorylation levels of TyrH in macrophages (Figure 3H and I). Moreover, inhibiting PKA abolished dbcAMP-induced phosphorylation of CaMKII, indicating that the activation of CaMKII and TyrH is driven by PKA signaling in macrophages. Given that macrophages from different sources have extraordinary degrees of heterogeneity, we performed a primary culture of adipose-resident macrophages isolated from 3-month-old male C56BL/6 mice. Flow cytometry analysis showed that the purity of sorted macrophages from adipose tissue was, on average, 92.3% (Figure 3J). Consistent with the study in RAW264.7 macrophage cells, inhibiting either PKA or CaMKII eliminated dbcAMP-stimulated phosphorylation of TyrH in adipose-resident macrophages (Figure 3K).

Figure 3.

Figure 3

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PKA signaling stimulates the phosphorylation of TyrH in CaMKII-dependent manner in macrophages. (A and B) Short-time treatment of dbcAMP stimulated the phosphorylation of TyrH and CaMKII in a time (A) and dose (B)-dependent manner in RAW264.7 macrophages. (C) Treatment of 100 μM dbcAMP for 22 h induced the expression of TyrH as well as the phosphorylation of TyrH and CaMKII in RAW264.7 macrophages. (D) False color images showing the fluorescence ratio increases over time following exposure to dbcAMP in RAW264.7 macrophages. (E) Averaged population data showing the time course of dbcAMP-stimulated accumulation of intracellular Ca2+ measured in macrophages (mean ± SEM, n = 10 cells). Data were expressed as both a background-corrected fluorescence ratio (left y-axis, red for 340 nm and blue for 380 nm) and the estimated Ca2+ (right y-axis). (F) Inhibiting CaMKII by 10 μM KN93 blocked dbcAMP-stimulated phosphorylation of TyrH in primary peritoneal macrophages. (G) Western blot analysis and quantification of P-TyrH in F. (H) Inhibiting PKA by 10 μM H89 markedly suppressed dbcAMP-stimulated phosphorylation of CaMKII and TyrH in primary peritoneal macrophages. (I) Western blot analysis and quantification of P-TyrH in H. (J) Flow cytometry analysis of primary macrophages isolated from adipose tissue using magnetic beads. F4/80 positive cells were considered as macrophages, and stromal vascular fraction (SVF) was used to indicate the purity of macrophage in SVF before isolation. The staining of isolated macrophages without primary antibody was used as a negative control. (K) dbcAMP-stimulated phosphorylation of TyrH was suppressed by inhibiting CaMKII with treatment of 10 μM KN93 or inhibiting PKA with treatment of 10 μM H89 in primary adipose-resident macrophages. The data in A−D, F, H, J, and K are the representative from at least three individual experiments with similar results. The data in E, G, and I are presented with mean ± SEM. *P < 0.05, **P < 0.01.

CaMKII signaling plays a critical role in regulating production of norepinephrine in primary macrophages

To further investigate the role of PKA/CaMKII pathway in catecholamine production in macrophages and thermogenesis in adipose tissue, adipose-resident macrophages were treated with KN93 for 1 h followed by the treatment of dbcAMP for 4 h. The cells were washed and then cultured in 1 ml fresh serum-free medium for 12 h, and the media was collected for ELISA analysis of norepinephrine and treatment of adipocytes. The secretion of norepinephrine was significantly increased by dbcAMP treatment in primary macrophages, while inhibiting CaMKII attenuated the stimulatory effect of dbcAMP on norepinephrine production in adipose tissue macrophages (Figure 4A). Moreover, dbcAMP-treated media of macrophages induced the expression of UCP1 and C/EBPβ in brown adipocytes compared to the control sample, while inhibition of CaMKII suppressed this promoting effect in brown adipocytes (Figure 4B and C), suggesting the importance of PKA/CaMKII signaling in catecholamine biosynthesis in macrophages. To further elucidate the involvement of CaMKII in the modulation of catecholamine biosynthesis, we isolated adipose macrophages from CaMKIIγ (a major isoform of CaMKII in macrophages)-deficient mice and wild-type control mice (Timmins et al., 2009; Hojabrpour et al., 2012). Consistently, CaMKIIγ deficiency suppressed dbcAMP-induced norepinephrine production in macrophages as well as activation of PKA and expression of UCP1 and C/EBPβ in brown adipocytes (Figure 4D–F), indicating that myeloid CaMKII plays a critical role in catecholamine biosynthesis and thermogenic function in adipose tissue.

Figure 4.

Figure 4

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CaMKII is required for PKA signaling-induced norepinephrine secretion in primary macrophages. Adipose-resident macrophages were treated with KN93 for 1 h followed by the treatment of dbcAMP for 4 h. The cells were then washed and cultured in 1 ml fresh serum-free medium for 12 h, and the media was collected to treat the differentiated brown adipocytes. (A) dbcAMP-induced norepinephrine secretion from adipose tissue macrophages were suppressed by CaMKII inhibitor KN93. Norepinephrine level was determined by ELISA. (B) CaMKII inhibition reversed the inducing effect of dbcAMP-treated macrophage media on UCP1 and C/EBPβ expression in brown adipocytes. (C) Western blot analysis and quantification of UCP1 and C/EBPβ in B. (D) CaMKIIγ deficiency suppressed dbcAMP-induced norepinephrine secretion from primary macrophages. (E) CaMKIIγ deficiency diminished the inducing effect of dbcAMP-treated macrophage media on phosphorylation of PKA and expression of UCP1 and C/EBPβ in brown adipocytes. (F) Western blot analysis and quantification of UCP1 and C/EBPβ in E. The data in A, B, D, and E are the representative from at least three individual experiments with similar results. The data in A, C, E, and F are presented with mean ± SEM. *P < 0.05, **P < 0.01.

CaMKII is required for adrenaline-induced catecholamine production in macrophages

As CaMKII signaling plays a critical role in catecholamine biosynthesis and thermogenesis, we hypothesize that CaMKII mediates the stimulatory effect of adrenaline on catecholamine production in macrophages. To investigate the mediatory effect of CaMKII on adrenaline stimulation of TyrH, RAW264.7 macrophage cells were pretreated with 10 μM KN93 before treatment of 100 nM adrenaline for 10 min. Similar to dbcAMP treatment, KN93 treatment had little effect on phosphorylation of CaMKII, while suppressed adrenaline-stimulated phosphorylation of TyrH in macrophages (Figure 5A and B). Consistent with this, overexpression of constitutively active (CA) mutant of CaMKII by infection of adenovirus increased the basal phosphorylation of TyrH compared to the overexpression of dominant-negative (DN) mutant of CaMKII in RAW264.7 macrophages (Figure 5C and D). In addition, overexpression of CA-CaMKII attenuated the stimulatory effect of adrenaline and the suppressing effect of KN93 on the phosphorylation of TyrH (Figure 5C and D). Our data suggest that CaMKII mediates adrenaline-induced activation of TyrH in macrophages. In line with this, the secreted factors from CA-CaMKII-overexpressed macrophages significantly upregulated expression levels of UCP1 and C/EBPβ as compared to DN-CaMKII control in brown adipocytes (Figure 5E and F). However, the inducing effect of adrenaline was diminished by overexpression of CA-CaMKII (Figure 5E and F), again suggesting that CaMKII plays an important role in adrenaline-induced catecholamine production and thermogenic program. Moreover, the deficiency of CaMKIIγ downregulated adrenaline-stimulated phosphorylation of TyrH, but had no significant effect on the expression levels of TyrH in primary peritoneal macrophages (Figure 5G and H). These results suggest that CaMKII plays an important role in adrenaline action in macrophages and thermogenic program in adipose tissue.

Figure 5.

Figure 5

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CaMKII is required for adrenaline-induced norepinephrine secretion in macrophages. (A) Inhibiting CaMKII by treatment of 10 μM KN93 suppressed adrenaline-stimulated phosphorylation of TyrH in RAW264.7 macrophages. (B) Western blot analysis and quantification of P-TyrH in A. (C) Overexpression of CA-CaMKII elevated basal P-TyrH and diminished the stimulatory effect of adrenaline on P-TyrH in RAW264.7 macrophages. (D) Western blot analysis and quantification of P-TyrH in C. (E) Myeloid overexpression of CA-CaMKII induced UCP1 and C/EBPβ expression and diminished the inducing effect of adrenaline in co-cultured brown adipocytes. Adipose-resident macrophages were infected with adenoviruses encoding DN-CaMKII and CA-CaMKII for 24 h, and then changed into the fresh medium and treated with KN93 for 1 h followed by the treatment of dbcAMP for 4 h. The cells were then cultured in 1 ml fresh serum-free medium for 12 h, and the media was collected to treat the differentiated brown adipocytes. (F) Western blot analysis and quantification of UCP1 in E. (G) CaMKIIγ deficiency diminished adrenaline-stimulated phosphorylation of TyrH in peritoneal macrophages. The primary macrophages were isolated from CaMKIIγ KO and wild-type (WT) mice. (H) Western blot analysis and quantification of P-TyrH in G. The data in A, C, E, and G are the representative from at least three individual experiments with similar results. The data in B, D, F, and H are presented with mean ± SEM. *P < 0.05, **P < 0.01.

CaMKII signaling plays an important role in IL-4 and IL-13-induced catecholamine production

Given that the IL-4rα pathway mediates the action of IL-4 and IL-13 in regulating catecholamine biosynthesis in macrophages (Nguyen et al., 2011), it is possible that the PKA/CaMKII pathway interacts with IL-4rα signaling in the activation of TyrH in macrophages. To determine if the CaMKII pathway is required for IL-4 and IL-13 (IL-4/IL-13)-induced catecholamine biosynthesis, peritoneal macrophages were isolated from 3-month-old C57BL/6 male mice. We found that short-time treatment of IL-4/IL-13 had little effect on the phosphorylation of TyrH (Figure 6A), while long-time treatment of IL-4/IL-13 induced expression and phosphorylation of TyrH in macrophages. In addition, inhibiting CaMKII with KN93 attenuated IL-4/IL-13-induced phosphorylation, but not the expression of TyrH in primary macrophages (Figure 6B and C). This suggests that CaMKII may not be required for IL-4rα pathway promoting TyrH expression, while it is critical for activation of TyrH in macrophages. In agreement with this, CaMKIIγ KO primary macrophages displayed the downregulation of IL-4/IL-13-induced TyrH phosphorylation and norepinephrine secretion despite no significant difference in expression of TyrH compared to wild-type (WT) cells (Figure 6D–F), suggesting that CaMKII signaling may cooperate with the IL-4rα pathway to regulate catecholamine biosynthesis in macrophages.

Figure 6.

Figure 6

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CaMKII plays an important role in IL-4/IL-13-induced catecholamine production in primary macrophages. The primary macrophages were cultured in serum-free medium containing 2% BSA for 4 h and then treated with or without KN93 for 1 h followed by the treatment of 10 ng/ml IL-4 and 10 ng/ml IL-13 for 22 h. (A) Phosphorylation of TyrH was stimulated by the treatment of adrenaline but not the treatment of IL-4 and IL-13 for 30 min in primary macrophages. (B) Expression and phosphorylation of TyrH were induced by 22 h treatment of IL-4/IL-13, and CaMKII inhibitor KN93 suppressed IL-4/IL-13-induced phosphorylation of TyrH in peritoneal macrophages. (C) Western blot analysis and quantification of P-TyrH in B. (D) CaMKIIγ deficiency diminished IL-4/IL-13-induced phosphorylation but not expression of TyrH in peritoneal macrophages. The primary macrophages were isolated from CaMKIIγ KO and WT mice. (E) Western blot analysis and quantification of P-TyrH and TyrH in D. (F) CaMKIIγ deficiency attenuated IL-4/IL-13-induced norepinephrine secretion from primary macrophages. The data in A, B, D, and F are the representative from at least three individual experiments with similar results. The data in C, E, and F are presented with mean ± SEM. *P < 0.05, **P < 0.01.

Myeloid CaMKII signaling is critical for cold-induced activation of beige adipocytes in vivo

To elucidate the physiological role of myeloid CaMKII in cold adaption, we investigated the relationship between adipose CaMKII signaling and cold adaption. The 3-month-old male C57BL/6 mice were exposed to cold stress (6°C) for 10 h. We found that acute cold stress stimulated the phosphorylation of CaMKII in inguinal fat (Figure 7A and B), suggesting that CaMKII may play an important role in regulation of cold response. Immunofluorescence staining results showed that cold stress stimulated phosphorylation of CaMKII in adipose-resident macrophages as well (Figure 7C). To gain the insight into the role of myeloid CaMKII signaling in cold-induced thermogenesis, we reconstituted wild-type mice with bone marrow cells from CaMKIIγ deficient (BMT-KO) and wild-type control mice (BMT-WT). PCR analysis indicated that genomic DNA from bone marrow-derived macrophages of BMT-KO mice only yielded PCR products for mutant alleles (Figure 7D). In contrast, genomic DNA from bone marrow cells and adipose tissue in MBT-KO mice showed PCR products for both WT and mutant alleles (Figure 7E). These data demonstrate the success of adoptive transfer of CaMKII-deficient myeloid cells to WT recipient mice. Six weeks post bone marrow transplantation, indirect calorimetry was performed under room temperature for 48 h followed by cold stress for another 48 h. We found that CaMKIIγ-deficient bone marrow chimeras displayed downregulated cold-induced UCP1 expression as well as TyrH phosphorylation in inguinal fat (Figure 7F and G). In contrast, the phosphorylation of TyrH and expression of UCP1 were not significantly affected by myeloid-ablation of CaMKIIγ in the interscapular brown fat (Figure 7H and I), suggesting the differential role of myeloid CaMKII in white and brown fat. This finding is consistent with selective myeloid regulation of thermogenic program in white fat (Brestoff et al., 2015; Lee et al., 2015). In addition, mRNA levels of UCP1, but not IL-4 and IL-13, were suppressed by myeloid-deficiency of CaMKII in inguinal fat, implying that myeloid CaMKII-driven adipose thermogenesis is independent of the IL-4/IL-13 pathway (Figure 7J). On the other hand, CaMKIIγ BMT-KO mice displayed a slight decrease of O2 consumption throughout the light and dark cycle under cold stress condition despite having similar O2 consumption under room temperature condition (data not shown). The difference in O2 consumption did not reach significance, indicating that in addition to adipose-resident macrophage, other types of bone marrow-derived cells may have compensatory effects on cold-induced energy expenditure. In addition, myeloid-specific CaMKIIγ ablation had little effect in locomotor activity, food intake, and body mass when compared to WT littermates (data not shown). These data together suggest that myeloid CaMKII signaling plays an important role in cold-induced adipose catecholamine production and browning of white fat.

Figure 7.

Figure 7

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CaMKII deficiency suppressed basal activity of TyrH and UCP1 expression in inguinal not brown fat. (A) Cold exposure stimulated the phosphorylation of CaMKII in inguinal fat. The 3-month-old male C57BL/6 mice were housed in the metabolic phenotyping system with a temperature-controllable chamber and exposed to cold stress (6°C) or room temperature (22°C) condition for 48 h. (B) The ratio of phosphorylation to protein level of CaMKII in A was quantified. (C) Immunofluorescence study of co-localization of macrophage marker F4/80 with P-CaMKII in inguinal fat of mice under room temperature or cold stress condition. Scale bar, 50 μm. (D−J) The 6-week-old C57BL/6 mice were reconstituted with bone marrow cells from CaMKIIγ KO (BMT-KO) and WT mice (BMT-WT). Six weeks post bone marrow transplantation, indirect calorimetry and cold exposure were performed followed by euthanasia and fat tissue collection. PCR analysis was performed for genomic DNA from bone marrow-derived macrophages (BMDM) (D), bone marrow cells (BMC) and adipose tissue (E). The amplified band of smaller size (250 bp, top panel) indicates CaMKIIγ KO alleles, whereas the PCR product of larger size (500 bp, bottom panel) denotes the WT band. (F) CaMKIIγ-deficient bone marrow chimeras (BMT-KO) displayed downregulated phosphorylation of TyrH and expression of UCP1 in inguinal fat compared to control mice (BMT-WT) under cold stress condition. (G) Western blot analysis and quantification of P-TyrH and UCP1 in F. (H) The levels of P-TyrH and UCP1 in brown fat were similar between BMT-KO and BMT-WT mice under cold stress condition. (I) Western blot analysis and quantification of P-TyrH and UCP1 in H. (J) mRNA levels of UCP1 but not TyrH, IL-4 and IL-13 were significantly suppressed by myeloid-deficiency of CaMKIIγ in inguinal fat. All data are presented with mean ± SEM. *P < 0.05, **P < 0.01.

Discussion

Alternative activation of macrophages has been linked to the induction of catecholamine biosynthesis and substantial promotion of thermogenesis in adipose tissue, providing a potential therapeutic approach for the treatment of obesity and its related disorders (Nguyen et al., 2011). T helper 2 (Th2) cytokines IL-4 and IL-13 drive the alternative activation of macrophages, browning of white adipose tissue and induction of thermogenesis (Qiu et al., 2014). However, the intracellular signaling events modulating catecholamine biosynthesis in macrophages are poorly defined. Our present study shows that the circulating level of adrenaline was elevated by 10 h cold stress (Figure 2). Moreover, treatment of adrenaline stimulated TyrH phosphorylation and promoted catecholamine biosynthesis through PKA/CaMKII-dependent mechanisms in macrophages (Figures 2–5). In addition, impaired myeloid CaMKII signaling attenuated cold stress-increased catecholamine production and UCP1 expression in vivo and the promoting effect of adrenaline on macrophage-dependent activation of brown adipocytes in vitro (Figures 5 and 7), whereas the PKA/CaMKII/TyrH/Catecholamine pathway was not found to exist in adipocytes. Our study suggests that myeloid adrenergic signaling via CaMKII mediates cold stress-induced activation of beige adipocytes, which forms a feedforward loop of catecholamine production in macrophages.

TyrH can be directly phosphorylated at multiple sites including Ser8, 19, 31, and 40 by multifunctional kinases (Haycock et al., 1982; Dunkley et al., 2004). The phosphorylation of tyrosine hydroxylase at Ser40 leads to the increase of its enzymatic activity in vitro, in situ and in vivo (Dunkley et al., 2004). However, the phosphorylation at Ser31 of TyrH increases its activity, albeit to a much lesser extent than Ser40 phosphorylation, and phosphorylation at Ser19 or Ser8 has no direct effect on its activity (Dunkley et al., 2004). It has been shown that PKA promotes catecholamine production by upregulating TyrH expression and direct phosphorylation at Ser40 in multiple cell types (Joh et al., 1978; Vulliet et al., 1980; Haycock et al., 1982; Kim et al., 1993). In addition, another type of kinase CaMKII has been shown to directly phosphorylate TyrH, leading to the activation of TyrH (Ames et al., 1978; Tachikawa et al., 1986; George et al., 1989; Funakoshi et al., 1991). Consistent with this, CaMKII activation is closely related to catecholamine secretion and tyrosine hydroxylase activation in cultured adrenal medullary cells as well as other cell types (Tsutsui et al., 1994; Yanagihara et al., 1996). Similar to PKA, CaMKII stimulates TyrH activity by phosphorylation at Ser40 and induces the expression of TyrH in different cell types (Chen et al., 1996; Kumar et al., 2003; Daubner et al., 2011). Although Tachikawa et al. (1986) showed that CaMKII-stimulated phosphorylation is not sufficient to activate TyrH, our data suggest that CaMKII plays a critical role in cAMP homolog-stimulated phosphorylation at Ser40 of TyrH as well as catecholamine production in macrophages. In addition, CaMKII phosphorylation at Thr286 is highly sensitive in response to cAMP analog or PKA activation in macrophages (Figure 4). However, whether PKA and CaMKII synergize to regulate the phosphorylation of TyrH and the mechanisms behind this regulation remains largely unknown. It has been shown that intermittent hypoxia-stimulated phosphorylation and activation of TyrH are mediated in part by PKA and CaMKII (Kumar et al., 2003). Consistent with this, Ca2+ or cyclic AMP in combination produces an additive increase in both phosphorylation of tyrosine hydroxylase and the activation of the enzyme (George et al., 1989), suggesting the important role of CaMKII and PKA in phosphorylation of TyrH. In addition, our study shows that blocking CaMKII abolishes PKA-induced phosphorylation of TyrH and catecholamine production in macrophages. Therefore, our data suggests that CaMKII acts as a downstream kinase to mediate the promoting effect of PKA in regulating TyrH phosphorylation and catecholamine production in macrophages.

In addition to direct activation of brown/beige adipocytes, our study suggests that adrenergic signaling in macrophages leads to feedforward activation of catecholamine production via myeloid CaMKII in white adipose tissue. In support of this, adrenaline stimulates the phosphorylation of TyrH and catecholamine production through activating CaMKII in macrophages (Figures 2 and 5). Furthermore, myeloid ablation of CaMKII downregulated the phosphorylation of TyrH and UCP1 expression in beige fat (Figures 4 and 5). However, this effect was not observed in brown fat. This differential role of myeloid CaMKII in various fat depots was suggested by the evidence of selective induction of alternative activation of macrophages on thermogenesis in white fat as well (Brestoff et al., 2015; Lee et al., 2015). One possibility is that brown adipose tissue is well innervated by sympathetic neurons compared to white adipose tissue (Slavin and Ballard, 1978; Himms-Hagen, 1990). Therefore, macrophage-derived catecholamine in brown fat is not a critical pathway for thermogenic program. Another possibility is due to less macrophage fraction in brown fat than that in beige fat (Ding et al., 2016). However, a recent study contradicts the previous findings that macrophages are able to produce catecholamine and thus to induce adipose thermogenesis (Nguyen et al., 2011; Qiu et al., 2014; Fischer et al., 2017). Our present study suggests that myeloid TyrH/catecholamine pathway appears to be present in adipose tissue (Figure 1). Despite the low expression level in macrophage, phosphorylation of TyrH is efficiently stimulated by β-adrenergic signaling pathway involving PKA and CaMKII, reflecting a feedforward loop of catecholamine in macrophage (Figures 3–5). In agreement with this, myeloid CaMKII signaling is required for cold induction of UCP1 in beige fat (Figure 7), whereas myeloid-deficiency of CaMKII has no significant contribution to cold-induced energy expenditure in vivo. One possibility is that the bone marrow-derived cells, other than macrophage, somehow neutralize the inhibitory effect on energy expenditure in CaMKII-deficient bone marrow chimeras. Another possibility is that other secreted factors, in addition to norepinephrine, are involved in myeloid CaMKII regulation of beige adipocytes. Therefore, the contribution of myeloid CaMKII signaling to cold-induced thermogenic program and energy expenditure needs to be clarified using CaMKII conditional knockout mice in the future.

Th2 cytokines IL-4 and IL-13 have been shown to mediate cold-induced catecholamine biosynthesis through activation of the IL-4rα/STAT6 pathway in adipose tissue macrophages (Nguyen et al., 2011). Although CaMKII modulates the JAK1/STAT1 pathway in macrophages (Wang et al., 2008), little is known whether the IL-4rα/STAT6 pathway interacts with other pathways to regulate catecholamine production in macrophages. We found that chronic treatment of IL-4/IL-13 induced the phosphorylation and expression of TyrH, while acute treatment has no significant effect (Figure 6A). In support of this, IL-4/IL-13 treatment has little effect on the phosphorylation of CaMKII. In addition, blocking CaMKII with a specific inhibitor, or through ablation of CaMKIIγ, partially diminishes IL-4/IL-13-induced catecholamine production in primary macrophages. Our data suggest that in addition to phosphorylation of TyrH, CaMKII also plays an important role in mediating the inducing effect of IL-4rα/STAT6 pathway on catecholamine production (Figure 6). Although blocking CaMKII has little effect on IL-4/IL-13-induced TyrH expression, CaMKII is required for activation of TyrH and substantial catecholamine biosynthesis (Figure 6), indicating a possibility that interaction between CaMKII and the IL-4rα/STAT6 pathway may be TyrH expression-independent. However, the mechanisms underlying the cross talk between these two pathways remain to be clarified in the future studies.

In summary, our data show that myeloid adrenergic signaling, forming a feedforward cycle, induces catecholamine production through PKA-CaMKII-dependent mechanisms. Moreover, CaMKII mediates PKA-stimulated catecholamine production in macrophages as well as myeloid secreted factors-induced thermogenic gene expression in brown adipocytes. In addition, CaMKII also plays an important role in mediating IL-4 and IL-13-induced catecholamine production in macrophages. Our study strongly suggests that myeloid CaMKII signaling plays a key role in regulating catecholamine biosynthesis and WAT browning in response to cold.

Materials and methods

Materials

Dibutyryl cyclic AMP (dbcAMP), CL316,243, KN-93, and H89 were from Sigma; adrenaline was from Cayman; fura-2 was from Invitrogen; Antibodies of p-CaMKII at Thr286, CaMKII, P-tyrosine hydroxylase (TyrH) at Ser40, β-actin, and p-PKA substrates were from Cell Signaling. Antibody of TyrH were from Millipore (MAB318). Antibodies of UCP1 and C/EBPβ were from Abcam. Antibodies of CaMKIIγ and p-CaMKII were from Santa Cruz. Mouse IgG was from Santa Cruz. Antibodies of F4/80-Biotin and SA-APC were from Biolegend. Antibody of rabbit IgG AF488 was from Invitrogen. IL-4 and IL-13 were from eBioscience. Noradrenaline ELISA kits were from Rocky Mountain Diagnostics. The adenoviruses encoding constitutive active (CA) mutant and kinase-dead (KD) mutant of CaMKII were kindly provided by Dr Lale Ozcan at Columbia University and Dr Harold Singer at Albany Medical College (Pfleiderer et al., 2004; Ozcan et al., 2012).

Statistics

Effects of various treatments on the expression levels of norepinephrine, UCP1, CaMKII phosphorylation, and TyrH phosphorylation in cells and in tissue were analyzed by T-test for comparison of two groups or ANOVA for comparison of more than two groups. Data are presented as means ± SEM. P < 0.05 was considered as significant.

More methods can be found in the Supplementary material.

Supplementary material

Supplementary material is available at Journal of Molecular Cell Biology online.

Funding

This work is supported by R01 Award (DK110439 to M.L.) from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Junior Faculty Research Award (1-13-JF-37 to M.L.) from the American Diabetes Association, Grant in Aid Award (#15GRNT24940018 to M.L.) from American Heart Association, Innovative Basic Science Award (1-17-IBS-261 to M.L.) from the American Diabetes Association, CoBRE Pilot Award associated with P30 (P30GM103400 (PI: J. Liu) to M.L.), RAC Pilot Award (to M.L.) and UNMCCC pilot Award (to M.L.) at the University of New Mexico Health Sciences Center (UNMHSC), as well as other awards of 1R21NS091600 and P20GM109089 from the National Institutes of Health, 14GRNT20380496 from American Heart Association, and 12.1223.2017/AP from RMSE to D.E.B.

Conflict of interest: none declared.

Supplementary Material

Supplementary Data
Click here for additional data file. (573.5KB, pdf)
Supplementary Data
Click here for additional data file. (18.7KB, docx)

Acknowledgements

We thank Dr Feng Liu (UTHSCSA) for C57BL/6 mice originally transferred from his laboratory, Dr Vojo Deretic (UNMHSC) for providing us RAW264.7 macrophages, and Dr Lale Ozcan at Columbia University and Dr Harold Singer at Albany Medical College for providing the adenoviruses encoding CA and KD mutants of CaMKII. We also thank Drs Jiandie Lin and Zhuoxian Meng at University of Michigan who generously offered the purified viruses of CA-CaMKII and DN-CaMKII for the present study. We thank Dr Curt Hines for his EVOS system that allowed us to take images for immunofluorescence staining and Dr John A. Connor for designing the Ca2+ imaging setup and initial help with experiments. In addition, we thank Dr Jesse Denson, Marco Santamaria, and Emily Johanson at the UNM for editing this manuscript.

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Associated Data

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Supplementary Materials

Supplementary Data
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Supplementary Data
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