Generation of Globular Fragment of Adiponectin by Leukocyte Elastase Secreted by Monocytic Cell Line THP-1
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内分泌学杂志 2005年第2期
Department of Metabolic Diseases (H.W., T.Y., J.K., S.K., Y.I., Y.H., S.U., A.T., S.T., T.K.), Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan; and Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST) (H.W., T.Y., T.K.), Kawaguchi 332-0012, Japan
Address all correspondence and requests for reprints to: Takashi Kadowaki, Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655. E-mail: kadowaki-3im@h.u-tokyo.ac.jp.
Abstract
Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin.
Introduction
ADIPONECTIN, ALSO KNOWN as 30-kDa adipocyte complement-related protein (ACRP30) or 28-kDa gelatin-binding protein (GBP28) (1, 2, 3, 4), is a hormone synthesized and secreted by adipocytes. It has been shown to play important roles in the regulation of glucose and lipid homeostasis and to be involved in the pathophysiology of atherosclerosis. Plasma adiponectin concentrations are inversely correlated with body mass index (5) and are lower in patients with type 2 diabetes (6). Reductions of the plasma adiponectin concentration by genetic and nutritional manipulations have been found to cause type 2 diabetes in mice (7, 8, 9), and supplementation reverses insulin resistance in rodent models of type 2 diabetes (9, 10). Adiponectin-deficient mice have been reported to exhibit increased neointima formation in response to mechanical vascular injury (7, 11), and adenovirus- or transgene-mediated elevation of plasma adiponectin suppresses the development of atherosclerosis (12, 13).
Adiponectin consists of a carboxyl-terminal globular domain and an amino-terminal collagen-like domain containing 22 Gly-X-Y repeats (1, 2, 3, 4). The presence of a small amount of a carboxyl-terminal fragment containing the globular domain of adiponectin has been demonstrated in human serum by immunoprecipitation (14). Many studies have shown that full-length adiponectin and the globular domain have distinct biological properties (9, 10, 14, 15). However, it is still unclear how the globular fragment of adiponectin is generated in vivo.
In this study, we show evidence that adiponectin is cleaved into carboxyl-terminal fragments containing the globular domain by leukocyte elastase secreted from the monocytic cell lines THP-1 and U937. These results indicate that adiponectin cleavage by leukocyte elastase could be a candidate for the mechanism of the generation of the globular fragment of adiponectin in plasma.
Materials and Methods
Materials
Phorbol 12-myristate 13-acetate (PMA), DMEM, and RPMI 1640 medium were purchased from Sigma (St. Louis, MO). Protease Inhibitor Set was purchased from Roche Diagnostics (Basel, Switzerland). Human neutrophil elastase and MeOSuc-AAPV-CMK were purchased from Calbiochem (La Jolla, CA). Microcon was from Millipore (Billerica, MA). Trichloroacetate was from Wako Pure Chemicals (Osaka, Japan).
Cell cultures
THP-1 and U937 monocytic cells were maintained at a cell density of 1–8 x 105/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. For differentiation, the cells were plated at a density of 2 x 105/cm2 in RPMI 1640 with 10% fetal bovine serum containing 100 ng/ml PMA for 3 d. Differentiated THP-1 cells adhered to the bottom of the well and extended pseudopods.
Adiponectin cleavage by conditioned medium of THP-1
THP-1 cells were washed with PBS three times and plated in 24-well plates at a density of 2 x 105/cm2 in serum-free RPMI 1640 medium containing 100 ng/ml PMA. After overnight incubation, the medium was collected and centrifuged at 1500 rpm for 10 min at 4 C, and the supernatant was used as the conditioned medium. Tris-HCl (1 M, pH 7.4) was added to the medium to a final concentration of 10 mM, and sodium azide was added to a final concentration of 0.02% (wt/vol). Microcon YM-10 was used to concentrate the conditioned medium into approximately one fifth the volume. Purified murine full-length adiponectin expressed in Escherichia coli was generated as described previously (9, 16), added to the conditioned medium to a concentration of 1.0 μg/ml, and incubated at 37 C for the indicated time. The reaction was stopped by the addition of SDS-PAGE sample buffer. Adiponectin cleavage was monitored by Western blotting as described in SDS-PAGE and Western blotting.
SDS-PAGE and Western blotting
Samples were diluted in Laemmli’s SDS-PAGE sample buffer (final 3% sodium dodecyl sulfate; 50 mM Tris-HCl, pH 6.8; 5% 2-mercaptoethanol; and 10% glycerol) and boiled at 95 C for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline and 0.1% Triton X-100 containing 3% skim milk and then incubated for 1 h at room temperature with 1:1000 diluted antiadiponectin globular domain antiserum (17) in Tris-buffered saline and 0.1% Triton X-100 containing 3% skim milk. In some experiments, anti-carboxyl-terminal and anti-amino-terminal peptide antibodies (17) were used. After washing the membranes, they were incubated with horseradish peroxidase-conjugated antirabbit antibody (1:4000) for 30 min at room temperature and then washed thoroughly. X-ray film (Fujifilm; Minamiashigara, Japan) was exposed to the membranes by using ECL Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ).
Effect of heat denaturation and protease inhibitors on adiponectin-cleaving activity by THP-1 cell-conditioned medium
For heat denaturation, the conditioned medium was boiled at 95 C for 15 min, and after centrifugation, the supernatant was used. Protease inhibitors were added to the earlier described reaction at the following concentration: EDTA, 5 mM; phenylmethylsulfonylfluoride (PMSF) 2+, 1 mM; PMSF+, 0.1 mM; antipain, 50 μg/ml; bestatin, 40 μg/ml; chymostatin, 10 μg/ml; E64, 1 μg/ml; leupeptin, 0.5 μg/ml; pepstatin, 0.7 μg/ml; phosphoramidon, 300 μg/ml; Pefabloc SC (Roche Diagnostics), 100 μg/ml; aprotinin, 1 μg/ml; and MeOSuc-AAPV-CMK, 1 mM.
Adiponectin cleavage by purified leukocyte elastase and amino-terminal sequencing of the cleaved products
Adiponectin (0.4 μg) and leukocyte elastase (0.048 U) were added to 200 μl of reaction buffer (0.2 M triethanolamine-HCl, pH 8.0; 1.0 M NaCl; 0.02% NaN3; and 0.1% PEG6000). After incubation for the time indicated, aliquots were collected, and the reaction solutions were subjected to trichloroacetate precipitation to remove the NaCl in the samples. Precipitated adiponectin was dissolved in the SDS-PAGE sample buffer and analyzed by Western blotting as described earlier. For amino-terminal sequencing, the sample was transferred to a Sequiblot PVDF membrane (Bio-Rad, Hercules, CA) after SDS-PAGE resolution. The band was visualized by Coomassie Brilliant Blue R-250 (Bio-Rad) staining, excised, and subjected to Edman degradation N-terminal analysis.
Results
Cleavage of adiponectin by monocytic cell line THP-1
To investigate the possibility that the globular fragment of adiponectin in plasma is generated by the proteolytic cleavage of adiponectin, we evaluated the activity to cleave adiponectin in culture medium of several cell lines that correspond to target tissues of adiponectin. No apparent adiponectin cleavage was observed in the medium of the Fao hepatocyte, 3T3-L1 adipocyte, and L6 myocyte cell lines (results not shown). By contrast, adiponectin was cleaved after 16-h incubation with conditioned medium of PMA-stimulated THP-1 (Fig. 1A). Concentration of the conditioned medium by ultrafiltration enhanced the cleavage of adiponectin and produced 25-, 20-, and 18-kDa fragments (Fig. 1A), which were detected by antiglobular domain antibody. The size of the 18-kDa fragment was almost the same as that of the globular domain prepared by trypsin treatment of full-length adiponectin (Fig. 1B) (14, 17). Another monocytic cell line, U937, also cleaved adiponectin in the same manner as THP-1 (results not shown). In a time course assay, the 25-kDa fragment initially appeared, and then the generation of 20- and 18-kDa fragments followed (Fig. 1C). The globular domain and the fragments generated by THP-1 were detected also by an antiadiponectin carboxyl-terminal peptide antibody but not by an anti-amino-terminal peptide antibody, suggesting that the fragments generated by THP-1 indeed contained the globular domain (results not shown).
FIG. 1. Adiponectin cleavage by monocytic cell line THP-1. A, The cell-free conditioned medium was collected after incubating THP-1 cells overnight in serum-free RPMI 1640 medium with or without 100 ng/ml PMA. Concentration of the conditioned medium was done by ultrafiltration. Adiponectin was incubated in the conditioned medium at 37 C for the indicated time, and the reaction was stopped by adding the SDS-PAGE sample buffer. Samples were subjected to SDS-PAGE and subsequent immunoblotting using the antiglobular domain antibody. B, Full-length adiponectin (fAd) and the globular domain (gAd) prepared by trypsin digestion (14 17 ) were run and detected by the antiglobular domain antibody. C, Time course of adiponectin cleavage by conditioned medium of THP-1. Adiponectin was incubated with conditioned medium prepared by using both PMA treatment and concentration as described in A. Reaction was stopped by adding SDS-PAGE sample buffer at the indicated time.
Serine protease secreted by THP-1 cleaves adiponectin
The cleavage of adiponectin by the conditioned medium of THP-1 was completely abrogated by heat treatment of the conditioned medium, suggesting that the cleavage was mediated by a protease (Fig. 2A). To identify the type of the protease, we analyzed the effect of various protease inhibitors on the adiponectin cleavage by THP-1-conditioned medium (Fig. 2B). The production of 18- and 20-kDa fragments by 16-h incubation with the conditioned medium was inhibited by the serine protease inhibitors PMSF, Pefabloc SC, and aprotinin, but not by leupeptin, chymostatin, or EDTA (Fig. 2B, lanes 1–20). Moreover, the initial appearance of the 25-kDa fragment after shorter 6-h incubation was clearly inhibited by the same series of serine proteases (Fig. 2B, lanes 21–25). These data suggest that the cleavage of adiponectin into these fragments was mediated by a secreted serine protease.
FIG. 2. Effect of heat treatment and various protease inhibitors on adiponectin cleavage by the conditioned medium of THP-1. A, The effect of heat treatment on adiponectin cleavage by conditioned medium. The conditioned medium of PMA-stimulated THP-1 was concentrated by ultrafiltration. The conditioned medium was heated at 95 C for 15 min before it was added to adiponectin. The detection of adiponectin was the same as in Fig. 1, A and B. Protease inhibitors were added to the concentrated conditioned medium of PMA-stimulated THP-1 at the following concentration: EDTA, 5 mM; PMSF2+, 1 mM; PMSF+, 0.1 mM; antipain, 50 μg/ml; bestatin, 40 μg/ml; chymostatin, 10 μg/ml; E64, 1 μg/ml; leupeptin, 0.5 μg/ml; pepstatin, 0.7 μg/ml; phosphoramidon, 300 μg/ml; Pefabloc SC, 100 μg/ml; and aprotinin, 1 μg/ml. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. Left panel shows the result of 16-h incubation of adiponectin. Right panel shows the result of 6-h incubation using selected protease inhibitors to show the inhibition of the initial appearance of the 25-kDa band. Cond. Med., Conditioned medium; DMSO, dimethylsulfoxide.
THP-1 cells lose adiponectin-cleaving activity as they differentiated into macrophages
THP-1 cells are known to differentiate into macrophage when they are stimulated by PMA. We next examined whether differentiated THP-1 cells after 3-d incubation with PMA still secrete the protease that cleaves adiponectin. Unexpectedly, no adiponectin-cleaving activity was detected in the medium of differentiated THP-1 cells (Fig. 3).
FIG. 3. Differentiation-dependent loss of adiponectin-cleaving activity by THP-1 cells. Left panel, Adiponectin was incubated with the concentrated conditioned medium of PMA-stimulated THP-1 at 37 C for 16 h as in Fig. 1. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. Right panel, THP-1 was cultured in culture medium (RPMI 1640 medium with fetal bovine serum) containing PMA (100 ng/ml) for 3 d to differentiate into macrophage. After differentiation, the cells were washed with PBS three times, and fresh serum-free RPMI medium containing PMA (100 ng/ml) was added; the cells were then incubated overnight at 37 C. The conditioned medium was collected the next day and used for the experiment in the same way as in the experiments using undifferentiated THP-1 cells shown in the left panel and in Figs. 1 and 2.
Leukocyte elastase is responsible for adiponectin cleavage by THP-1
We hypothesized that leukocyte elastase is responsible for the adiponectin cleavage by the conditioned medium in the THP-1 for the following reasons. First, leukocyte elastase is a serine protease that is present in monocytes (18) and the monocytic cell lines THP-1 (19) and U937 (18). Second, leukocyte elastase is released into the medium in response to PMA stimulation within several hours (20). Third, monocytes and U937 cells lose their leukocyte elastase as they differentiate into macrophages (21). Fourth, the series of responses to protease inhibitors matched; leukocyte elastase is effectively inhibited by PMSF and aprotinin but not by chymostatin and EDTA.
To determine whether leukocyte elastase cleaves adiponectin, we allowed adiponectin to react with purified leukocyte elastase. As expected, the leukocyte elastase cleaved adiponectin (Fig. 4). The cleavage pattern produced by purified leukocyte elastase was exactly the same as that produced by THP-1-conditioned medium (compare Fig. 4 with Fig. 3). The leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK completely blocked the cleavage of adiponectin by leukocyte elastase (Fig. 4). We then examined whether MeOSuc-AAPV-CMK inhibits the cleavage of adiponectin by the conditioned medium of THP-1. As shown in Fig. 5, adiponectin cleavage by THP-1-conditioned medium was completely blocked by MeOSuc-AAPV-CMK, suggesting that the adiponectin-cleaving activity of THP-1-conditioned medium is attributable to leukocyte elastase.
FIG. 4. Adiponectin cleavage by leukocyte elastase. Left panel, Leukocyte elastase (0.048 U) was added to 200 μl of reaction buffer containing 400 ng adiponectin and incubated at 37 C for the indicated time. The reaction was stopped by the addition of trichloroacetate. Precipitated adiponectin was directly dissolved in the SDS-PAGE sample buffer. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. The cleavage pattern by leukocyte elastase was identical to that by THP-1-conditioned medium (Fig. 3, left). Right panel, The same reaction was performed in the presence of 1 mM of the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK.
FIG. 5. Effect of the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK on adiponectin cleavage by THP-1-conditioned medium. MeOSuc-AAPV-CMK (final concentration, 1 mM) was added to the THP-1-conditioned medium before the incubation with adiponectin. Incubation and detection of the cleaved fragment was performed in the same way shown in Fig. 1.
Neutrophils are a well-known source of leukocyte elastase (also known as neutrophil elastase). Adiponectin was also cleaved in the same manner by the conditioned medium of neutrophils collected from human blood and activated by PMA or N-formyl-Met-Leu-Phe (results not shown). This cleavage was completely blocked by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK (results not shown). These data suggest that adiponectin was cleaved also by leukocyte elastase secreted from activated neutrophils.
Amino-terminal sequencing of leukocyte elastase-cleaved adiponectin
To identify the sites of cleavage of adiponectin by leukocyte elastase, we performed Edman amino-terminal sequencing of the three major proteolytic bands, with molecular masses of 25, 20, and 18 kDa (Fig. 4). The 25-kDa band was a three-protein mixture that had amino termini at positions 39 (39CAGWM), 41 (41GWMAG), and 45 (45GIPGH) (Table 1, Fig. 6). The sequence of 92EGPRG was detected in the 20-kDa band, and 111AYMYR was detected in the 18-kDa band (Table 1, Fig. 6). These findings suggest that the cleavage occurred in the amino-terminal collagenous domain, and smaller fragments containing the globular domain were generated.
TABLE 1. Amino-terminal sequences of products of adiponectin by leukocyte elastase
FIG. 6. The site of the cleavage of adiponectin by leukocyte elastase. Three major proteolytic fragments (25, 20, and 18 kDa) by leukocyte elastase (Fig. 4) were subjected to the Edman amino-terminal microsequencing. The 25-kDa band was a three-protein mixture that had amino termini at positions 39 (39CAGWM), 41 (41GWMAG), and 45 (45GIPGH). The 20-kDa band had its amino terminus at position 92 (92EGPRG), and the 18-kDa band had its amino terminus at position 111 (111AYMYR) (Table 1).
Discussion
Full-length adiponectin and the globular domain of adiponectin have different biological effects (9, 10, 14, 15), and it has been hypothesized that adiponectin is cleaved and its activity is regulated (14, 22). In this study, we analyzed adiponectin-cleaving activity in the medium of several cell lines in vitro, and found that the monocytic cell lines THP-1 and U937 efficiently cleaved adiponectin into carboxyl-terminal fragments containing the globular domain (Fig. 1). We demonstrated that this activity is attributable to leukocyte elastase secreted from these cells.
Both conditioned medium of THP-1 and purified leukocyte elastase cleave adiponectin into several fragments containing the globular domain (Figs. 3 and 4). Although we cannot completely exclude the possibility that protease(s) other than leukocyte elastase cleaves adiponectin in conditioned medium of THP-1, we think that major fragments are produced by leukocyte elastase. This is because the cleavage patterns by THP-1 and leukocyte elastase are identical including minor bands (compare Figs. 3 and 4), and the generation of all fragments by THP-1 are inhibited by the same series of serine protease inhibitors, including leukocyte elastase-specific MeOSuc-AAPV-CMK. The fact that the 25-kDa band in the experiments in Fig. 2B was relatively resistant to various protease inhibitors raises the possibility that protease responsible for generation of the 25-kDa band and the protease responsible for the 20- and 18-kDa bands may not be the same. We think, however, that this is unlikely because the 25-kDa band seems to be the initial product (Figs. 4 and 1C) and, therefore, tends to remain when the efficiency of protease inhibitor is partial. In fact, protease inhibitors that blocked the production of the 20- and 18-kDa bands also inhibited the initial production of 25 kDa in a shorter incubation (6 h; Fig. 2B). The leukocyte elastase-specific inhibitor MeOSuc-AAPV-CMK showed complete inhibition of all the fragments seen in the THP-1 experiment (Fig. 5). These data support our notion that at least all three major products are generated by leukocyte elastase.
Adiponectin has been found to colocalize with macrophages in atherosclerotic lesion (12) and in injured arteries (23). Leukocyte elastase is also present in atheromatous plaques and is thought to play a role in the adaptive remodeling of vessels and in the pathogenesis of arterial diseases (24). The antiinflammatory effect of adiponectin on monocytes/macrophages is thought to be important in the atheroprotective properties of adiponectin in vivo. Adiponectin is known to inhibit phagocytotic activity and lipopolysaccharide-induced TNF- production in macrophage (25). Adiponectin also reduces cholesterol ester accumulation through suppression of the expression of macrophage scavenger receptor, class A (SR-A) and subsequent reduction of binding and uptake of SR-A ligand in macrophage (26).
The result that leukocyte elastase secreted from THP-1 cleaves adiponectin raises the possibility that adiponectin in atherosclerotic lesion might be cleaved into globular fragment and the activity of adiponectin is modulated through the cleavage. From this point of view, we examined whether the globular domain has different effects on macrophage from those of full-length adiponectin. Our preliminary experiments, however, showed that the globular domain had similar effects on macrophage as compared with full-length adiponectin as far as we examined the suppression of SR-A and lipopolysaccharide-induced TNF- expression in macrophage (our unpublished data).
Pajvani et al. (22) previously reported that adiponectin is cleaved by HEK 293-T cells and primary hepatocytes in vitro. We did not detect adiponectin cleavage by hepatocyte cell line Fao. This difference might be due to the difference between primary cells and cell lines. Although the responsible protease and the physiological relevance of adiponectin cleavage by these cells have not been determined, the cleavage by hepatocytes is noteworthy because the liver is one of the major target organs of adiponectin.
It is an unanswered question whether the globular proteolytic fragment reported in human plasma (8, 9, 14) has the same amino-terminal cleavage site as the fragments produced by leukocyte elastase. Identification of the cleavage site of the globular fragment in human serum is under investigation in our laboratory. To directly test the hypothesis that leukocyte elastase is responsible for the generation of the globular fragment in plasma, it would be worthwhile to examine whether the globular fragment is observed in plasma of leukocyte elastase-deficient mice (27).
Monocytes/macrophages, neutrophils, and leukocyte elastase are activated and exert their biological functions in restricted areas of inflammation. Although the amount of globular fragment of adiponectin in plasma is considerably low, it is reasonable to speculate that cleavage of adiponectin takes place locally and the cleaved product is abundant in such regions. Several lines of evidence that adiponectin accumulates locally in inflammatory lesion have been reported. Adiponectin has been detected in the walls of the catheter-injured arteries (23) and colocalizes with macrophages in injured human aorta (26). Adiponectin also localizes in the interstitial spaces in carbon tetrachloride-injured liver (28) and is distributed in the interstitium in myocardial infarct lesions (29). It would be interesting to determine whether the adiponectin that accumulates is cleaved or intact.
Leukocyte elastase (also know as neutrophil elastase) is abundant in the primary granules of neutrophils and has been shown to be involved in acute inflammatory responses, such as acute host defense against microorganisms (27), arthritis (30), and endotoxin shock (31). We observed that adiponectin is efficiently cleaved also by activated neutrophils (results not shown). Considering that adiponectin has antiinflammatory effects on many type of cells (32, 33, 34), it is also possible that adiponectin has similar biological effects on neutrophils and the effect is modulated by the cleavage of adiponectin by leukocyte elastase.
In conclusion, we have demonstrated that adiponectin is cleaved by monocytic cell lines THP-1 and U937 and that leukocyte elastase is responsible for the cleavage of adiponectin. Although the pathophysiological importance of adiponectin cleavage by leukocyte elastase in vivo remains to be determined, our data indicate that adiponectin can be cleaved by leukocyte elastase secreted from activated monocytes and/or neutrophils and that this cleavage could be a candidate for the mechanism of the generation of the globular fragment of adiponectin in plasma.
Acknowledgments
We thank Drs. Sadaaki Iwanaga, Shohei Mitani, Ken Oofusa, Saku Miyamoto, Junji Matsui, Eiji Majima, and Kenji Yamamoto for their helpful suggestions and K. Kirii, M. Shibata, A. Okano, and A. Itoh for their excellent technical assistance.
References
Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF 1995 A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270:26746–26749
Hu E, Liang P, Spiegelman BM 1996 AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697–10703
Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K 1996 cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). Biochem Biophys Res Commun 221:286–289
Nakano Y, Tobe T, Choi-Miura NH, Mazda T, Tomita M 1996 Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J Biochem (Tokyo) 120:803–812
Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y 1999 Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257:79–83
Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y 2000 Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20:1595–1599
Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M, Matsui J, Eto K, Yamashita T, Kamon J, Satoh H, Yano W, Froguel P, Nagai R, Kimura S, Kadowaki T, Noda T 2002 Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem 277:25863–25866
Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama N, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takeda S, Aoyama T, Funahashi T, Matsuzawa Y 2002 Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 8:731–737
Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T 2001 The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941–946
Berg AH, Combs TP, Du X, Brownlee M, Scherer PE 2001 The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7:947–953
Matsuda M, Shimomura I, Sata M, Arita Y, Nishida M, Maeda N, Kumada M, Okamoto Y, Nagaretani H, Nishizawa H, Kishida K, Komuro R, Ouchi N, Kihara S, Nagai R, Funahashi T, Matsuzawa Y 2002 Role of adiponectin in preventing vascular stenosis. The missing link of adipo-vascular axis. J Biol Chem 277:37487–37491
Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y, Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y 2002 Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation 106:2767–2770
Yamauchi T, Kamon J, Waki H, Imai Y, Shimozawa N, Hioki K, Uchida S, Ito Y, Takakuwa K, Matsui J, Takata M, Eto K, Terauchi Y, Komeda K, Tsunoda M, Murakami K, Ohnishi Y, Naitoh T, Yamamura K, Ueyama Y, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. J Biol Chem 278:2461–2468
Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF 2001 Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010
Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T 2002 Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8:1288–1295
Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T 2003 Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762–769
Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352–40363
Campbell EJ, Cury JD, Shapiro SD, Goldberg GI, Welgus HG 1991 Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. J Immunol 146:1286–1293
Abrink M, Gobl AE, Huang R, Nilsson K, Hellman L 1994 Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage. Leukemia 8:1579–1584
Campbell EJ, Silverman EK, Campbell MA 1989 Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity. J Immunol 143:2961–2968
Welgus HG, Senior RM, Parks WC, Kahn AJ, Ley TJ, Shapiro SD, Campbell EJ 1992 Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation. Matrix Suppl 1:363–367
Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T, Engel J, Brownlee M, Scherer PE 2003 Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 278:9073–9085
Okamoto Y, Arita Y, Nishida M, Muraguchi M, Ouchi N, Takahashi M, Igura T, Inui Y, Kihara S, Nakamura T, Yamashita S, Miyagawa J, Funahashi T, Matsuzawa Y 2000 An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. Horm Metab Res 32:47–50
Dollery CM, Owen CA, Sukhova GK, Krettek A, Shapiro SD, Libby P 2003 Neutrophil elastase in human atherosclerotic plaques: production by macrophages. Circulation 107:2829–2836
Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, Kihara S, Funahashi T, Tenner AJ, Tomiyama Y, Matsuzawa Y 2000 Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 96:1723–1732
Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y, Ishigami M, Kuriyama H, Kishida K, Nishizawa H, Hotta K, Muraguchi M, Ohmoto Y, Yamashita S, Funahashi T, Matsuzawa Y 2001 Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 103:1057–1063
Belaaouaj A, Kim KS, Shapiro SD 2000 Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289:1185–1188
Yoda-Murakami M, Taniguchi M, Takahashi K, Kawamata S, Saito K, Choi-Miura NH, Tomita M 2001 Change in expression of GBP28/adiponectin in carbon tetrachloride-administrated mouse liver. Biochem Biophys Res Commun 285:372–377
Ishikawa Y, Akasaka Y, Ishii T, Yoda-Murakami M, Choi-Miura NH, Tomita M, Ito K, Zhang L, Akishima Y, Ishihara M, Muramatsu M, Taniyama M 2003 Changes in the distribution pattern of gelatin-binding protein of 28 kDa (adiponectin) in myocardial remodelling after ischaemic injury. Histopathology 42:43–52
Adkison AM, Raptis SZ, Kelley DG, Pham CT 2002 Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest 109:363–371
Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J 2000 Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201–210
Yokota T, Meka CS, Medina KL, Igarashi H, Comp PC, Takahashi M, Nishida M, Oritani K, Miyagawa J, Funahashi T, Tomiyama Y, Matsuzawa Y, Kincade PW 2002 Paracrine regulation of fat cell formation in bone marrow cultures via adiponectin and prostaglandins. J Clin Invest 109:1303–1310
Tsao TS, Murrey HE, Hug C, Lee DH, Lodish HF 2002 Oligomerization state-dependent activation of NF- B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem 277:29359–29362
Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 2000 Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-B signaling through a cAMP-dependent pathway. Circulation 102:1296–1301(Hironori Waki, Toshimasa )
Address all correspondence and requests for reprints to: Takashi Kadowaki, Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655. E-mail: kadowaki-3im@h.u-tokyo.ac.jp.
Abstract
Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin.
Introduction
ADIPONECTIN, ALSO KNOWN as 30-kDa adipocyte complement-related protein (ACRP30) or 28-kDa gelatin-binding protein (GBP28) (1, 2, 3, 4), is a hormone synthesized and secreted by adipocytes. It has been shown to play important roles in the regulation of glucose and lipid homeostasis and to be involved in the pathophysiology of atherosclerosis. Plasma adiponectin concentrations are inversely correlated with body mass index (5) and are lower in patients with type 2 diabetes (6). Reductions of the plasma adiponectin concentration by genetic and nutritional manipulations have been found to cause type 2 diabetes in mice (7, 8, 9), and supplementation reverses insulin resistance in rodent models of type 2 diabetes (9, 10). Adiponectin-deficient mice have been reported to exhibit increased neointima formation in response to mechanical vascular injury (7, 11), and adenovirus- or transgene-mediated elevation of plasma adiponectin suppresses the development of atherosclerosis (12, 13).
Adiponectin consists of a carboxyl-terminal globular domain and an amino-terminal collagen-like domain containing 22 Gly-X-Y repeats (1, 2, 3, 4). The presence of a small amount of a carboxyl-terminal fragment containing the globular domain of adiponectin has been demonstrated in human serum by immunoprecipitation (14). Many studies have shown that full-length adiponectin and the globular domain have distinct biological properties (9, 10, 14, 15). However, it is still unclear how the globular fragment of adiponectin is generated in vivo.
In this study, we show evidence that adiponectin is cleaved into carboxyl-terminal fragments containing the globular domain by leukocyte elastase secreted from the monocytic cell lines THP-1 and U937. These results indicate that adiponectin cleavage by leukocyte elastase could be a candidate for the mechanism of the generation of the globular fragment of adiponectin in plasma.
Materials and Methods
Materials
Phorbol 12-myristate 13-acetate (PMA), DMEM, and RPMI 1640 medium were purchased from Sigma (St. Louis, MO). Protease Inhibitor Set was purchased from Roche Diagnostics (Basel, Switzerland). Human neutrophil elastase and MeOSuc-AAPV-CMK were purchased from Calbiochem (La Jolla, CA). Microcon was from Millipore (Billerica, MA). Trichloroacetate was from Wako Pure Chemicals (Osaka, Japan).
Cell cultures
THP-1 and U937 monocytic cells were maintained at a cell density of 1–8 x 105/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. For differentiation, the cells were plated at a density of 2 x 105/cm2 in RPMI 1640 with 10% fetal bovine serum containing 100 ng/ml PMA for 3 d. Differentiated THP-1 cells adhered to the bottom of the well and extended pseudopods.
Adiponectin cleavage by conditioned medium of THP-1
THP-1 cells were washed with PBS three times and plated in 24-well plates at a density of 2 x 105/cm2 in serum-free RPMI 1640 medium containing 100 ng/ml PMA. After overnight incubation, the medium was collected and centrifuged at 1500 rpm for 10 min at 4 C, and the supernatant was used as the conditioned medium. Tris-HCl (1 M, pH 7.4) was added to the medium to a final concentration of 10 mM, and sodium azide was added to a final concentration of 0.02% (wt/vol). Microcon YM-10 was used to concentrate the conditioned medium into approximately one fifth the volume. Purified murine full-length adiponectin expressed in Escherichia coli was generated as described previously (9, 16), added to the conditioned medium to a concentration of 1.0 μg/ml, and incubated at 37 C for the indicated time. The reaction was stopped by the addition of SDS-PAGE sample buffer. Adiponectin cleavage was monitored by Western blotting as described in SDS-PAGE and Western blotting.
SDS-PAGE and Western blotting
Samples were diluted in Laemmli’s SDS-PAGE sample buffer (final 3% sodium dodecyl sulfate; 50 mM Tris-HCl, pH 6.8; 5% 2-mercaptoethanol; and 10% glycerol) and boiled at 95 C for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with Tris-buffered saline and 0.1% Triton X-100 containing 3% skim milk and then incubated for 1 h at room temperature with 1:1000 diluted antiadiponectin globular domain antiserum (17) in Tris-buffered saline and 0.1% Triton X-100 containing 3% skim milk. In some experiments, anti-carboxyl-terminal and anti-amino-terminal peptide antibodies (17) were used. After washing the membranes, they were incubated with horseradish peroxidase-conjugated antirabbit antibody (1:4000) for 30 min at room temperature and then washed thoroughly. X-ray film (Fujifilm; Minamiashigara, Japan) was exposed to the membranes by using ECL Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ).
Effect of heat denaturation and protease inhibitors on adiponectin-cleaving activity by THP-1 cell-conditioned medium
For heat denaturation, the conditioned medium was boiled at 95 C for 15 min, and after centrifugation, the supernatant was used. Protease inhibitors were added to the earlier described reaction at the following concentration: EDTA, 5 mM; phenylmethylsulfonylfluoride (PMSF) 2+, 1 mM; PMSF+, 0.1 mM; antipain, 50 μg/ml; bestatin, 40 μg/ml; chymostatin, 10 μg/ml; E64, 1 μg/ml; leupeptin, 0.5 μg/ml; pepstatin, 0.7 μg/ml; phosphoramidon, 300 μg/ml; Pefabloc SC (Roche Diagnostics), 100 μg/ml; aprotinin, 1 μg/ml; and MeOSuc-AAPV-CMK, 1 mM.
Adiponectin cleavage by purified leukocyte elastase and amino-terminal sequencing of the cleaved products
Adiponectin (0.4 μg) and leukocyte elastase (0.048 U) were added to 200 μl of reaction buffer (0.2 M triethanolamine-HCl, pH 8.0; 1.0 M NaCl; 0.02% NaN3; and 0.1% PEG6000). After incubation for the time indicated, aliquots were collected, and the reaction solutions were subjected to trichloroacetate precipitation to remove the NaCl in the samples. Precipitated adiponectin was dissolved in the SDS-PAGE sample buffer and analyzed by Western blotting as described earlier. For amino-terminal sequencing, the sample was transferred to a Sequiblot PVDF membrane (Bio-Rad, Hercules, CA) after SDS-PAGE resolution. The band was visualized by Coomassie Brilliant Blue R-250 (Bio-Rad) staining, excised, and subjected to Edman degradation N-terminal analysis.
Results
Cleavage of adiponectin by monocytic cell line THP-1
To investigate the possibility that the globular fragment of adiponectin in plasma is generated by the proteolytic cleavage of adiponectin, we evaluated the activity to cleave adiponectin in culture medium of several cell lines that correspond to target tissues of adiponectin. No apparent adiponectin cleavage was observed in the medium of the Fao hepatocyte, 3T3-L1 adipocyte, and L6 myocyte cell lines (results not shown). By contrast, adiponectin was cleaved after 16-h incubation with conditioned medium of PMA-stimulated THP-1 (Fig. 1A). Concentration of the conditioned medium by ultrafiltration enhanced the cleavage of adiponectin and produced 25-, 20-, and 18-kDa fragments (Fig. 1A), which were detected by antiglobular domain antibody. The size of the 18-kDa fragment was almost the same as that of the globular domain prepared by trypsin treatment of full-length adiponectin (Fig. 1B) (14, 17). Another monocytic cell line, U937, also cleaved adiponectin in the same manner as THP-1 (results not shown). In a time course assay, the 25-kDa fragment initially appeared, and then the generation of 20- and 18-kDa fragments followed (Fig. 1C). The globular domain and the fragments generated by THP-1 were detected also by an antiadiponectin carboxyl-terminal peptide antibody but not by an anti-amino-terminal peptide antibody, suggesting that the fragments generated by THP-1 indeed contained the globular domain (results not shown).
FIG. 1. Adiponectin cleavage by monocytic cell line THP-1. A, The cell-free conditioned medium was collected after incubating THP-1 cells overnight in serum-free RPMI 1640 medium with or without 100 ng/ml PMA. Concentration of the conditioned medium was done by ultrafiltration. Adiponectin was incubated in the conditioned medium at 37 C for the indicated time, and the reaction was stopped by adding the SDS-PAGE sample buffer. Samples were subjected to SDS-PAGE and subsequent immunoblotting using the antiglobular domain antibody. B, Full-length adiponectin (fAd) and the globular domain (gAd) prepared by trypsin digestion (14 17 ) were run and detected by the antiglobular domain antibody. C, Time course of adiponectin cleavage by conditioned medium of THP-1. Adiponectin was incubated with conditioned medium prepared by using both PMA treatment and concentration as described in A. Reaction was stopped by adding SDS-PAGE sample buffer at the indicated time.
Serine protease secreted by THP-1 cleaves adiponectin
The cleavage of adiponectin by the conditioned medium of THP-1 was completely abrogated by heat treatment of the conditioned medium, suggesting that the cleavage was mediated by a protease (Fig. 2A). To identify the type of the protease, we analyzed the effect of various protease inhibitors on the adiponectin cleavage by THP-1-conditioned medium (Fig. 2B). The production of 18- and 20-kDa fragments by 16-h incubation with the conditioned medium was inhibited by the serine protease inhibitors PMSF, Pefabloc SC, and aprotinin, but not by leupeptin, chymostatin, or EDTA (Fig. 2B, lanes 1–20). Moreover, the initial appearance of the 25-kDa fragment after shorter 6-h incubation was clearly inhibited by the same series of serine proteases (Fig. 2B, lanes 21–25). These data suggest that the cleavage of adiponectin into these fragments was mediated by a secreted serine protease.
FIG. 2. Effect of heat treatment and various protease inhibitors on adiponectin cleavage by the conditioned medium of THP-1. A, The effect of heat treatment on adiponectin cleavage by conditioned medium. The conditioned medium of PMA-stimulated THP-1 was concentrated by ultrafiltration. The conditioned medium was heated at 95 C for 15 min before it was added to adiponectin. The detection of adiponectin was the same as in Fig. 1, A and B. Protease inhibitors were added to the concentrated conditioned medium of PMA-stimulated THP-1 at the following concentration: EDTA, 5 mM; PMSF2+, 1 mM; PMSF+, 0.1 mM; antipain, 50 μg/ml; bestatin, 40 μg/ml; chymostatin, 10 μg/ml; E64, 1 μg/ml; leupeptin, 0.5 μg/ml; pepstatin, 0.7 μg/ml; phosphoramidon, 300 μg/ml; Pefabloc SC, 100 μg/ml; and aprotinin, 1 μg/ml. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. Left panel shows the result of 16-h incubation of adiponectin. Right panel shows the result of 6-h incubation using selected protease inhibitors to show the inhibition of the initial appearance of the 25-kDa band. Cond. Med., Conditioned medium; DMSO, dimethylsulfoxide.
THP-1 cells lose adiponectin-cleaving activity as they differentiated into macrophages
THP-1 cells are known to differentiate into macrophage when they are stimulated by PMA. We next examined whether differentiated THP-1 cells after 3-d incubation with PMA still secrete the protease that cleaves adiponectin. Unexpectedly, no adiponectin-cleaving activity was detected in the medium of differentiated THP-1 cells (Fig. 3).
FIG. 3. Differentiation-dependent loss of adiponectin-cleaving activity by THP-1 cells. Left panel, Adiponectin was incubated with the concentrated conditioned medium of PMA-stimulated THP-1 at 37 C for 16 h as in Fig. 1. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. Right panel, THP-1 was cultured in culture medium (RPMI 1640 medium with fetal bovine serum) containing PMA (100 ng/ml) for 3 d to differentiate into macrophage. After differentiation, the cells were washed with PBS three times, and fresh serum-free RPMI medium containing PMA (100 ng/ml) was added; the cells were then incubated overnight at 37 C. The conditioned medium was collected the next day and used for the experiment in the same way as in the experiments using undifferentiated THP-1 cells shown in the left panel and in Figs. 1 and 2.
Leukocyte elastase is responsible for adiponectin cleavage by THP-1
We hypothesized that leukocyte elastase is responsible for the adiponectin cleavage by the conditioned medium in the THP-1 for the following reasons. First, leukocyte elastase is a serine protease that is present in monocytes (18) and the monocytic cell lines THP-1 (19) and U937 (18). Second, leukocyte elastase is released into the medium in response to PMA stimulation within several hours (20). Third, monocytes and U937 cells lose their leukocyte elastase as they differentiate into macrophages (21). Fourth, the series of responses to protease inhibitors matched; leukocyte elastase is effectively inhibited by PMSF and aprotinin but not by chymostatin and EDTA.
To determine whether leukocyte elastase cleaves adiponectin, we allowed adiponectin to react with purified leukocyte elastase. As expected, the leukocyte elastase cleaved adiponectin (Fig. 4). The cleavage pattern produced by purified leukocyte elastase was exactly the same as that produced by THP-1-conditioned medium (compare Fig. 4 with Fig. 3). The leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK completely blocked the cleavage of adiponectin by leukocyte elastase (Fig. 4). We then examined whether MeOSuc-AAPV-CMK inhibits the cleavage of adiponectin by the conditioned medium of THP-1. As shown in Fig. 5, adiponectin cleavage by THP-1-conditioned medium was completely blocked by MeOSuc-AAPV-CMK, suggesting that the adiponectin-cleaving activity of THP-1-conditioned medium is attributable to leukocyte elastase.
FIG. 4. Adiponectin cleavage by leukocyte elastase. Left panel, Leukocyte elastase (0.048 U) was added to 200 μl of reaction buffer containing 400 ng adiponectin and incubated at 37 C for the indicated time. The reaction was stopped by the addition of trichloroacetate. Precipitated adiponectin was directly dissolved in the SDS-PAGE sample buffer. Cleavage of adiponectin was monitored by immunoblotting using antiglobular domain antibody. The cleavage pattern by leukocyte elastase was identical to that by THP-1-conditioned medium (Fig. 3, left). Right panel, The same reaction was performed in the presence of 1 mM of the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK.
FIG. 5. Effect of the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK on adiponectin cleavage by THP-1-conditioned medium. MeOSuc-AAPV-CMK (final concentration, 1 mM) was added to the THP-1-conditioned medium before the incubation with adiponectin. Incubation and detection of the cleaved fragment was performed in the same way shown in Fig. 1.
Neutrophils are a well-known source of leukocyte elastase (also known as neutrophil elastase). Adiponectin was also cleaved in the same manner by the conditioned medium of neutrophils collected from human blood and activated by PMA or N-formyl-Met-Leu-Phe (results not shown). This cleavage was completely blocked by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK (results not shown). These data suggest that adiponectin was cleaved also by leukocyte elastase secreted from activated neutrophils.
Amino-terminal sequencing of leukocyte elastase-cleaved adiponectin
To identify the sites of cleavage of adiponectin by leukocyte elastase, we performed Edman amino-terminal sequencing of the three major proteolytic bands, with molecular masses of 25, 20, and 18 kDa (Fig. 4). The 25-kDa band was a three-protein mixture that had amino termini at positions 39 (39CAGWM), 41 (41GWMAG), and 45 (45GIPGH) (Table 1, Fig. 6). The sequence of 92EGPRG was detected in the 20-kDa band, and 111AYMYR was detected in the 18-kDa band (Table 1, Fig. 6). These findings suggest that the cleavage occurred in the amino-terminal collagenous domain, and smaller fragments containing the globular domain were generated.
TABLE 1. Amino-terminal sequences of products of adiponectin by leukocyte elastase
FIG. 6. The site of the cleavage of adiponectin by leukocyte elastase. Three major proteolytic fragments (25, 20, and 18 kDa) by leukocyte elastase (Fig. 4) were subjected to the Edman amino-terminal microsequencing. The 25-kDa band was a three-protein mixture that had amino termini at positions 39 (39CAGWM), 41 (41GWMAG), and 45 (45GIPGH). The 20-kDa band had its amino terminus at position 92 (92EGPRG), and the 18-kDa band had its amino terminus at position 111 (111AYMYR) (Table 1).
Discussion
Full-length adiponectin and the globular domain of adiponectin have different biological effects (9, 10, 14, 15), and it has been hypothesized that adiponectin is cleaved and its activity is regulated (14, 22). In this study, we analyzed adiponectin-cleaving activity in the medium of several cell lines in vitro, and found that the monocytic cell lines THP-1 and U937 efficiently cleaved adiponectin into carboxyl-terminal fragments containing the globular domain (Fig. 1). We demonstrated that this activity is attributable to leukocyte elastase secreted from these cells.
Both conditioned medium of THP-1 and purified leukocyte elastase cleave adiponectin into several fragments containing the globular domain (Figs. 3 and 4). Although we cannot completely exclude the possibility that protease(s) other than leukocyte elastase cleaves adiponectin in conditioned medium of THP-1, we think that major fragments are produced by leukocyte elastase. This is because the cleavage patterns by THP-1 and leukocyte elastase are identical including minor bands (compare Figs. 3 and 4), and the generation of all fragments by THP-1 are inhibited by the same series of serine protease inhibitors, including leukocyte elastase-specific MeOSuc-AAPV-CMK. The fact that the 25-kDa band in the experiments in Fig. 2B was relatively resistant to various protease inhibitors raises the possibility that protease responsible for generation of the 25-kDa band and the protease responsible for the 20- and 18-kDa bands may not be the same. We think, however, that this is unlikely because the 25-kDa band seems to be the initial product (Figs. 4 and 1C) and, therefore, tends to remain when the efficiency of protease inhibitor is partial. In fact, protease inhibitors that blocked the production of the 20- and 18-kDa bands also inhibited the initial production of 25 kDa in a shorter incubation (6 h; Fig. 2B). The leukocyte elastase-specific inhibitor MeOSuc-AAPV-CMK showed complete inhibition of all the fragments seen in the THP-1 experiment (Fig. 5). These data support our notion that at least all three major products are generated by leukocyte elastase.
Adiponectin has been found to colocalize with macrophages in atherosclerotic lesion (12) and in injured arteries (23). Leukocyte elastase is also present in atheromatous plaques and is thought to play a role in the adaptive remodeling of vessels and in the pathogenesis of arterial diseases (24). The antiinflammatory effect of adiponectin on monocytes/macrophages is thought to be important in the atheroprotective properties of adiponectin in vivo. Adiponectin is known to inhibit phagocytotic activity and lipopolysaccharide-induced TNF- production in macrophage (25). Adiponectin also reduces cholesterol ester accumulation through suppression of the expression of macrophage scavenger receptor, class A (SR-A) and subsequent reduction of binding and uptake of SR-A ligand in macrophage (26).
The result that leukocyte elastase secreted from THP-1 cleaves adiponectin raises the possibility that adiponectin in atherosclerotic lesion might be cleaved into globular fragment and the activity of adiponectin is modulated through the cleavage. From this point of view, we examined whether the globular domain has different effects on macrophage from those of full-length adiponectin. Our preliminary experiments, however, showed that the globular domain had similar effects on macrophage as compared with full-length adiponectin as far as we examined the suppression of SR-A and lipopolysaccharide-induced TNF- expression in macrophage (our unpublished data).
Pajvani et al. (22) previously reported that adiponectin is cleaved by HEK 293-T cells and primary hepatocytes in vitro. We did not detect adiponectin cleavage by hepatocyte cell line Fao. This difference might be due to the difference between primary cells and cell lines. Although the responsible protease and the physiological relevance of adiponectin cleavage by these cells have not been determined, the cleavage by hepatocytes is noteworthy because the liver is one of the major target organs of adiponectin.
It is an unanswered question whether the globular proteolytic fragment reported in human plasma (8, 9, 14) has the same amino-terminal cleavage site as the fragments produced by leukocyte elastase. Identification of the cleavage site of the globular fragment in human serum is under investigation in our laboratory. To directly test the hypothesis that leukocyte elastase is responsible for the generation of the globular fragment in plasma, it would be worthwhile to examine whether the globular fragment is observed in plasma of leukocyte elastase-deficient mice (27).
Monocytes/macrophages, neutrophils, and leukocyte elastase are activated and exert their biological functions in restricted areas of inflammation. Although the amount of globular fragment of adiponectin in plasma is considerably low, it is reasonable to speculate that cleavage of adiponectin takes place locally and the cleaved product is abundant in such regions. Several lines of evidence that adiponectin accumulates locally in inflammatory lesion have been reported. Adiponectin has been detected in the walls of the catheter-injured arteries (23) and colocalizes with macrophages in injured human aorta (26). Adiponectin also localizes in the interstitial spaces in carbon tetrachloride-injured liver (28) and is distributed in the interstitium in myocardial infarct lesions (29). It would be interesting to determine whether the adiponectin that accumulates is cleaved or intact.
Leukocyte elastase (also know as neutrophil elastase) is abundant in the primary granules of neutrophils and has been shown to be involved in acute inflammatory responses, such as acute host defense against microorganisms (27), arthritis (30), and endotoxin shock (31). We observed that adiponectin is efficiently cleaved also by activated neutrophils (results not shown). Considering that adiponectin has antiinflammatory effects on many type of cells (32, 33, 34), it is also possible that adiponectin has similar biological effects on neutrophils and the effect is modulated by the cleavage of adiponectin by leukocyte elastase.
In conclusion, we have demonstrated that adiponectin is cleaved by monocytic cell lines THP-1 and U937 and that leukocyte elastase is responsible for the cleavage of adiponectin. Although the pathophysiological importance of adiponectin cleavage by leukocyte elastase in vivo remains to be determined, our data indicate that adiponectin can be cleaved by leukocyte elastase secreted from activated monocytes and/or neutrophils and that this cleavage could be a candidate for the mechanism of the generation of the globular fragment of adiponectin in plasma.
Acknowledgments
We thank Drs. Sadaaki Iwanaga, Shohei Mitani, Ken Oofusa, Saku Miyamoto, Junji Matsui, Eiji Majima, and Kenji Yamamoto for their helpful suggestions and K. Kirii, M. Shibata, A. Okano, and A. Itoh for their excellent technical assistance.
References
Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF 1995 A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270:26746–26749
Hu E, Liang P, Spiegelman BM 1996 AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271:10697–10703
Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K 1996 cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (AdiPose Most abundant Gene transcript 1). Biochem Biophys Res Commun 221:286–289
Nakano Y, Tobe T, Choi-Miura NH, Mazda T, Tomita M 1996 Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J Biochem (Tokyo) 120:803–812
Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y 1999 Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257:79–83
Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y 2000 Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20:1595–1599
Kubota N, Terauchi Y, Yamauchi T, Kubota T, Moroi M, Matsui J, Eto K, Yamashita T, Kamon J, Satoh H, Yano W, Froguel P, Nagai R, Kimura S, Kadowaki T, Noda T 2002 Disruption of adiponectin causes insulin resistance and neointimal formation. J Biol Chem 277:25863–25866
Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama N, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takeda S, Aoyama T, Funahashi T, Matsuzawa Y 2002 Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med 8:731–737
Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K, Mori Y, Ide T, Murakami K, Tsuboyama-Kasaoka N, Ezaki O, Akanuma Y, Gavrilova O, Vinson C, Reitman ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T 2001 The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941–946
Berg AH, Combs TP, Du X, Brownlee M, Scherer PE 2001 The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7:947–953
Matsuda M, Shimomura I, Sata M, Arita Y, Nishida M, Maeda N, Kumada M, Okamoto Y, Nagaretani H, Nishizawa H, Kishida K, Komuro R, Ouchi N, Kihara S, Nagai R, Funahashi T, Matsuzawa Y 2002 Role of adiponectin in preventing vascular stenosis. The missing link of adipo-vascular axis. J Biol Chem 277:37487–37491
Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y, Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y 2002 Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation 106:2767–2770
Yamauchi T, Kamon J, Waki H, Imai Y, Shimozawa N, Hioki K, Uchida S, Ito Y, Takakuwa K, Matsui J, Takata M, Eto K, Terauchi Y, Komeda K, Tsunoda M, Murakami K, Ohnishi Y, Naitoh T, Yamamura K, Ueyama Y, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis. J Biol Chem 278:2461–2468
Fruebis J, Tsao TS, Javorschi S, Ebbets-Reed D, Erickson MR, Yen FT, Bihain BE, Lodish HF 2001 Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010
Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T 2002 Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8:1288–1295
Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T 2003 Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762–769
Waki H, Yamauchi T, Kamon J, Ito Y, Uchida S, Kita S, Hara K, Hada Y, Vasseur F, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin. J Biol Chem 278:40352–40363
Campbell EJ, Cury JD, Shapiro SD, Goldberg GI, Welgus HG 1991 Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases. J Immunol 146:1286–1293
Abrink M, Gobl AE, Huang R, Nilsson K, Hellman L 1994 Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage. Leukemia 8:1579–1584
Campbell EJ, Silverman EK, Campbell MA 1989 Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity. J Immunol 143:2961–2968
Welgus HG, Senior RM, Parks WC, Kahn AJ, Ley TJ, Shapiro SD, Campbell EJ 1992 Neutral proteinase expression by human mononuclear phagocytes: a prominent role of cellular differentiation. Matrix Suppl 1:363–367
Pajvani UB, Du X, Combs TP, Berg AH, Rajala MW, Schulthess T, Engel J, Brownlee M, Scherer PE 2003 Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications for metabolic regulation and bioactivity. J Biol Chem 278:9073–9085
Okamoto Y, Arita Y, Nishida M, Muraguchi M, Ouchi N, Takahashi M, Igura T, Inui Y, Kihara S, Nakamura T, Yamashita S, Miyagawa J, Funahashi T, Matsuzawa Y 2000 An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. Horm Metab Res 32:47–50
Dollery CM, Owen CA, Sukhova GK, Krettek A, Shapiro SD, Libby P 2003 Neutrophil elastase in human atherosclerotic plaques: production by macrophages. Circulation 107:2829–2836
Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N, Kihara S, Funahashi T, Tenner AJ, Tomiyama Y, Matsuzawa Y 2000 Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood 96:1723–1732
Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y, Ishigami M, Kuriyama H, Kishida K, Nishizawa H, Hotta K, Muraguchi M, Ohmoto Y, Yamashita S, Funahashi T, Matsuzawa Y 2001 Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 103:1057–1063
Belaaouaj A, Kim KS, Shapiro SD 2000 Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289:1185–1188
Yoda-Murakami M, Taniguchi M, Takahashi K, Kawamata S, Saito K, Choi-Miura NH, Tomita M 2001 Change in expression of GBP28/adiponectin in carbon tetrachloride-administrated mouse liver. Biochem Biophys Res Commun 285:372–377
Ishikawa Y, Akasaka Y, Ishii T, Yoda-Murakami M, Choi-Miura NH, Tomita M, Ito K, Zhang L, Akishima Y, Ishihara M, Muramatsu M, Taniyama M 2003 Changes in the distribution pattern of gelatin-binding protein of 28 kDa (adiponectin) in myocardial remodelling after ischaemic injury. Histopathology 42:43–52
Adkison AM, Raptis SZ, Kelley DG, Pham CT 2002 Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest 109:363–371
Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J 2000 Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201–210
Yokota T, Meka CS, Medina KL, Igarashi H, Comp PC, Takahashi M, Nishida M, Oritani K, Miyagawa J, Funahashi T, Tomiyama Y, Matsuzawa Y, Kincade PW 2002 Paracrine regulation of fat cell formation in bone marrow cultures via adiponectin and prostaglandins. J Clin Invest 109:1303–1310
Tsao TS, Murrey HE, Hug C, Lee DH, Lodish HF 2002 Oligomerization state-dependent activation of NF- B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30). J Biol Chem 277:29359–29362
Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 2000 Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-B signaling through a cAMP-dependent pathway. Circulation 102:1296–1301(Hironori Waki, Toshimasa )