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Cannabinoid receptor 2 promotes the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage
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Cannabinoid receptor 2 promotes the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage Huiting Zhouc,1, Rao Dua,1, Gang Lic,1, Zhenjiang Baid, Jin Maa, Chenmei Maoa, Jian Wangb, , ⁎ Huan Guia, ⁎
a
Department of Pharmacology, Children’s Hospital of Soochow University, Suzhou 215025, China Department of Pediatric Surgery, Children's Hospital of Soochow University, Suzhou 215025, China c Institute of Pediatric Research, Children's Hospital of Soochow University, Suzhou 215025, China d Intensive Care Unit, Children's Hospital of Soochow University, Suzhou 215025, China b
ARTICLE INFO
ABSTRACT
Keywords: Cannabinoid receptor Ⅱ High mobility group box 1 Autophagy Macrophages
High mobility group box 1 (HMGB1) is a late phase inflammatory mediator in many inflammatory diseases. Extracellular HMGB1 could bind to many membrane receptors to activate downstream signaling molecules and promote inflammation resulting in cell and tissue damage. In our previous work, we found cannabinoid receptor Ⅱ(CB2R) inhibited the expression of HMGB1 in lipopolysaccharide (LPS)-induced septic models in vivo and in vitro, but the underlying mechanism is still unclear. The present study was aimed to explore the possible pathway through which CB2R suppressed HMGB1. Here, we found that the specific agonist of CB2R, GW405833 (GW) could induce intracellular HMGB1 degradation without influencing HMGB1 mRNA in peritoneal macrophages. Then we observed that autophagy inhibitor 3-methyladenine (3-MA) but not proteasome inhibitor MG-132 (MG) could block GW-induced HMGB1 degradation, which indicated that the autophagy-lysosome but not the ubiquitination pathway was involved in this process. Further study showed that GW could promote the integrity of autophagy flux in macrophages in terms of increased level of LC3Ⅱand decreased expression of p62 protein. It also observed that inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophages. GW could up-regulate expression of Cathepsin B (CTSB), and inhibition of CTSB blocked GW-induced HMGB1 degradation. In summary, all the data showed that activation of CB2R could promote the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage.
1. Introduction High mobility group box1 (HMGB1) is a highly conserved nuclear protein with an abundance of approximately one-tenth of the histidine in the nucleu [1]. In most resting cells, HMGB1 binds loosely to DNA and is primarily involved in the transcriptional regulation of the glucocorticoid receptor. Macrophages are the main source of HMGB1 in vivo [2]. Wang H, et al. found that serum HMGB1, as a late phase inflammatory mediator, is highly correlated with the endotoxic death in mice [2]. In our previous study, we found that cannabinoid receptor Ⅱ(CB2R) inhibits the expression of inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and HMGB1 in lipopolysaccharide (LPS)-induced septic models in vivo and in vitro [3]. It’s found that activation of CB2R reduces protein level of HMGB1 in animal serum and cell supernatants without influencing HMGB1 mRNA. The underlying mechanism of this phenomenon is still not
understood. Autophagy is a process of lysosome-mediated phagocytosis of autologous cytoplasmic proteins and organelles. It facilitates the nutrient metabolism of cells and the renewal of organelles so as to maintain important physiological processes of homeostasis. Several reports showed a close relation between autophagy and HMGB1. Autophagy promotes the release of HMGB1. Meanwhile, HMGB1 could translocate to the cytosol, compete with Bcl-2 to bind to Beclin1 and promote autophagy. In response to inflammation, enhanced autophagy can inhibit the activation of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes, and ultimately inhibit IL-1β and IL-18 release, thereby inhibit inflammation [4]. However, whether there is a similar outcome for HMGB1 after nuclear translocation is unknown. Increasing evidence shows that CB2R activation promotes autophagy to protect against certain disease conditions. Shao et al. found that
Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (H. Gui). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.intimp.2019.106007 Received 19 September 2019; Received in revised form 25 September 2019; Accepted 25 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Huiting Zhou, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106007
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the autophagy-associated protein LC3II/LC3I ratio and Beclin1 were decreased in the spinal cords of CB2R knockout mice with experimental autoimmune encephalomyelitis (EAE). The CB2R-specific agonist HU308 can promote autophagy in BV2 microglia to inhibit the expression and activation of NLRP3 inflammasome [5]. Denaes reported that CB2R activation inhibited inflammation of Kupffer cells in alcoholic fatty liver via an autophagy-dependent pathway [6]. HU308 also enhances the level of autophagy in the heart tissue of mice with diabetic cardiomyopathy (DCM) as well as in isolated cardiomyocytes challenged with high glucose (HG), thus improving cardiac function in DCM as well as cell viability in HG-challenged cardiomyocytes [7]. It’s well known that LPS is a powerful stimulator for HMGB1 secretion and autophagy progression in macrophages [8]. Our previous data and other reports indicated that activation of CB2R could not only decrease LPS-induced HMGB1 secretion but also promote autophagy in vitro. To clearly investigate the role of autophagy in CB2R-mediated inhibition, we need to exclude the influence of LPS on autophagy progression. So in most experiments of the present study, CB2R agonist is the only treatment reagent. Macrophages, as a key cell type of the innate immune system, are involved in development, homeostasis, tissue repair and immunity [9]. The autophagy-lysosome pathway is utilized following conventional phagocytosis in macrophages to clear invading bacteria and other pathogens, and also to digest portions of the cytoplasm [10]. In lysosomes, a series of acidic hydrolase enzymes is surrounded by alipoprotein membrane. Cathepsin is an important functional enzyme. Cathepsin B (CTSB), a member of the cysteine cathepsin family, has been reported to regulate the number of lysosomes and autophagosomes in mouse bone marrow-derived macrophages (BMDMs), thereby leading to enhanced survival of pathogens and increased susceptibility of the host to infection [11]. However, the role of CTSB in CB2R-mediated suppression of HMGB1 is unclear. Here, based on our previous study, we explored the underlying mechanism through which CB2R suppresses HMGB1 in macrophages. For the first time, we studied the possibility of the autophagolysosomal regulating HMGB1 metabolism under noninflammatory conditions.
with the university guidelines and approved by the Ethical Committee for Animal Care and the Use of Laboratory Animals of Children’s Hospital of Soochow University. 2.3. Culture of mouse peritoneal macrophages Mouse peritoneal macrophages were collected 3 days after intraperitoneal (i.p.) injection of sterilized broth culture (1 mL) in mice as described previously [12]. The cells were washed twice with PBS, resuspended in RPMI-1640 containing 10% FCS, and seeded at a density of 1–3 × 106/mL in plates. Two hours later, the culture medium was replaced to remove the non-adherent cells and then incubated at 37 °C in a humidified 5% CO2 atmosphere overnight for the subsequent procedures. 2.4. Quantitative Real-time RT-PCR Total RNA was extracted from cultured peritoneal macrophages with TRIzol. RNA was evaluated spectrophotometrically for quantity and purity. After reverse transcription, complementary DNA was used as a template for PCR. PCR amplification was performed using specific primers as follows: mouse HMGB1 sense 5′- TTG TGC AAA CTT GCC GGG AGG A-3′, mouse HMGB1 antisense 5′- ACT TCT CCT TCA GCT TGG CAG C-3′, mouse actin sense 5′-AGT GTG ACG TTG ACA TCC GT3′,and mouse actin antisense 5′-GCA GCT CAG TAA CAG TCC GC-3′. Quantitative real-time RT-PCR was carried out on a Roche LightCycler® 480ⅡReal-Time PCR System (Roche Life Science). The housekeeping gene actin was used to normalize all tested genes, and quantification of the mRNA level was performed using the ΔΔCt method. The value of each control sample was set at one and used to calculate the fold change of target genes. 2.5. Cell variability assay Macrophages were seeded in 96-well plates at 1 × 106 cells/mL in 100 μL of complete RPMI containing GW with/without LPS (10 ng/mL) and cultured for different times in a humidified, 5% CO2 atmosphere at 37 °C. Cell variability was measured as previously described using CCK8 purchased from Dojindo Laboratories (Kumamoto, Japan) [13]. Briefly, 10 μL of CCK-8 reagent was added to each well 3 h in advance, and the absorbance at 450 nm was determined by a Multiskan FCELISA plate reader.
2. Materials and methods 2.1. Reagents GW405833 (GW) and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-Methyladenine (3-MA), MG-132 (MG), and CA-074 Me (CA) were purchased from APExBIO Technology (Houston, TX, USA). RPMI-1640 medium and phosphate-buffered saline (PBS) free of Ca2+ and Mg2+ were obtained from Life Technologies (GIBCO, CA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). An ELISA kit for mouse HMGB1 was purchased from Westang Biological Technology Co., Ltd. (Shanghai, China). Anti-HMGB1 and anti-Cathepsin B antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-p62 and anti-LC3B antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against glyceraldehyde dehydrogenase (GAPDH), α-tubulin and β-actin, and horseradish peroxidase (HRP)labeled goat anti-rabbit/mouse IgG secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 565- and Alexa Fluor 488-conjugated donkey anti-rabbit IgG was purchased from Biolegend (San Diego, CA, USA).
2.6. Enzyme-linked immuno sorbent assay (ELISA) HMGB1 levels in supernatants were determined by ELISA according to the manufacturer’s instructions. A total of 500 μL/well cell suspension was seeded in a 24-well plate and treated with LPS and/or GW for different times. Samples were collected and centrifuged at 12,000 g for 5 min to obtain the supernatants. The concentrations of HMGB1 were determined. 2.7. Western blotting Cultured cells were lysed with lysis buffer. Protein concentrations were determined using the Bradford method. The lysates were fractionated by Tris-glycine-buffered 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibodies against HMGB1, p62, LC3B, CTSB, GAPDH, α-tubulin or β-actin. After washing, the membranes were incubated with HRP-conjugated secondary antibodies and then scanned using an Amersham Imager 600. Densitometry of the signal bands was analyzed with ImageJ software.
2.2. Animals Male C57BL/6J mice (8 weeks old, 18–22 g) were obtained from Lingchang Biotechnology Co., Ltd. (Shanghai, China). All animals were fed standard mouse chow and water freely and maintained under constant conditions (temperature: 20–25 °C; humidity: 40%–60%; light/dark cycle:12 h). All procedures were conducted in accordance 2
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2.8. Immunofluorescence
treatment for 24 h did not affect cell viability at doses ranging from 1 to 10 μM (Fig. 1C). Furthermore, even when the treatment time was prolonged to 42 h, there was still no effect of extracelluar HMGB1 expression in the GW-treated group (Fig. 1D). These data suggested that a posttranscriptional mechanism is involved in CB2R-regulated inhibition of HMGB1 in macrophages.
Peritoneal macrophages were washed with phosphate-buffered saline three times and then fixed and permeabilized with 4% paraformaldehyde containing 0.3% Triton X-100 for 15 min. Cells were blocked with 5% bovine serum albumin for 30 min and incubated with primary an anti-HMGB1 or LC3B antibody overnight at 4 °C followed by incubation with secondary Alexa Fluor 488- or 565-conjugated donkey anti-rabbit IgG for 1 h. Finally, cells were counterstained with 4′,6diamidino-2-phenylindole (DAPI) for 3 min. Photographs were taken using an Olympus micro FV10-ASW 2.1 viewer.
3.2. The autophagy-lysosome pathway but not ubiquitination pathway is involved in CB2R-mediated HMGB1 degradation
3. Results
Next, the level of intracellular HMGB1 protein was assessed. It was found that activation of CB2R by GW directly promoted the degradation of intracellular HMGB1 over time (Fig. 2A and B). Because the autophagy-lysosomal and ubiquitination pathways are the two main pathways mediating the degradation of endogenous proteins, we further explored the exact approach mediating HMGB1 degradation by specific inhibitors of these two pathways (3-MA and MG). It was observed that 3-MA but not MG could block GW-induced HMGB1 degradation (Fig. 2C-F). These results showed that the autophagy-lysosome but not the ubiquitination pathway is involved in CB2R-mediated HMGB1 degradation.
3.1. CB2R activation decreased LPS-induced HMGB1 secretion without influencing HMGB1 mRNA in peritoneal macrophages
3.3. Activation of CB2R promoted the integrity of autophagyic flux in macrophages
First, the effect of CB2R on the extracellular level of HMGB1 was assessed. At a regular anti-inflammatory dose of 10 μM, it was observed that the specific CB2R agonist GW could suppress extracellular HMGB1 as early as 12 h after LPS stimulation, and the suppression was stronger at 24 h after LPS stimulation (Fig. 1A). Second, we detected HMGB1 mRNA by RT-PCR. As shown in Fig. 1B, neither LPS nor GW treatment changed the expression of HMGB1 mRNA. To exclude the possible effect of cell number on the level of HMGB1 in the supernatant, cell viability was tested with CCK-8. Compared with the control group, GW
Next, the role of CB2R on autophagy function was explored. As shown in Fig. 3A-D, the CB2R agonist GW increased the ratio of LC3II/ LC3I in a time- and dose-dependent manner. Using laser confocal technology, it was observed that treatment with GW could markedly increase the number of mature autophagosomes containing LC3II (Fig. 3E). Furthermore, GW treatment also decreased the expression of p62 protein in a time-dependent manner in macrophages (Fig. 3F and G). Taken together, these data indicated that activation of CB2R promoted the integrity of autophagic flux in macrophages.
2.9. Statistical analysis Analysis of paired data was completed using a two-tailed Wilcoxon signed-rank test or paired Student’s t-test. Unpaired groups were analyzed by an unpaired Student’s t-test. All tests were performed, including a 95% confidence interval, using GraphPad Prism Software v8.0.
Fig. 1. Agonist of CB2R decreased LPS-induced HMGB1 secretion without influencing HMGB1 mRNA in peritoneal macrophages. (A) Primary peritoneal macrophages isolated from C57BL/6J mice were treated with LPS (10 ng/mL) in the absence or presence of GW (10 μM) for indicated time. Levels of HMGB1 was detected by ELISA. Data are presented as means ± SD (n = 3) and ∗∗ P < 0.01. (B) Peritoneal macrophages were incubated with LPS (10 ng/mL) or GW (1–10 μM) for 6 h. Total RNA was extracted and subjected to quantitative real-time RT-PCR for analysis of HMGB1 mRNA expression. (C) Peritoneal macrophages were incubated with GW (1–10 μM) for 24 h, cell viability was detected by CCK-8. Data are means ± SD (n = 4). (D) Primary peritoneal macrophages were treated GW (10 μM) for indicated time. Levels of HMGB1 was detected by ELISA. Data are presented as means ± SD (n = 3).
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Fig. 2. Autophagy-lysosome but not ubiquitination pathway was involved in CB2R-mediated HMGB1 degradation. (A) Peritoneal macrophages were treated with GW (10 μM) for indicated time, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (B), Quantification of the result of (A), HMGB1 expression was normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05. (C) Peritoneal macrophages were treated with GW in the presence or absence of 3-MA (5 μM) for 18 h, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (D), Quantification of the result of (C), HMGB1 expression was normalized to GAPDH. Data are means ± SD (n = 3) and * P < 0.05, vs control group; ## P < 0.01, vs GW group. (E) Peritoneal macrophages were treated with GW in the presence or absence of MG (1 μM) for 18 h, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (F), Quantification of the result of (E), HMGB1 expression was normalized to β-actin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05.
HMGB1 and subsequent development of broad inflammatory injury. This result will be very useful as a therapeutic approach in many inflammation-related diseases. Here, for the first time, we explored the underlying mechanism of CB2R-regulated inhibition of HMGB1 in macrophages. We also demonstrated that the autophagy-lysosome pathway played an important role in CB2R-induced intracellular degradation of HMGB1. LPS is a powerful stimulator of inflammation. LPS has also been reported to induce autophagy through multiple manners, such as Tollinterleukin 1 receptor domain-containing adaptor-inducing interferonβ (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling in macrophages [8]; LPS-regulated autophagy may present at least 8–16 h after LPS treatment. In our experiment, GW, the agonist of CB2R, inhibited LPS-induced expression of inflammatory factors and boosted autophagy in as little as 3 h after GW treatment, even without the presence of LPS. Specifically, GW induced autophagy faster and to a greater extent than LPS in terms of LC3II formation, which represents mature autophagosomes. Therefore, CB2R can induce autophagy in an LPS-independent manner. Few articles have mentioned the signaling pathway mediating CB2R-regulated autophagy. Vara D reported that another specific CB2R agonist, JWH-015, induced autophagy in the hepatocellular carcinoma cell line HepG2 via tribble homolog 3 (TRB3) upregulation and subsequent inhibition of the serine-threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation [14]. Dando also demonstrated that GW inhibited energetic metabolism and induced AMPK-dependent autophagy in pancreatic cancer cells [15]. In our previous study (data not shown), we did not observe any influence of GW on the phosphorylation of AMPK in macrophages. However, the exact signaling pathway underlying CB2R-induced autophagy requires further investigation. Normally, HMGB1 mainly exists in the nucleus but can also shuttle between the nucleus and cytoplasm to achieve homeostasis. Acetylation, phosphorylation and N-terminal glycosylation of protein molecules regulate homeostasis. Excessive posttranscriptional
3.4. Inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophages To assess the role of autophagy in CB2R-mediated HMGB1 regulation, we studied the nuclear translocation of HMGB1. In the control group, HMGB1 mainly remained in nucleus. After 18 h of treatment with GW, the amount of HMGB1 scattered in the cytoplasm increased. Most HMGB1 redistributed from the nucleus to the cytoplasm after GW treatment for 24 h (Fig. 4A). However, 3-MA suppressed GW-induced HMGB1 translocation at 18 h (Fig. 4B). This result demonstrated that autophagy is also involved in GW-regulated HMGB1 translocation. 3.5. CTSB was involved in the degradation of HMGB1 Last but not least, we wanted to understand how HMGB1 was degraded in the cytoplasm. Because CTSB is an important cysteine endopeptidase in lysosomes and is extensively expressed in macrophages. We explored whether HMGB1 was a substrate of CTSB. We found that the expression of CTSB protein increased at 12 h after GW addition (Fig. 5A), but treatment with a specific inhibitor of CTSB, CA, prevented GW-induced degradation of HMGB1 in macrophages, indicating HMGB1 was likely a substrate of CTSB (Fig. 5B). 4. Discussion As a danger-associated molecular pattern (DAMP), it is generally believed that extracellular HMGB1 binds to many membrane receptors to activate downstream signaling molecules and promote inflammation resulting in cell and tissue damage. To date, most studies have mainly focused on the functions and underlying mechanisms of extracellular HMGB1. This strategy appears to have extensive limitations because targets that could bind to HMGB1 are so diverse that one single inhibitor does not cover all the combinations. In the present study, we found that activation of CB2R in macrophages distinctly decreased the level of intracellular HMGB1 protein, thus preventing the secretion of 4
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Fig. 3. Activation of CB2R promoted the integrity of autophagic flux in macrophages (A) Peritoneal macrophages were treated with GW (10 μM) for indicated time, LC3Ⅰand LC3Ⅱprotein were detected by Western blot. Data are representative of three independent experiments. (B), Quantification of the result of (A), Ratio of LC3Ⅱ/LC3Ⅰwas obtained after been normalized to βactin separately. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05, vs control group. (C) Peritoneal macrophages were incubated with GW (1–10 μM) for 12 h, LC3Ⅰand LC3Ⅱprotein were detected by Western blotting. Data are representative of three independent experiments. (D), Quantification of the **result of (C), Ratio of LC3Ⅱ/LC3Ⅰwas obtained after been normalized to tubulin separately. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05. (E) LC3Ⅰand LC3Ⅱ was detected by confocal technology. LC3Ⅰand LC3Ⅱ was marked in red fluorescence and nucleus was marked with DAPI in blue. Data are representative of three independent experiment. (F) Macrophages were treated with GW (10 μM) for indicated time, p62 protein were detected by Western blotting. Data are representative of three independent experiments. (G), Quantification of the result of (A), p62 expression was normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05.
Fig. 4. Inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophage. (A) Macrophages were treated with GW (10 μM) for indicated time. Confocal images were taken after cells were stained with the anti-HMGB1 Ab and Alexa Fluor 488-conjugated secondary Ab (green). Data are representative of three independent experiments. (B) Macrophages were treated with GW (10 μM) in the absence or presence of 3-MA (5 μM) for 18 h. Confocal images were taken after cells stained with the anti-HMGB1 Ab and Alexa Fluor 488-conjugated secondary Ab (green). Data are representative of three independent experiments. 5
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Fig. 5. CTSB was involved in the degradation of HMGB1. (A) Macrophages were treated with GW (10 μM) for indicated time. CTSB expression was detected by Western blot. Data are representative of three independent experiments. (B) Quantification of the result of (A), Ratio of CTSB was obtained after been normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01. (C) Macrophages were treated with GW (10 μM) in the absence or presence of CA (10 μM) for 18 h, HMGB1 protein were detected by Western blot. Data are representative of three independent experiments. (D), Quantification of (C), HMGB1 expression was normalized to GAPDH. Data are means ± SD (n = 3) and ∗∗ P < 0.01, vs control group; ## P < 0.01, vs GW group.
modification can cause HMGB1 molecules to detach from DNA and bind to the nuclear transporter CRM1, resulting in unidirectional nuclear translocation [16]. Inhibition of CRM1 prevents its distribution to the nucleus and suppresses HMGB1 translocation to the cytoplasm [17]. In the present study, GW induced HMGB1 nuclear translocation, which was observed 18 h after GW challenge. In addition, GW-induced CRM1 translocation occurred as early as 12 h after GW treatment. However, Western blotting analysis revealed that GW decreased the expression of CRM1 protein (data not shown). It seems that the role of CRM1 in the process of GW-regulated HMGB1 nuclear translocation requires further study. It is well known that autophagy is closely associated with HMGB1. On the one hand, extracellular HMGB1 triggers RAGE to promote autophagy in cancer cells [18]. On the other hand, intracellular HMGB1 translocated to the cytoplasm binds to Beclin1 to initiate autophagosome formation [19]. However, our research showed the possibility that autophagy induced HMGB1 redistribution. Because in chronological order, CB2R agonist-induced autophagy occurred much earlier than the induced HMGB1 intracellular redistribution and degradation (3 h vs 18 h). The inhibition of autophagy by 3-MA suppressed HMGB1 translocation and prevented HMGB1 degradation. This interesting finding provides additional evidence indicating a new form of interaction between autophagy and HMGB1within macrophages. However, there is a need for further investigation. Unlike the classic endoplasmic reticulum-Golgi secretion model, HMGB1 does not enter the endoplasmic reticulum after translocation to the cytoplasm [20]. In monocytes, LPS-induced nuclear translocation of HMGB1 is mainly distributed in secretory lysosomes and rapidly secreted extracellularly by exocytosis under the stimulation of lysophosphatidylcholine (LPC) [21]. These data indicate that HMGB1 has a temporary interaction with lysosomes in the cytoplasm during inflammation. However, in the absence of inflammation, related evidence is limited. Here, our results shed light on the possible pathway of HMGB1 degradation inside the cytoplasm, in which the autophagy-lysosomes pathway may play an important role. Cathepsins are important functional enzymes in lysosomes. CTSB has been reported to regulate the number of lysosomes and autophagosomes in BMDMs. Specifically, CTSB directly cleaves the lysosomal calcium channel MCOLN1/ TRPML1 and negatively regulates the efflux of calcium and activation of the serine/threonine phosphatase PPP3/calcineurin, thus inhibiting transcription of lysosomal and autophagy-related genes. CTSB-deficient cells have increased number and volume of lysosomes and
autophagosomes [11]. These data did not provide direct evidence that CTSB was involved in intracellular HMGB1 regulation but provided us with a clue that CTSB interacts with autophagy and lysosomes. Here, we focused on the enzymatic function of CTSB. GW treatment did not affect the expression of CTSB in macrophages, but an inhibitor of CTSB, CA-074, prevented the GW-induced degradation of HMGB1 in macrophages, indicating that HMGB1 is likely a substrate of CTSB. In the current study, we demonstrated that CB2R promoted HMGB1 degradation through the autophagy-lysosome pathway in macrophages. For the first time, we found that HMGB1 could be degraded intracellularly by CB2R activation, and another step further, we presented data showing that autophagy could induce HMGB1 nuclear translocation and mediate the intracellular degradation of HMGB1. These findings could be helpful to understand the complex interaction between autophagy and HMGB1. Additionally, these data suggest that CB2R is a powerful potential target for treatment of multiple inflammatory diseases. Author contributions Huan Gui and Jan Wang designed the experiments and research project. Huan Gui, Huiting Zhou and Gang Li performed the experiments and analyzed the data. Zhenjiang Bai, Chenmei Mao and Jing Ma participated in the discussion. Huiting Zhou and Rao Du wrote the paper. Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (Grant 81501364, 81501703, 81671967, 81871594, 81571551 and 81801559), the Natural Science Foundation of Jiangsu Province (Grant BK20150294, BK20190053), the Science and Technology Program of the Bureau of Traditional Chinese Medicine of Jiangsu Province (Grant YB2017096), the Science and Technology Program of Suzhou (Grant SYS2018067). 6
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Cannabinoid receptor 2 promotes the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage Huiting Zhouc,1, Rao Dua,1, Gang Lic,1, Zhenjiang Baid, Jin Maa, Chenmei Maoa, Jian Wangb, , ⁎ Huan Guia, ⁎
a
Department of Pharmacology, Children’s Hospital of Soochow University, Suzhou 215025, China Department of Pediatric Surgery, Children's Hospital of Soochow University, Suzhou 215025, China c Institute of Pediatric Research, Children's Hospital of Soochow University, Suzhou 215025, China d Intensive Care Unit, Children's Hospital of Soochow University, Suzhou 215025, China b
ARTICLE INFO
ABSTRACT
Keywords: Cannabinoid receptor Ⅱ High mobility group box 1 Autophagy Macrophages
High mobility group box 1 (HMGB1) is a late phase inflammatory mediator in many inflammatory diseases. Extracellular HMGB1 could bind to many membrane receptors to activate downstream signaling molecules and promote inflammation resulting in cell and tissue damage. In our previous work, we found cannabinoid receptor Ⅱ(CB2R) inhibited the expression of HMGB1 in lipopolysaccharide (LPS)-induced septic models in vivo and in vitro, but the underlying mechanism is still unclear. The present study was aimed to explore the possible pathway through which CB2R suppressed HMGB1. Here, we found that the specific agonist of CB2R, GW405833 (GW) could induce intracellular HMGB1 degradation without influencing HMGB1 mRNA in peritoneal macrophages. Then we observed that autophagy inhibitor 3-methyladenine (3-MA) but not proteasome inhibitor MG-132 (MG) could block GW-induced HMGB1 degradation, which indicated that the autophagy-lysosome but not the ubiquitination pathway was involved in this process. Further study showed that GW could promote the integrity of autophagy flux in macrophages in terms of increased level of LC3Ⅱand decreased expression of p62 protein. It also observed that inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophages. GW could up-regulate expression of Cathepsin B (CTSB), and inhibition of CTSB blocked GW-induced HMGB1 degradation. In summary, all the data showed that activation of CB2R could promote the intracellular degradation of HMGB1 via the autophagy-lysosome pathway in macrophage.
1. Introduction High mobility group box1 (HMGB1) is a highly conserved nuclear protein with an abundance of approximately one-tenth of the histidine in the nucleu [1]. In most resting cells, HMGB1 binds loosely to DNA and is primarily involved in the transcriptional regulation of the glucocorticoid receptor. Macrophages are the main source of HMGB1 in vivo [2]. Wang H, et al. found that serum HMGB1, as a late phase inflammatory mediator, is highly correlated with the endotoxic death in mice [2]. In our previous study, we found that cannabinoid receptor Ⅱ(CB2R) inhibits the expression of inflammatory mediators such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and HMGB1 in lipopolysaccharide (LPS)-induced septic models in vivo and in vitro [3]. It’s found that activation of CB2R reduces protein level of HMGB1 in animal serum and cell supernatants without influencing HMGB1 mRNA. The underlying mechanism of this phenomenon is still not
understood. Autophagy is a process of lysosome-mediated phagocytosis of autologous cytoplasmic proteins and organelles. It facilitates the nutrient metabolism of cells and the renewal of organelles so as to maintain important physiological processes of homeostasis. Several reports showed a close relation between autophagy and HMGB1. Autophagy promotes the release of HMGB1. Meanwhile, HMGB1 could translocate to the cytosol, compete with Bcl-2 to bind to Beclin1 and promote autophagy. In response to inflammation, enhanced autophagy can inhibit the activation of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes, and ultimately inhibit IL-1β and IL-18 release, thereby inhibit inflammation [4]. However, whether there is a similar outcome for HMGB1 after nuclear translocation is unknown. Increasing evidence shows that CB2R activation promotes autophagy to protect against certain disease conditions. Shao et al. found that
Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (H. Gui). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.intimp.2019.106007 Received 19 September 2019; Received in revised form 25 September 2019; Accepted 25 October 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Huiting Zhou, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106007
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the autophagy-associated protein LC3II/LC3I ratio and Beclin1 were decreased in the spinal cords of CB2R knockout mice with experimental autoimmune encephalomyelitis (EAE). The CB2R-specific agonist HU308 can promote autophagy in BV2 microglia to inhibit the expression and activation of NLRP3 inflammasome [5]. Denaes reported that CB2R activation inhibited inflammation of Kupffer cells in alcoholic fatty liver via an autophagy-dependent pathway [6]. HU308 also enhances the level of autophagy in the heart tissue of mice with diabetic cardiomyopathy (DCM) as well as in isolated cardiomyocytes challenged with high glucose (HG), thus improving cardiac function in DCM as well as cell viability in HG-challenged cardiomyocytes [7]. It’s well known that LPS is a powerful stimulator for HMGB1 secretion and autophagy progression in macrophages [8]. Our previous data and other reports indicated that activation of CB2R could not only decrease LPS-induced HMGB1 secretion but also promote autophagy in vitro. To clearly investigate the role of autophagy in CB2R-mediated inhibition, we need to exclude the influence of LPS on autophagy progression. So in most experiments of the present study, CB2R agonist is the only treatment reagent. Macrophages, as a key cell type of the innate immune system, are involved in development, homeostasis, tissue repair and immunity [9]. The autophagy-lysosome pathway is utilized following conventional phagocytosis in macrophages to clear invading bacteria and other pathogens, and also to digest portions of the cytoplasm [10]. In lysosomes, a series of acidic hydrolase enzymes is surrounded by alipoprotein membrane. Cathepsin is an important functional enzyme. Cathepsin B (CTSB), a member of the cysteine cathepsin family, has been reported to regulate the number of lysosomes and autophagosomes in mouse bone marrow-derived macrophages (BMDMs), thereby leading to enhanced survival of pathogens and increased susceptibility of the host to infection [11]. However, the role of CTSB in CB2R-mediated suppression of HMGB1 is unclear. Here, based on our previous study, we explored the underlying mechanism through which CB2R suppresses HMGB1 in macrophages. For the first time, we studied the possibility of the autophagolysosomal regulating HMGB1 metabolism under noninflammatory conditions.
with the university guidelines and approved by the Ethical Committee for Animal Care and the Use of Laboratory Animals of Children’s Hospital of Soochow University. 2.3. Culture of mouse peritoneal macrophages Mouse peritoneal macrophages were collected 3 days after intraperitoneal (i.p.) injection of sterilized broth culture (1 mL) in mice as described previously [12]. The cells were washed twice with PBS, resuspended in RPMI-1640 containing 10% FCS, and seeded at a density of 1–3 × 106/mL in plates. Two hours later, the culture medium was replaced to remove the non-adherent cells and then incubated at 37 °C in a humidified 5% CO2 atmosphere overnight for the subsequent procedures. 2.4. Quantitative Real-time RT-PCR Total RNA was extracted from cultured peritoneal macrophages with TRIzol. RNA was evaluated spectrophotometrically for quantity and purity. After reverse transcription, complementary DNA was used as a template for PCR. PCR amplification was performed using specific primers as follows: mouse HMGB1 sense 5′- TTG TGC AAA CTT GCC GGG AGG A-3′, mouse HMGB1 antisense 5′- ACT TCT CCT TCA GCT TGG CAG C-3′, mouse actin sense 5′-AGT GTG ACG TTG ACA TCC GT3′,and mouse actin antisense 5′-GCA GCT CAG TAA CAG TCC GC-3′. Quantitative real-time RT-PCR was carried out on a Roche LightCycler® 480ⅡReal-Time PCR System (Roche Life Science). The housekeeping gene actin was used to normalize all tested genes, and quantification of the mRNA level was performed using the ΔΔCt method. The value of each control sample was set at one and used to calculate the fold change of target genes. 2.5. Cell variability assay Macrophages were seeded in 96-well plates at 1 × 106 cells/mL in 100 μL of complete RPMI containing GW with/without LPS (10 ng/mL) and cultured for different times in a humidified, 5% CO2 atmosphere at 37 °C. Cell variability was measured as previously described using CCK8 purchased from Dojindo Laboratories (Kumamoto, Japan) [13]. Briefly, 10 μL of CCK-8 reagent was added to each well 3 h in advance, and the absorbance at 450 nm was determined by a Multiskan FCELISA plate reader.
2. Materials and methods 2.1. Reagents GW405833 (GW) and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-Methyladenine (3-MA), MG-132 (MG), and CA-074 Me (CA) were purchased from APExBIO Technology (Houston, TX, USA). RPMI-1640 medium and phosphate-buffered saline (PBS) free of Ca2+ and Mg2+ were obtained from Life Technologies (GIBCO, CA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). An ELISA kit for mouse HMGB1 was purchased from Westang Biological Technology Co., Ltd. (Shanghai, China). Anti-HMGB1 and anti-Cathepsin B antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-p62 and anti-LC3B antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against glyceraldehyde dehydrogenase (GAPDH), α-tubulin and β-actin, and horseradish peroxidase (HRP)labeled goat anti-rabbit/mouse IgG secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Alexa Fluor 565- and Alexa Fluor 488-conjugated donkey anti-rabbit IgG was purchased from Biolegend (San Diego, CA, USA).
2.6. Enzyme-linked immuno sorbent assay (ELISA) HMGB1 levels in supernatants were determined by ELISA according to the manufacturer’s instructions. A total of 500 μL/well cell suspension was seeded in a 24-well plate and treated with LPS and/or GW for different times. Samples were collected and centrifuged at 12,000 g for 5 min to obtain the supernatants. The concentrations of HMGB1 were determined. 2.7. Western blotting Cultured cells were lysed with lysis buffer. Protein concentrations were determined using the Bradford method. The lysates were fractionated by Tris-glycine-buffered 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibodies against HMGB1, p62, LC3B, CTSB, GAPDH, α-tubulin or β-actin. After washing, the membranes were incubated with HRP-conjugated secondary antibodies and then scanned using an Amersham Imager 600. Densitometry of the signal bands was analyzed with ImageJ software.
2.2. Animals Male C57BL/6J mice (8 weeks old, 18–22 g) were obtained from Lingchang Biotechnology Co., Ltd. (Shanghai, China). All animals were fed standard mouse chow and water freely and maintained under constant conditions (temperature: 20–25 °C; humidity: 40%–60%; light/dark cycle:12 h). All procedures were conducted in accordance 2
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2.8. Immunofluorescence
treatment for 24 h did not affect cell viability at doses ranging from 1 to 10 μM (Fig. 1C). Furthermore, even when the treatment time was prolonged to 42 h, there was still no effect of extracelluar HMGB1 expression in the GW-treated group (Fig. 1D). These data suggested that a posttranscriptional mechanism is involved in CB2R-regulated inhibition of HMGB1 in macrophages.
Peritoneal macrophages were washed with phosphate-buffered saline three times and then fixed and permeabilized with 4% paraformaldehyde containing 0.3% Triton X-100 for 15 min. Cells were blocked with 5% bovine serum albumin for 30 min and incubated with primary an anti-HMGB1 or LC3B antibody overnight at 4 °C followed by incubation with secondary Alexa Fluor 488- or 565-conjugated donkey anti-rabbit IgG for 1 h. Finally, cells were counterstained with 4′,6diamidino-2-phenylindole (DAPI) for 3 min. Photographs were taken using an Olympus micro FV10-ASW 2.1 viewer.
3.2. The autophagy-lysosome pathway but not ubiquitination pathway is involved in CB2R-mediated HMGB1 degradation
3. Results
Next, the level of intracellular HMGB1 protein was assessed. It was found that activation of CB2R by GW directly promoted the degradation of intracellular HMGB1 over time (Fig. 2A and B). Because the autophagy-lysosomal and ubiquitination pathways are the two main pathways mediating the degradation of endogenous proteins, we further explored the exact approach mediating HMGB1 degradation by specific inhibitors of these two pathways (3-MA and MG). It was observed that 3-MA but not MG could block GW-induced HMGB1 degradation (Fig. 2C-F). These results showed that the autophagy-lysosome but not the ubiquitination pathway is involved in CB2R-mediated HMGB1 degradation.
3.1. CB2R activation decreased LPS-induced HMGB1 secretion without influencing HMGB1 mRNA in peritoneal macrophages
3.3. Activation of CB2R promoted the integrity of autophagyic flux in macrophages
First, the effect of CB2R on the extracellular level of HMGB1 was assessed. At a regular anti-inflammatory dose of 10 μM, it was observed that the specific CB2R agonist GW could suppress extracellular HMGB1 as early as 12 h after LPS stimulation, and the suppression was stronger at 24 h after LPS stimulation (Fig. 1A). Second, we detected HMGB1 mRNA by RT-PCR. As shown in Fig. 1B, neither LPS nor GW treatment changed the expression of HMGB1 mRNA. To exclude the possible effect of cell number on the level of HMGB1 in the supernatant, cell viability was tested with CCK-8. Compared with the control group, GW
Next, the role of CB2R on autophagy function was explored. As shown in Fig. 3A-D, the CB2R agonist GW increased the ratio of LC3II/ LC3I in a time- and dose-dependent manner. Using laser confocal technology, it was observed that treatment with GW could markedly increase the number of mature autophagosomes containing LC3II (Fig. 3E). Furthermore, GW treatment also decreased the expression of p62 protein in a time-dependent manner in macrophages (Fig. 3F and G). Taken together, these data indicated that activation of CB2R promoted the integrity of autophagic flux in macrophages.
2.9. Statistical analysis Analysis of paired data was completed using a two-tailed Wilcoxon signed-rank test or paired Student’s t-test. Unpaired groups were analyzed by an unpaired Student’s t-test. All tests were performed, including a 95% confidence interval, using GraphPad Prism Software v8.0.
Fig. 1. Agonist of CB2R decreased LPS-induced HMGB1 secretion without influencing HMGB1 mRNA in peritoneal macrophages. (A) Primary peritoneal macrophages isolated from C57BL/6J mice were treated with LPS (10 ng/mL) in the absence or presence of GW (10 μM) for indicated time. Levels of HMGB1 was detected by ELISA. Data are presented as means ± SD (n = 3) and ∗∗ P < 0.01. (B) Peritoneal macrophages were incubated with LPS (10 ng/mL) or GW (1–10 μM) for 6 h. Total RNA was extracted and subjected to quantitative real-time RT-PCR for analysis of HMGB1 mRNA expression. (C) Peritoneal macrophages were incubated with GW (1–10 μM) for 24 h, cell viability was detected by CCK-8. Data are means ± SD (n = 4). (D) Primary peritoneal macrophages were treated GW (10 μM) for indicated time. Levels of HMGB1 was detected by ELISA. Data are presented as means ± SD (n = 3).
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Fig. 2. Autophagy-lysosome but not ubiquitination pathway was involved in CB2R-mediated HMGB1 degradation. (A) Peritoneal macrophages were treated with GW (10 μM) for indicated time, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (B), Quantification of the result of (A), HMGB1 expression was normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05. (C) Peritoneal macrophages were treated with GW in the presence or absence of 3-MA (5 μM) for 18 h, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (D), Quantification of the result of (C), HMGB1 expression was normalized to GAPDH. Data are means ± SD (n = 3) and * P < 0.05, vs control group; ## P < 0.01, vs GW group. (E) Peritoneal macrophages were treated with GW in the presence or absence of MG (1 μM) for 18 h, HMGB1 protein were detected by Western blotting. Data are representative of three independent experiments. (F), Quantification of the result of (E), HMGB1 expression was normalized to β-actin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05.
HMGB1 and subsequent development of broad inflammatory injury. This result will be very useful as a therapeutic approach in many inflammation-related diseases. Here, for the first time, we explored the underlying mechanism of CB2R-regulated inhibition of HMGB1 in macrophages. We also demonstrated that the autophagy-lysosome pathway played an important role in CB2R-induced intracellular degradation of HMGB1. LPS is a powerful stimulator of inflammation. LPS has also been reported to induce autophagy through multiple manners, such as Tollinterleukin 1 receptor domain-containing adaptor-inducing interferonβ (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling in macrophages [8]; LPS-regulated autophagy may present at least 8–16 h after LPS treatment. In our experiment, GW, the agonist of CB2R, inhibited LPS-induced expression of inflammatory factors and boosted autophagy in as little as 3 h after GW treatment, even without the presence of LPS. Specifically, GW induced autophagy faster and to a greater extent than LPS in terms of LC3II formation, which represents mature autophagosomes. Therefore, CB2R can induce autophagy in an LPS-independent manner. Few articles have mentioned the signaling pathway mediating CB2R-regulated autophagy. Vara D reported that another specific CB2R agonist, JWH-015, induced autophagy in the hepatocellular carcinoma cell line HepG2 via tribble homolog 3 (TRB3) upregulation and subsequent inhibition of the serine-threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation [14]. Dando also demonstrated that GW inhibited energetic metabolism and induced AMPK-dependent autophagy in pancreatic cancer cells [15]. In our previous study (data not shown), we did not observe any influence of GW on the phosphorylation of AMPK in macrophages. However, the exact signaling pathway underlying CB2R-induced autophagy requires further investigation. Normally, HMGB1 mainly exists in the nucleus but can also shuttle between the nucleus and cytoplasm to achieve homeostasis. Acetylation, phosphorylation and N-terminal glycosylation of protein molecules regulate homeostasis. Excessive posttranscriptional
3.4. Inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophages To assess the role of autophagy in CB2R-mediated HMGB1 regulation, we studied the nuclear translocation of HMGB1. In the control group, HMGB1 mainly remained in nucleus. After 18 h of treatment with GW, the amount of HMGB1 scattered in the cytoplasm increased. Most HMGB1 redistributed from the nucleus to the cytoplasm after GW treatment for 24 h (Fig. 4A). However, 3-MA suppressed GW-induced HMGB1 translocation at 18 h (Fig. 4B). This result demonstrated that autophagy is also involved in GW-regulated HMGB1 translocation. 3.5. CTSB was involved in the degradation of HMGB1 Last but not least, we wanted to understand how HMGB1 was degraded in the cytoplasm. Because CTSB is an important cysteine endopeptidase in lysosomes and is extensively expressed in macrophages. We explored whether HMGB1 was a substrate of CTSB. We found that the expression of CTSB protein increased at 12 h after GW addition (Fig. 5A), but treatment with a specific inhibitor of CTSB, CA, prevented GW-induced degradation of HMGB1 in macrophages, indicating HMGB1 was likely a substrate of CTSB (Fig. 5B). 4. Discussion As a danger-associated molecular pattern (DAMP), it is generally believed that extracellular HMGB1 binds to many membrane receptors to activate downstream signaling molecules and promote inflammation resulting in cell and tissue damage. To date, most studies have mainly focused on the functions and underlying mechanisms of extracellular HMGB1. This strategy appears to have extensive limitations because targets that could bind to HMGB1 are so diverse that one single inhibitor does not cover all the combinations. In the present study, we found that activation of CB2R in macrophages distinctly decreased the level of intracellular HMGB1 protein, thus preventing the secretion of 4
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Fig. 3. Activation of CB2R promoted the integrity of autophagic flux in macrophages (A) Peritoneal macrophages were treated with GW (10 μM) for indicated time, LC3Ⅰand LC3Ⅱprotein were detected by Western blot. Data are representative of three independent experiments. (B), Quantification of the result of (A), Ratio of LC3Ⅱ/LC3Ⅰwas obtained after been normalized to βactin separately. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05, vs control group. (C) Peritoneal macrophages were incubated with GW (1–10 μM) for 12 h, LC3Ⅰand LC3Ⅱprotein were detected by Western blotting. Data are representative of three independent experiments. (D), Quantification of the **result of (C), Ratio of LC3Ⅱ/LC3Ⅰwas obtained after been normalized to tubulin separately. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05. (E) LC3Ⅰand LC3Ⅱ was detected by confocal technology. LC3Ⅰand LC3Ⅱ was marked in red fluorescence and nucleus was marked with DAPI in blue. Data are representative of three independent experiment. (F) Macrophages were treated with GW (10 μM) for indicated time, p62 protein were detected by Western blotting. Data are representative of three independent experiments. (G), Quantification of the result of (A), p62 expression was normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01, * P < 0.05.
Fig. 4. Inhibition of autophagy blocked GW-induced nuclear translocation of HMGB1 in macrophage. (A) Macrophages were treated with GW (10 μM) for indicated time. Confocal images were taken after cells were stained with the anti-HMGB1 Ab and Alexa Fluor 488-conjugated secondary Ab (green). Data are representative of three independent experiments. (B) Macrophages were treated with GW (10 μM) in the absence or presence of 3-MA (5 μM) for 18 h. Confocal images were taken after cells stained with the anti-HMGB1 Ab and Alexa Fluor 488-conjugated secondary Ab (green). Data are representative of three independent experiments. 5
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Fig. 5. CTSB was involved in the degradation of HMGB1. (A) Macrophages were treated with GW (10 μM) for indicated time. CTSB expression was detected by Western blot. Data are representative of three independent experiments. (B) Quantification of the result of (A), Ratio of CTSB was obtained after been normalized to tubulin. Data are means ± SD (n = 3) and ∗∗ P < 0.01. (C) Macrophages were treated with GW (10 μM) in the absence or presence of CA (10 μM) for 18 h, HMGB1 protein were detected by Western blot. Data are representative of three independent experiments. (D), Quantification of (C), HMGB1 expression was normalized to GAPDH. Data are means ± SD (n = 3) and ∗∗ P < 0.01, vs control group; ## P < 0.01, vs GW group.
modification can cause HMGB1 molecules to detach from DNA and bind to the nuclear transporter CRM1, resulting in unidirectional nuclear translocation [16]. Inhibition of CRM1 prevents its distribution to the nucleus and suppresses HMGB1 translocation to the cytoplasm [17]. In the present study, GW induced HMGB1 nuclear translocation, which was observed 18 h after GW challenge. In addition, GW-induced CRM1 translocation occurred as early as 12 h after GW treatment. However, Western blotting analysis revealed that GW decreased the expression of CRM1 protein (data not shown). It seems that the role of CRM1 in the process of GW-regulated HMGB1 nuclear translocation requires further study. It is well known that autophagy is closely associated with HMGB1. On the one hand, extracellular HMGB1 triggers RAGE to promote autophagy in cancer cells [18]. On the other hand, intracellular HMGB1 translocated to the cytoplasm binds to Beclin1 to initiate autophagosome formation [19]. However, our research showed the possibility that autophagy induced HMGB1 redistribution. Because in chronological order, CB2R agonist-induced autophagy occurred much earlier than the induced HMGB1 intracellular redistribution and degradation (3 h vs 18 h). The inhibition of autophagy by 3-MA suppressed HMGB1 translocation and prevented HMGB1 degradation. This interesting finding provides additional evidence indicating a new form of interaction between autophagy and HMGB1within macrophages. However, there is a need for further investigation. Unlike the classic endoplasmic reticulum-Golgi secretion model, HMGB1 does not enter the endoplasmic reticulum after translocation to the cytoplasm [20]. In monocytes, LPS-induced nuclear translocation of HMGB1 is mainly distributed in secretory lysosomes and rapidly secreted extracellularly by exocytosis under the stimulation of lysophosphatidylcholine (LPC) [21]. These data indicate that HMGB1 has a temporary interaction with lysosomes in the cytoplasm during inflammation. However, in the absence of inflammation, related evidence is limited. Here, our results shed light on the possible pathway of HMGB1 degradation inside the cytoplasm, in which the autophagy-lysosomes pathway may play an important role. Cathepsins are important functional enzymes in lysosomes. CTSB has been reported to regulate the number of lysosomes and autophagosomes in BMDMs. Specifically, CTSB directly cleaves the lysosomal calcium channel MCOLN1/ TRPML1 and negatively regulates the efflux of calcium and activation of the serine/threonine phosphatase PPP3/calcineurin, thus inhibiting transcription of lysosomal and autophagy-related genes. CTSB-deficient cells have increased number and volume of lysosomes and
autophagosomes [11]. These data did not provide direct evidence that CTSB was involved in intracellular HMGB1 regulation but provided us with a clue that CTSB interacts with autophagy and lysosomes. Here, we focused on the enzymatic function of CTSB. GW treatment did not affect the expression of CTSB in macrophages, but an inhibitor of CTSB, CA-074, prevented the GW-induced degradation of HMGB1 in macrophages, indicating that HMGB1 is likely a substrate of CTSB. In the current study, we demonstrated that CB2R promoted HMGB1 degradation through the autophagy-lysosome pathway in macrophages. For the first time, we found that HMGB1 could be degraded intracellularly by CB2R activation, and another step further, we presented data showing that autophagy could induce HMGB1 nuclear translocation and mediate the intracellular degradation of HMGB1. These findings could be helpful to understand the complex interaction between autophagy and HMGB1. Additionally, these data suggest that CB2R is a powerful potential target for treatment of multiple inflammatory diseases. Author contributions Huan Gui and Jan Wang designed the experiments and research project. Huan Gui, Huiting Zhou and Gang Li performed the experiments and analyzed the data. Zhenjiang Bai, Chenmei Mao and Jing Ma participated in the discussion. Huiting Zhou and Rao Du wrote the paper. Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (Grant 81501364, 81501703, 81671967, 81871594, 81571551 and 81801559), the Natural Science Foundation of Jiangsu Province (Grant BK20150294, BK20190053), the Science and Technology Program of the Bureau of Traditional Chinese Medicine of Jiangsu Province (Grant YB2017096), the Science and Technology Program of Suzhou (Grant SYS2018067). 6
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