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Glycated albumin (Amadori product) induces activation of MAP kinases in monocyte-like MonoMac 6 cells
Biochimica et Biophysica Acta 1760 (2006) 1749 – 1753 www.elsevier.com/locate/bbagen
Glycated albumin (Amadori product) induces activation of MAP kinases in monocyte-like MonoMac 6 cells Rowena Brandt ⁎, Sven Krantz Institute of Medical Biochemistry and Molecular Biology, Ernst Moritz Arndt University, Klinikum Sauerbruchstrasse, D-17487 Greifswald, Germany Received 11 July 2006; received in revised form 4 September 2006; accepted 7 September 2006 Available online 15 September 2006
Abstract Increased levels of glycated, Amadori-modified albumin are a risk factor for diabetic vascular disorders. Glycated albumin binds to specific receptors and induces cellular signaling pathways, the complexity of which is largely unknown. Binding of glycated albumin to MonoMac 6 cells leads to an activation of MAPK p44/42 (ERK1/2) and p38 with subsequent translocation of NF-κB into the nucleus. The activation of MAPK is in part mediated by protein kinase C activation, but a PKC-independent pathway via MEK-1 is also involved. Protein tyrosine kinases do not play a role in the activation of NF-κB. The results may have pathophysiological significance, because the MonoMac 6 cell line is not greatly different from blood monocytes. © 2006 Elsevier B.V. All rights reserved. Keywords: Glycated albumin; Fructoselysine; Receptor; Signal transduction; MonoMac 6
1. Introduction Chronic hyperglycemia in diabetes leads to an increased formation of Amadori products through the nonenzymatic glycation reaction between glucose and free amino groups in proteins. Initially, a Schiff base is formed which rearranges into an aminoketose (Amadori product). The main Amadori product in plasma proteins is fructoselysine. These Amadori adducts undergo further irreversible reactions to form advanced glycation end products (AGEs). It has been established that these AGEs play an important role in aging of tissues and in the pathogenesis of late diabetic complications, in chronic renal failure, in disturbances of peritoneal dialysis and in some degenerative brain diseases, for instance Alzheimer's disease [1,2]. In addition, several studies have demonstrated that Abbreviations: AGEs, advanced glycation end products; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; GA, glycated albumin; IL-1β, interleukin 1β; MAPK, mitogen-activated protein kinases; NA, native, nonglycated albumin; NF-κB, nuclear factor-κB; PKC, protein kinase C; PTK, protein tyrosine kinase; ROS, reactive oxygen species; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TNF, tumor necrosis factor ⁎ Corresponding author. Tel.: +49 3834 865414; fax: +49 3834 5402. E-mail address: [email protected] (R. Brandt). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.09.004
Amadori products mainly in the form of glycated albumin are also associated with diabetic macro- and microangiopathies [3–7]. Glycation induces alterations in the properties of proteins. Thus for example, glycated, Amadori-modified albumin exerts its effects by binding to specific receptors on several cell types. Such sites were found on monocytes, peritoneal and alveolar macrophages, the monocytic cell lines MonoMac6 (MM6), U937, THP-1, and HL-60, as well as on endothelial cells and fibroblasts [8–10]. Membrane-bound nucleolin, nucleophosmin and a myosin heavy chain derivative with molecular masses of 110, 150 and 200 kDa respectively have recently been shown to interact with glycated albumin through the fructoselysine moieties. Binding of glycated albumin to MM6 cells induced secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factorα (TNF-α). These cells binding of glycated albumin led to activation of protein kinase C-ε (PKC) and this was linked to translocation of activator protein-1 (AP-1) and nuclear factorκB (NF-κB) into the nucleus. Glycated albumin also stimulated activation of protein tyrosine kinases (PTK) and the PKC inhibitor GÖ̈6976 prevented all of these effects. Genistein, an inhibitor of tyrosine kinases prevented the activation of AP-1, but not the activation of NF-κB, which was only dependent on
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PKC activity [8–14]. Recently, Naitoh et al. [15] also showed the release of TNF-α through glycated albumin in human monocyte-like THP-1 cells. Cohen and Ziyadeh and Chen et al. [16,17] demonstrated binding sites for an Amadori-modified amino acid sequence in glycated albumin on endothelial and mesangial cells. Glycated albumin stimulated PKC-β activation, TGF-β1 production and increased collagen synthesis in mesangial and glomerular endothelial cells, which may be important for the development of diabetic nephropathy. Activation of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) 1/2 by glycated albumin is required for the inhibition of cell growth and enhanced matrix protein synthesis in mesangial cells [18]. Glycated albumin also activated ERK and NF-κB in macrophages and induced oxidative stress, which is partially responsible for NF-κB translocation [19]. The glycated protein is involved in the development of insulin resistance in skeletal muscle cells due to inhibition of the PI3K/PKB insulin signal transduction pathway through activation of PKC-α, while leaving ERK pathway unchanged [20]. The existence of fructoselysine-specific receptors, which bind glycated albumin on monocytes/macrophages supports the hypothesis that glycated albumin may stimulate monocytes and activate various signal transduction pathways and transcription factors. The activation of PKC and PTK in MM6 results in a translocation of AP-1 and NF-κB into the nucleus and an increased synthesis of proinflammatory cytokines [9,13]. It is not known, whether activated PKC directly stimulates NF-κB activation and which role MAPK and oxidative stress may have in eliciting this pathophysiologically important reaction. We demonstrate here that glycated albumin promotes activation of the MAPK ERK1/2 (p44/42) and p38 in the presence of the antioxidant N-acetyl-L-cysteine in the monocyte-like cell line MonoMac 6. 2. Materials and methods 2.1. Albumins Glycated and nonglycated human serum albumins were purchased from Sigma (Steinheim, Germany). Glycated albumin contained 2.7–3.5 mol fructoselysine per mol albumin. In glycated and nonglycated albumin fluorescent AGEs and bacterial lipopolysaccharide were not detectable, carboxymethyllysine was present in only minute amounts. The protein is not borohydride reduced [20].
2.2. Cell culture MM6 cells (DSMZ, Braunschweig, Germany) were seeded into 6-well plates at density of 1 × 106/well and grown overnight in RPMI 1640 (PAA, Pasching, Austria) supplemented with 10% fetal calf sera, 100 μg/ml kanamycin, 1 mM sodium pyruvate, 2 mM glutamine, 1× non-essential amino acids (Biochrom Seromed, Berlin, Germany) and 500 μM N-acetyl-L-cysteine for prevention of oxidative stress. The cells were cultivated for a further 24 h before the indicated amounts of native or glycated albumin were added for 30 min. In selected experiments, cells were preincubated for 30 min with the MEK-1 inhibitor PD 98059, the specific inhibitor of p38 MAPK SB 203580, the specific inhibitor of PKC-ε Ro-32-0432, or genistein, a protein tyrosine kinase (PTK) inhibitor (Calbiochem, Darmstadt, Germany).
2.3. Activation of p38 MAPK and ERK MM6 cells were washed with ice-cold PBS pH 7.4 and solubilized in lysis buffer (25 mM Tris–HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 5 mM EGTA, 1 mM EDTA, 1% (v/v) triton, 92 mg/ml saccharose, 1 mM sodium vanadate, 1 mM benzamidine, 0.1 mM PMSF, 2 μM microcystin, 0.1% (v/v) mercaptoethanol). Cellular lysates (50 μg protein) were fractionated on SDSPAGE (10%) and analyzed by immunoblotting. Immunoblot analysis was performed using a phospho-p38 MAPK antibody, which recognizes phosphorylated threonine 180 and tyrosine 182 residues of p38 MAPK, or using a phospho-p44/42 MAPK antibody, which recognizes endogeneous levels of p42/p44 MAPK (ERK1/2) only when phosphorylated at threonine 202 and tyrosine 204 of human ERK (Cell Signaling, www.cellsignal. com). The primary antibody against total p38 MAPK was from Calbiochem, San Diego, CA, U.S.A. The primary antibody against ERK1/2 was from Sigma, Steinheim, Germany. Blots were washed and then incubated with the corresponding anti-rabbit or anti-mouse–horseradish-peroxidase conjugate (1:3000 dilution) and detected by enhanced chemiluminescence reagent (ECL, Amersham, Buckinghamshire, UK). Appropriate exposures were quantitated by densitometry.
2.4. Electrophoretic mobility shift assay 1 × 107 MM6 cells were incubated in the presence of the indicated amounts of ligands and 500 μM N-acetyl-cysteine for 2 h at 37 °C. Cells were collected and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin and incubated on ice for 10 min. The samples were then centrifuged at 1000×g for 5 min at 4 °C. The nuclear pellet was resuspended in 50 μl HEPES (pH 7.9), 0.42 M NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol and protease inhibitors as above and mixed on ice for 15 min. The nuclear extract was centrifuged at 15,000×g for 10 min at 4 °C. The supernatant (nuclear proteins) was collected and stored at − 70 °C. NF-κB consensus oligonucleotide (5′-AGT TGA GGG TTT CCC AGG C-3′) was 5′-end labeled with 32P γ-ATP (Amersham) using T4 poly-nucleotide kinase (Promega) to specific activity of 5000 to 20,000 cpm/100 fmol. Nonincorporated radioactivity was removed using Microspin G25 columns (Amersham). 6 μg of nuclear protein was incubated with 0.5 ng of labeled oligonucleotide in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, 0.05 mg/ml poly(dI–dC)) for 20 min at room temperature in a final volume of 10 μl. Free oligonucleotide and oligonucleotide protein complexes were separated by electrophoresis on a native 6% polyacrylamide gel. The gel was dried and exposed to a X-ray film with intensifying screen overnight at −70 °C. Specificity of binding was ascertained by competition with a 100-fold excess of unlabeled consensus oligonucleotide.
2.5. Detection of activated transcription factor NF-κB p65 1 × 107 MM6 cells were incubated in the presence of the indicated ligands and 500 μM N-acetyl-L-cysteine for 2 h at 37 °C. For inhibition experiments cells were pretreated for 30 min with either the PTK inhibitor genistein (100 μM), the PKC inhibitor Ro-32-0432 (200 nM), the specific MAPK p38 inhibitor SB 203580 (10 μM) or the specific MEK-1 inhibitor PD 98059 (50 μM), respectively. Cells were collected and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 5 μg/ml antipain, 2 μg/ml leupeptin and incubated on ice for 10 min. The samples were then centrifuged at 1000×g for 10 min at 4 °C. The nuclear pellet was resuspended in 50 μl PBS (pH 7.4), 1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 1 mM dithiothreitol and the protease inhibitors as above and mixed on ice for 15 min. The nuclear extract was centrifuged at 15,000 × g for 10 min. The supernatant (nuclear proteins) was collected and stored at − 70 °C. Protein content was quantified using a BioRad protein assay. NF-κB quantification was performed with an NF-κB p65 ELISA Kit from Stressgen Bioreagents Corporation (Victoria, Canada). Equal amounts of
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nuclear protein (10 μg) were applied to streptavidin-coated plates with bound NF-κB biotinylated consensus sequence to capture only the active form of NF-κB. The captured active form was then incubated with a specific NF-κB p65 antibody, which was detected using an HRP conjugated secondary antibody. The assay was developed with a chemiluminescent substrate and the signal was measured with a luminometer.
2.6. Statistical analysis For statistical comparisons of the values between NA and GA treated cells a t-test was performed; p < 0.05 was considered significant.
3. Results MM6 cells treated with glycated albumin (GA) activated NF-κB as demonstrated with an electrophoretic mobility shift assay while those treated with nonglycated protein (NA) did not (Fig. 1). The GA increased NF-κB DNA binding compared with untreated control or NA. The specificity of the binding was demonstrated by the inhibition of the gel shift by a 100-fold excess of unlabeled oligonucleotide. We next performed experiments to determine whether MAPK were involved in this process. We examined MAPK activation by Western blotting using antibodies specific for the active forms of the kinases. To diminish effects of oxidative stress MM6 cells were preincubated with the antioxidant Nacetyl-L-cysteine. GA treatment activated MAPK family member p38 in MM6 cells. Samples were taken after 30-min exposure to the indicated amounts of NA or GA and subjected to SDS-PAGE and Western blot analysis. Immunoblot analysis using a phospho-p38 MAPK antibody, which recognizes phosphorylated threonine 180 and
Fig. 1. Electrophoretic mobility shift assay for binding of NF-κB to DNA. 400 μg/ml glycated albumin (lane 3) increases NF-κB DNA binding over untreated control or native albumin (400 μg/ml) (lanes 1 and 2). 100-fold excess of unlabeled NF-κB specific oligonucleotide was able to compete for binding (lane 4). The arrow indicates specific binding of NF-κB to the probe.
Fig. 2. Activation of MAPK p38 by glycated albumin in MM6 cells. Upper panel: p38 probed with an antibody that specifically recognizes the phosphorylated activated form. Lower panel: p38 probed with an antibody against total p38, showing equal protein loading of all lanes. NA = nonglycated albumin; GA = glycated albumin; Gen = genistein, PTK inhibitor; SB = p38 inhibitor; RO = PKC inhibitor. Results shown are representative of three independent experiments. Densitometric analysis for kinase activation by GA compared with NA (200 μg/ml for 30 min, set as 1.0) is indicated.
tyrosine 182 residues of p38 MAPK showed enhanced phosphorylation of p38, when cells were incubated with increasing amounts of GA. The difference between stimulations with NA and GA (400 μg/ml each) was statistically significant (p < 0.02). To test the effect of the inhibitors genistein (100 μM), SB203580 (10 μM) or Ro-32-0423 (200 nM), cells were pretreated with either inhibitor for 30 min followed by exposure for another 30 min to GA at a concentration of 400 μg/ml (Fig. 2). The increased phosphorylation of p38 was abolished, when MM6 cells were incubated in the presence of SB 203580, a specific inhibitor of p38 MAPK activation. Similarly, the specific inhibitor of PKC-ε Ro-32-0432 inhibited the phosphorylation of p38, whereas genistein, a PTK inhibitor, had only a slight effect (Fig. 2). Together these results suggest a role for p38 in the signaling cascade which follows GA binding to MM6 cells. To determine whether GA treatment activates MAPK family member ERK1/2 (p44/42) in MM6 cells, samples were taken 30 min after exposure to the indicated amounts of NA or GA and subjected to SDS-PAGE and Western blotting analysis. Immunoblot analysis using a phospho-p44/42 MAPK antibody, which recognizes endogeneous levels of p44/p42 (ERK1/2) only when phosphorylated at threonine 202 and tyrosine 204 showed enhanced phosphorylation of p44/42 when cells were incubated with increasing amounts of glycated albumin. The differences in activation by NA and GA (400 μg/ml each) were statistically significant (p < 0.03). The increased phosphorylation was prevented by the MEK-1 inhibitor PD 98059 (an upstream kinase for p44/42). Similarly, as in activation of p38 the specific inhibitor of PKC-ε Ro-32-0432 prevented the phosphorylation of p44/42, whereas genistein, the protein tyrosine kinase inhibitor showed no effect (Fig. 3). To examine the activation and nuclear translocation of NF-κB in MM6 cells by GA in the presence of N-acetyl-cysteine activated NF-κB in the nuclei was quantitated by ELISA. To test the effect of the inhibitors genistein and Ro-32-0423, cells were pretreated with either inhibitor for 30 min followed by exposure to the indicated amounts of ligands for 2 h. The PKC inhibitor Ro-32-0423 was able to diminish the effect of glycated albumin to activation of NF-κB. To test the effect of MAPK inhibitors
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Fig. 3. Activation of MAPK p44/42 (ERK1/2) by glycated albumin. Results shown are representative of three independent experiments. Densitometric analysis for kinase activation by GA compared with NA (400 μg/ml for 30 min, set as 1.0) is indicated. GA = glycated albumin; NA = native albumin; PD = MEK-1 inhibitor for ERK; RO = specific inhibitor of PKC-ε; Gen = genistein, PTK inhibitor. Upper panel: probed with an antibody that specifically recognizes the phosphorylated form of ERK1/2. Lower panel: the membrane was probed with an antibody against total ERK1/2 showing equal protein loading of all lanes.
SB203580 or PD98059 cells were pretreated with either inhibitor for 30 min followed by GA exposure for 2 h. Both inhibitors were able to reduce activation of p65 subunit of NF-κB and hence its translocation to the nucleus (Fig. 4). 4. Discussion Glucose-dependent Amadori product formation in proteins is thought to provoke vascular complications in diabetes, but it is not known, in which way glycated albumin influences injurious pathways in cells. Although the importance of AGEs is established, increasing evidence shows that Amadori-modified proteins have biological effects which are very similar to those of AGEs. Their biological reactivity results mainly from binding to specific cellular receptors whose structures and functions remain to be clarified. These receptors are clearly different from all described AGE-binding proteins. Calnexin, a binding protein for an Amadori-modified sequence in glycated albumin on mesangial cells [21] as well as nucleolin, nucleophosmin and cellular myosin heavy chain as specific binding proteins for fructoselysine on cell membranes of human monocytes, U937, MM6, THP-1 and HL-60 cells as monocytelike cells have been reported [10,12,14]. The cell signaling pathways by which glycated albumin induces alterations in the vasculature are not yet fully elucidated. It is known that the expression of the fructoselysine-specific receptor on monocytes varied between individuals and is different in diabetics and nondiabetics. Its expression is correlated with an early appearance and the severity of microangiopathy in diabetic humans and rats [11]. The results described here identify a signal transduction process in MM6 cells in which after binding of Amadorimodified albumin phosphorylation and activation of p38 and of p44/42 MAPK takes place together with NF-κB activation. It is accepted that MAPK activation plays a role in cell proliferation and differentiation, which is associated with activation of vascular cells.
Amadori-modified albumin may also stimulate MAPK p44/ 42 and p38 phosphorylation by activation of PKC-ε. In support of this observation, we previously demonstrated activation of this PKC isoform [14] which we now show can be specifically inactivated by Ro32-0432. This finding suggests that Amadorimodified albumins stimulate MAPK activation via a PKC. In addition there is also evidence from the experiments with the MEK-1 inhibitor PD-98059, which blocked p44/42 activation that other signaling pathways are implicated in the activation of MAPK. We conclude that an upstream MAPK activator of the MEK family may be involved. PTK activation is not required in the MAPK activation, since the activation is unaffected by the PTK inhibitor genistein. Since all experiments were performed in the presence of an adequate concentration of the antioxidant N-acetyl-L-cysteine, we consider it unlikely that activation of NADPH oxidase and the formation of reactive oxygen species play a role for the observed effects which has been described for endothelial cells after binding of glycated albumin [22]. The cellular mechanisms, by which glycated albumin stimulates signal transduction are cell-type-dependent. In vascular smooth muscle cells GA induces the expression of monocyte chemoattractant proteins, activation of p44/42 and of NF-κB and AP-1 [7,23,24]. GA also stimulates tissue plasminogen activator inhibitor synthesis in mesothelial cells by activation of NF-κB and AP-1 [3] and in macrophage RAW cells after NADPH oxidase stimulation a p44/42dependent activation of NF-κB [19]. GA activates NADPH oxidase, protein kinase B and JNK resulting in increased Eselectin expression, which in endothelial cells is upregulated by NF-κB and AP-1 [22]. GA stimulates cardiomyocyte ROS production through a protein kinase C-dependent activation of NADPH oxidase, which results in NF-κB activation and
Fig. 4. Activation of NF-κB by glycated albumin determined by ELISA. NA = native albumin; GA = glycated albumin; Gen = genistein, PTK inhibitor; RO = PKC inhibitor; PD = MEK-1 inhibitor; SB = MAPK inhibitor. MAPK and PKC inhibitors prevented GA stimulated activation of NF-κB. Genistein was ineffective. The results are means of three independent experiments. *p < 0.005 compared with the level of activation by NA, **p < 0.005 compared with the level of activation by GA.
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upregulation of atrial natriuretic factor mRNA. These findings suggest that early glycation may play a role in the pathogenesis of diabetic heart disease [25]. Reduction of Amadori-glycated albumin levels in diabetic animals ameliorates the progression of nephropathy and retinopathy, indicating a causal role of glycated albumin in the pathogenesis of diabetic microangiopathies [5]. Inhibition of GA may be a target for prevention of its cellular interactions and of diabetic complications [5,17]. These and our results show that GA induces different signaling pathways in various cells, which activate the transcription factors NF-κB and AP-1. Further studies are necessary to characterize the receptors for Amadori-modified proteins and the signaling pathways. These experiments have significance for the pathophysiology of diabetic sequelae, because the MM6 cell line is very similar to human monocytes. MM6 constitutively expresses phenotypic and functional characteristics of mature monocytes [26], which also bind GA [11]. Fructoselysine adducts are the principal forms, in which glycated albumin exists in vivo and may be involved in the pathogenesis of diabetic vascular disorders, when formed in excess during chronic hyperglycemia. Acknowledgements We thank Professor Dr. R. Jack for valuable comments and critical reading of the manuscript. This study was financially supported by the Medical Faculty of the Ernst Moritz Arndt University Greifswald.
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
References [20] [1] R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetologia 44 (2001) 129–146. [2] G. Suji, S. Sivakami, Glucose, glycation and aging, Biogerontology 5 (2004) 365–373. [3] Y. Hattori, M. Suzuki, S. Hattori, K. Kasai, Vascular smooth muscle cell activation by glycated albumin (Amadori adducts), Hypertension 39 (2002) 22–28. [4] D. Ruggiero-Lopez, N. Rellier, M. Lecomte, M. Lagarde, N. Wiensperger, Growth modulation of retinal microvascular cells by early and advanced glycation products, Diabetes Res. Clin. Pract. 34 (1997) 135–142. [5] C.G. Schalkwijk, M. Lieuw-a-Fa, V.W.M. van Hinsbergh, C.D.A. Stehouwer, Pathophysiological role of Amadori-glycated proteins in diabetic microangiopathy, Semin. Vasc. Med. 2 (2002) 191–197. [6] M.P. Cohen, F.N. Ziyadeh, Role of Amadori-modified nonenzymatically glycated serum proteins in the pathogenesis of diabetic nephropathy, J. Am. Soc. Nephrol. 7 (1996) 183–190. [7] S. Mandl-Weber, B. Haslinger, C.G. Schalkwijk, T. Sitter, Early glycated albumin, but not advanced glycated albumin, methylglyoxal, or 3-deoxyglucosone increases the expression of PAI-1 in human peritoneal mesothelial cells, Perit. Dial. Int. 21 (2001) 487–494. [8] S. Krantz, R. Brandt, B. Gromoll, Binding sites for short-term glycated albumin on peritoneal cells of the rat, Biochim. Biophys. Acta 1177 (1993) 15–24. [9] R. Salazar, R. Brandt, S. Krantz, Expression of fructosyllysine receptors on
[21]
[22]
[23]
[24]
[25]
[26]
1753
human monocytes and monocyte-like cell lines, Biochim. Biophys. Acta 1266 (1995) 57–63. S. Krantz, R. Salazar, R. Brandt, J. Kellermann, F. Lottspeich, Purification and partial amino acid sequencing of a fructoselysine-specific binding protein from cell membranes of the monocyte-like cell line U937, Biochim. Biophys. Acta 1266 (1995) 109–112. R. Brandt, C. Landmesser, L. Vogt, B. Hehmke, R. Hanschke, J. Kasbohm, K. Hartmann, B. Jäger, S. Krantz, D. Michaelis, Differential expression of fructosyllysine-specific receptors on monocytes and macrophages and possible pathophysiological significance, Diabetologia 39 (1996) 1140–1147. R. Salazar, R. Brandt, J. Kellermann, S. Krantz, Purification and characterization of a 200 kDa fructosyllysine-specific binding protein from cell membranes of U937 cells, Glycoconj. J. 17 (2000) 713–716. R. Salazar, R. Brandt, S. Krantz, Binding of Amadori glucose-modified albumin by the monocytic cell line MonoMac 6 activates protein kinase Cε, protein tyrosine kinases and the transcription factors AP-1 and NF-κB, Glycoconj. J. 18 (2001) 769–777. R. Brandt, M. Nawka, J. Kellermann, R. Salazar, D. Becher, S. Krantz, Nucleophosmin is a component of the fructoselysine-specific receptor in cell membranes of Mono Mac 6 and U937 monocyte-like cells, Biochim. Biophys. Acta 1670 (2004) 132–136. T. Naitoh, M. Kitahara, N. Tsuruzoe, Tumor necrosis factor-α is induced through phorbol ester- and glycated albumin-dependent pathway in THP-1 cells, Cell Signalling 13 (2001) 331–334. M.P. Cohen, F.N. Ziyadeh, Amadori glucose adducts modulate mesangial cell growth and collagen gene expression, Kidney Int. 45 (1994) 475–484. S. Chen, M.P. Cohen, F.N. Ziyadeh, Amadori-glycated albumin in diabetic nephropathy: pathophysiologic connections, Kidney Int. 58 (2000) S40–S44. M.P. Cohen, E. Shea, C.W. Shearman, ERK mediates effects of glycated albumin in mesangial cells, Biochem. Biophys. Res. Commun. 283 (2001) 641–643. M.P. Cohen, E. Shea, S. Chen, C.W. Shearman, Glycated albumin increases oxidative stress, activates NF-κB and extracellular signalregulated kinase (ERK), and stimulates ERK-dependent transforming growth factor-β1 production in macrophage RAW cells, J. Lab. Clin. Med. 141 (2003) 242–249. C. Miele, A. Riboulet, M.A. Maitan, F. Oriente, C. Romano, P. Formisano, J. Giudicelli, F. Beguinot, E. van Obberghen, Human glycated albumin effects glucose metabolism in L6 skeletal muscle cells by impairing insulininduced insulin receptor substrate (IRS) signaling through a protein kinase C α-mediated mechanism, J. Biol. Chem. 278 (2003) 47376–47387. V.Y. Wu, C.W. Sherman, M.P. Cohen, Identification of calnexin as a binding protein for Amadori-modified glycated albumin, Biochem. Biophys. Res. Commun. 284 (2001) 602–606. K. Higai, A. Shimamura, K. Matsumoto, Amadori-modified glycated albumin predominantly induces E-selectin expression on human umbilical vein endothelial cells through NADP oxidase activation, Clin. Chim. Acta 367 (2006) 137–143. T. Ichiki, Y. Funakoshi, K. Ito, A. Takeshita, Expression of monocyte chemoattractant protein-1 by nonenzymatically glycated albumin (Amadori adducts) in vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 269 (2000) 666–670. Y. Hattori, M. Suzuki, S. Hattori, K. Kasai, Vascular smooth muscle cell activation by glycated albumin (Amadori adducts), Hypertension 39 (2002) 22–28. M. Zhang, A.L. Kho, N. Anilkumar, R. Chibber, P.J. Pagano, A.M. Shah, A.C. Cave, Glycated proteins stimulate reactive oxygen species production in cardiac myocytes, Circulation 113 (2006) 1235–1243. H.W.L. Ziegler-Heitbrock, E. Thiel, A. Fütterer, V. Herzog, A. Wirtz, G. Riethmüller, Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes, Int. J. Cancer 41 (1988) 456–461.
Glycated albumin (Amadori product) induces activation of MAP kinases in monocyte-like MonoMac 6 cells Rowena Brandt ⁎, Sven Krantz Institute of Medical Biochemistry and Molecular Biology, Ernst Moritz Arndt University, Klinikum Sauerbruchstrasse, D-17487 Greifswald, Germany Received 11 July 2006; received in revised form 4 September 2006; accepted 7 September 2006 Available online 15 September 2006
Abstract Increased levels of glycated, Amadori-modified albumin are a risk factor for diabetic vascular disorders. Glycated albumin binds to specific receptors and induces cellular signaling pathways, the complexity of which is largely unknown. Binding of glycated albumin to MonoMac 6 cells leads to an activation of MAPK p44/42 (ERK1/2) and p38 with subsequent translocation of NF-κB into the nucleus. The activation of MAPK is in part mediated by protein kinase C activation, but a PKC-independent pathway via MEK-1 is also involved. Protein tyrosine kinases do not play a role in the activation of NF-κB. The results may have pathophysiological significance, because the MonoMac 6 cell line is not greatly different from blood monocytes. © 2006 Elsevier B.V. All rights reserved. Keywords: Glycated albumin; Fructoselysine; Receptor; Signal transduction; MonoMac 6
1. Introduction Chronic hyperglycemia in diabetes leads to an increased formation of Amadori products through the nonenzymatic glycation reaction between glucose and free amino groups in proteins. Initially, a Schiff base is formed which rearranges into an aminoketose (Amadori product). The main Amadori product in plasma proteins is fructoselysine. These Amadori adducts undergo further irreversible reactions to form advanced glycation end products (AGEs). It has been established that these AGEs play an important role in aging of tissues and in the pathogenesis of late diabetic complications, in chronic renal failure, in disturbances of peritoneal dialysis and in some degenerative brain diseases, for instance Alzheimer's disease [1,2]. In addition, several studies have demonstrated that Abbreviations: AGEs, advanced glycation end products; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; GA, glycated albumin; IL-1β, interleukin 1β; MAPK, mitogen-activated protein kinases; NA, native, nonglycated albumin; NF-κB, nuclear factor-κB; PKC, protein kinase C; PTK, protein tyrosine kinase; ROS, reactive oxygen species; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TNF, tumor necrosis factor ⁎ Corresponding author. Tel.: +49 3834 865414; fax: +49 3834 5402. E-mail address: [email protected] (R. Brandt). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.09.004
Amadori products mainly in the form of glycated albumin are also associated with diabetic macro- and microangiopathies [3–7]. Glycation induces alterations in the properties of proteins. Thus for example, glycated, Amadori-modified albumin exerts its effects by binding to specific receptors on several cell types. Such sites were found on monocytes, peritoneal and alveolar macrophages, the monocytic cell lines MonoMac6 (MM6), U937, THP-1, and HL-60, as well as on endothelial cells and fibroblasts [8–10]. Membrane-bound nucleolin, nucleophosmin and a myosin heavy chain derivative with molecular masses of 110, 150 and 200 kDa respectively have recently been shown to interact with glycated albumin through the fructoselysine moieties. Binding of glycated albumin to MM6 cells induced secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factorα (TNF-α). These cells binding of glycated albumin led to activation of protein kinase C-ε (PKC) and this was linked to translocation of activator protein-1 (AP-1) and nuclear factorκB (NF-κB) into the nucleus. Glycated albumin also stimulated activation of protein tyrosine kinases (PTK) and the PKC inhibitor GÖ̈6976 prevented all of these effects. Genistein, an inhibitor of tyrosine kinases prevented the activation of AP-1, but not the activation of NF-κB, which was only dependent on
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PKC activity [8–14]. Recently, Naitoh et al. [15] also showed the release of TNF-α through glycated albumin in human monocyte-like THP-1 cells. Cohen and Ziyadeh and Chen et al. [16,17] demonstrated binding sites for an Amadori-modified amino acid sequence in glycated albumin on endothelial and mesangial cells. Glycated albumin stimulated PKC-β activation, TGF-β1 production and increased collagen synthesis in mesangial and glomerular endothelial cells, which may be important for the development of diabetic nephropathy. Activation of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) 1/2 by glycated albumin is required for the inhibition of cell growth and enhanced matrix protein synthesis in mesangial cells [18]. Glycated albumin also activated ERK and NF-κB in macrophages and induced oxidative stress, which is partially responsible for NF-κB translocation [19]. The glycated protein is involved in the development of insulin resistance in skeletal muscle cells due to inhibition of the PI3K/PKB insulin signal transduction pathway through activation of PKC-α, while leaving ERK pathway unchanged [20]. The existence of fructoselysine-specific receptors, which bind glycated albumin on monocytes/macrophages supports the hypothesis that glycated albumin may stimulate monocytes and activate various signal transduction pathways and transcription factors. The activation of PKC and PTK in MM6 results in a translocation of AP-1 and NF-κB into the nucleus and an increased synthesis of proinflammatory cytokines [9,13]. It is not known, whether activated PKC directly stimulates NF-κB activation and which role MAPK and oxidative stress may have in eliciting this pathophysiologically important reaction. We demonstrate here that glycated albumin promotes activation of the MAPK ERK1/2 (p44/42) and p38 in the presence of the antioxidant N-acetyl-L-cysteine in the monocyte-like cell line MonoMac 6. 2. Materials and methods 2.1. Albumins Glycated and nonglycated human serum albumins were purchased from Sigma (Steinheim, Germany). Glycated albumin contained 2.7–3.5 mol fructoselysine per mol albumin. In glycated and nonglycated albumin fluorescent AGEs and bacterial lipopolysaccharide were not detectable, carboxymethyllysine was present in only minute amounts. The protein is not borohydride reduced [20].
2.2. Cell culture MM6 cells (DSMZ, Braunschweig, Germany) were seeded into 6-well plates at density of 1 × 106/well and grown overnight in RPMI 1640 (PAA, Pasching, Austria) supplemented with 10% fetal calf sera, 100 μg/ml kanamycin, 1 mM sodium pyruvate, 2 mM glutamine, 1× non-essential amino acids (Biochrom Seromed, Berlin, Germany) and 500 μM N-acetyl-L-cysteine for prevention of oxidative stress. The cells were cultivated for a further 24 h before the indicated amounts of native or glycated albumin were added for 30 min. In selected experiments, cells were preincubated for 30 min with the MEK-1 inhibitor PD 98059, the specific inhibitor of p38 MAPK SB 203580, the specific inhibitor of PKC-ε Ro-32-0432, or genistein, a protein tyrosine kinase (PTK) inhibitor (Calbiochem, Darmstadt, Germany).
2.3. Activation of p38 MAPK and ERK MM6 cells were washed with ice-cold PBS pH 7.4 and solubilized in lysis buffer (25 mM Tris–HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 5 mM EGTA, 1 mM EDTA, 1% (v/v) triton, 92 mg/ml saccharose, 1 mM sodium vanadate, 1 mM benzamidine, 0.1 mM PMSF, 2 μM microcystin, 0.1% (v/v) mercaptoethanol). Cellular lysates (50 μg protein) were fractionated on SDSPAGE (10%) and analyzed by immunoblotting. Immunoblot analysis was performed using a phospho-p38 MAPK antibody, which recognizes phosphorylated threonine 180 and tyrosine 182 residues of p38 MAPK, or using a phospho-p44/42 MAPK antibody, which recognizes endogeneous levels of p42/p44 MAPK (ERK1/2) only when phosphorylated at threonine 202 and tyrosine 204 of human ERK (Cell Signaling, www.cellsignal. com). The primary antibody against total p38 MAPK was from Calbiochem, San Diego, CA, U.S.A. The primary antibody against ERK1/2 was from Sigma, Steinheim, Germany. Blots were washed and then incubated with the corresponding anti-rabbit or anti-mouse–horseradish-peroxidase conjugate (1:3000 dilution) and detected by enhanced chemiluminescence reagent (ECL, Amersham, Buckinghamshire, UK). Appropriate exposures were quantitated by densitometry.
2.4. Electrophoretic mobility shift assay 1 × 107 MM6 cells were incubated in the presence of the indicated amounts of ligands and 500 μM N-acetyl-cysteine for 2 h at 37 °C. Cells were collected and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 2 μg/ml leupeptin and incubated on ice for 10 min. The samples were then centrifuged at 1000×g for 5 min at 4 °C. The nuclear pellet was resuspended in 50 μl HEPES (pH 7.9), 0.42 M NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol and protease inhibitors as above and mixed on ice for 15 min. The nuclear extract was centrifuged at 15,000×g for 10 min at 4 °C. The supernatant (nuclear proteins) was collected and stored at − 70 °C. NF-κB consensus oligonucleotide (5′-AGT TGA GGG TTT CCC AGG C-3′) was 5′-end labeled with 32P γ-ATP (Amersham) using T4 poly-nucleotide kinase (Promega) to specific activity of 5000 to 20,000 cpm/100 fmol. Nonincorporated radioactivity was removed using Microspin G25 columns (Amersham). 6 μg of nuclear protein was incubated with 0.5 ng of labeled oligonucleotide in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, 0.05 mg/ml poly(dI–dC)) for 20 min at room temperature in a final volume of 10 μl. Free oligonucleotide and oligonucleotide protein complexes were separated by electrophoresis on a native 6% polyacrylamide gel. The gel was dried and exposed to a X-ray film with intensifying screen overnight at −70 °C. Specificity of binding was ascertained by competition with a 100-fold excess of unlabeled consensus oligonucleotide.
2.5. Detection of activated transcription factor NF-κB p65 1 × 107 MM6 cells were incubated in the presence of the indicated ligands and 500 μM N-acetyl-L-cysteine for 2 h at 37 °C. For inhibition experiments cells were pretreated for 30 min with either the PTK inhibitor genistein (100 μM), the PKC inhibitor Ro-32-0432 (200 nM), the specific MAPK p38 inhibitor SB 203580 (10 μM) or the specific MEK-1 inhibitor PD 98059 (50 μM), respectively. Cells were collected and washed twice with ice-cold PBS. The cell pellet was resuspended in 1 ml 10 mM HEPES (pH 7.6), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% NP-40, 1 mM PMSF, 10 μg/ml aprotinin, 5 μg/ml antipain, 2 μg/ml leupeptin and incubated on ice for 10 min. The samples were then centrifuged at 1000×g for 10 min at 4 °C. The nuclear pellet was resuspended in 50 μl PBS (pH 7.4), 1% NP-40, 0.5% sodiumdesoxycholate, 0.1% SDS, 1 mM dithiothreitol and the protease inhibitors as above and mixed on ice for 15 min. The nuclear extract was centrifuged at 15,000 × g for 10 min. The supernatant (nuclear proteins) was collected and stored at − 70 °C. Protein content was quantified using a BioRad protein assay. NF-κB quantification was performed with an NF-κB p65 ELISA Kit from Stressgen Bioreagents Corporation (Victoria, Canada). Equal amounts of
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nuclear protein (10 μg) were applied to streptavidin-coated plates with bound NF-κB biotinylated consensus sequence to capture only the active form of NF-κB. The captured active form was then incubated with a specific NF-κB p65 antibody, which was detected using an HRP conjugated secondary antibody. The assay was developed with a chemiluminescent substrate and the signal was measured with a luminometer.
2.6. Statistical analysis For statistical comparisons of the values between NA and GA treated cells a t-test was performed; p < 0.05 was considered significant.
3. Results MM6 cells treated with glycated albumin (GA) activated NF-κB as demonstrated with an electrophoretic mobility shift assay while those treated with nonglycated protein (NA) did not (Fig. 1). The GA increased NF-κB DNA binding compared with untreated control or NA. The specificity of the binding was demonstrated by the inhibition of the gel shift by a 100-fold excess of unlabeled oligonucleotide. We next performed experiments to determine whether MAPK were involved in this process. We examined MAPK activation by Western blotting using antibodies specific for the active forms of the kinases. To diminish effects of oxidative stress MM6 cells were preincubated with the antioxidant Nacetyl-L-cysteine. GA treatment activated MAPK family member p38 in MM6 cells. Samples were taken after 30-min exposure to the indicated amounts of NA or GA and subjected to SDS-PAGE and Western blot analysis. Immunoblot analysis using a phospho-p38 MAPK antibody, which recognizes phosphorylated threonine 180 and
Fig. 1. Electrophoretic mobility shift assay for binding of NF-κB to DNA. 400 μg/ml glycated albumin (lane 3) increases NF-κB DNA binding over untreated control or native albumin (400 μg/ml) (lanes 1 and 2). 100-fold excess of unlabeled NF-κB specific oligonucleotide was able to compete for binding (lane 4). The arrow indicates specific binding of NF-κB to the probe.
Fig. 2. Activation of MAPK p38 by glycated albumin in MM6 cells. Upper panel: p38 probed with an antibody that specifically recognizes the phosphorylated activated form. Lower panel: p38 probed with an antibody against total p38, showing equal protein loading of all lanes. NA = nonglycated albumin; GA = glycated albumin; Gen = genistein, PTK inhibitor; SB = p38 inhibitor; RO = PKC inhibitor. Results shown are representative of three independent experiments. Densitometric analysis for kinase activation by GA compared with NA (200 μg/ml for 30 min, set as 1.0) is indicated.
tyrosine 182 residues of p38 MAPK showed enhanced phosphorylation of p38, when cells were incubated with increasing amounts of GA. The difference between stimulations with NA and GA (400 μg/ml each) was statistically significant (p < 0.02). To test the effect of the inhibitors genistein (100 μM), SB203580 (10 μM) or Ro-32-0423 (200 nM), cells were pretreated with either inhibitor for 30 min followed by exposure for another 30 min to GA at a concentration of 400 μg/ml (Fig. 2). The increased phosphorylation of p38 was abolished, when MM6 cells were incubated in the presence of SB 203580, a specific inhibitor of p38 MAPK activation. Similarly, the specific inhibitor of PKC-ε Ro-32-0432 inhibited the phosphorylation of p38, whereas genistein, a PTK inhibitor, had only a slight effect (Fig. 2). Together these results suggest a role for p38 in the signaling cascade which follows GA binding to MM6 cells. To determine whether GA treatment activates MAPK family member ERK1/2 (p44/42) in MM6 cells, samples were taken 30 min after exposure to the indicated amounts of NA or GA and subjected to SDS-PAGE and Western blotting analysis. Immunoblot analysis using a phospho-p44/42 MAPK antibody, which recognizes endogeneous levels of p44/p42 (ERK1/2) only when phosphorylated at threonine 202 and tyrosine 204 showed enhanced phosphorylation of p44/42 when cells were incubated with increasing amounts of glycated albumin. The differences in activation by NA and GA (400 μg/ml each) were statistically significant (p < 0.03). The increased phosphorylation was prevented by the MEK-1 inhibitor PD 98059 (an upstream kinase for p44/42). Similarly, as in activation of p38 the specific inhibitor of PKC-ε Ro-32-0432 prevented the phosphorylation of p44/42, whereas genistein, the protein tyrosine kinase inhibitor showed no effect (Fig. 3). To examine the activation and nuclear translocation of NF-κB in MM6 cells by GA in the presence of N-acetyl-cysteine activated NF-κB in the nuclei was quantitated by ELISA. To test the effect of the inhibitors genistein and Ro-32-0423, cells were pretreated with either inhibitor for 30 min followed by exposure to the indicated amounts of ligands for 2 h. The PKC inhibitor Ro-32-0423 was able to diminish the effect of glycated albumin to activation of NF-κB. To test the effect of MAPK inhibitors
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Fig. 3. Activation of MAPK p44/42 (ERK1/2) by glycated albumin. Results shown are representative of three independent experiments. Densitometric analysis for kinase activation by GA compared with NA (400 μg/ml for 30 min, set as 1.0) is indicated. GA = glycated albumin; NA = native albumin; PD = MEK-1 inhibitor for ERK; RO = specific inhibitor of PKC-ε; Gen = genistein, PTK inhibitor. Upper panel: probed with an antibody that specifically recognizes the phosphorylated form of ERK1/2. Lower panel: the membrane was probed with an antibody against total ERK1/2 showing equal protein loading of all lanes.
SB203580 or PD98059 cells were pretreated with either inhibitor for 30 min followed by GA exposure for 2 h. Both inhibitors were able to reduce activation of p65 subunit of NF-κB and hence its translocation to the nucleus (Fig. 4). 4. Discussion Glucose-dependent Amadori product formation in proteins is thought to provoke vascular complications in diabetes, but it is not known, in which way glycated albumin influences injurious pathways in cells. Although the importance of AGEs is established, increasing evidence shows that Amadori-modified proteins have biological effects which are very similar to those of AGEs. Their biological reactivity results mainly from binding to specific cellular receptors whose structures and functions remain to be clarified. These receptors are clearly different from all described AGE-binding proteins. Calnexin, a binding protein for an Amadori-modified sequence in glycated albumin on mesangial cells [21] as well as nucleolin, nucleophosmin and cellular myosin heavy chain as specific binding proteins for fructoselysine on cell membranes of human monocytes, U937, MM6, THP-1 and HL-60 cells as monocytelike cells have been reported [10,12,14]. The cell signaling pathways by which glycated albumin induces alterations in the vasculature are not yet fully elucidated. It is known that the expression of the fructoselysine-specific receptor on monocytes varied between individuals and is different in diabetics and nondiabetics. Its expression is correlated with an early appearance and the severity of microangiopathy in diabetic humans and rats [11]. The results described here identify a signal transduction process in MM6 cells in which after binding of Amadorimodified albumin phosphorylation and activation of p38 and of p44/42 MAPK takes place together with NF-κB activation. It is accepted that MAPK activation plays a role in cell proliferation and differentiation, which is associated with activation of vascular cells.
Amadori-modified albumin may also stimulate MAPK p44/ 42 and p38 phosphorylation by activation of PKC-ε. In support of this observation, we previously demonstrated activation of this PKC isoform [14] which we now show can be specifically inactivated by Ro32-0432. This finding suggests that Amadorimodified albumins stimulate MAPK activation via a PKC. In addition there is also evidence from the experiments with the MEK-1 inhibitor PD-98059, which blocked p44/42 activation that other signaling pathways are implicated in the activation of MAPK. We conclude that an upstream MAPK activator of the MEK family may be involved. PTK activation is not required in the MAPK activation, since the activation is unaffected by the PTK inhibitor genistein. Since all experiments were performed in the presence of an adequate concentration of the antioxidant N-acetyl-L-cysteine, we consider it unlikely that activation of NADPH oxidase and the formation of reactive oxygen species play a role for the observed effects which has been described for endothelial cells after binding of glycated albumin [22]. The cellular mechanisms, by which glycated albumin stimulates signal transduction are cell-type-dependent. In vascular smooth muscle cells GA induces the expression of monocyte chemoattractant proteins, activation of p44/42 and of NF-κB and AP-1 [7,23,24]. GA also stimulates tissue plasminogen activator inhibitor synthesis in mesothelial cells by activation of NF-κB and AP-1 [3] and in macrophage RAW cells after NADPH oxidase stimulation a p44/42dependent activation of NF-κB [19]. GA activates NADPH oxidase, protein kinase B and JNK resulting in increased Eselectin expression, which in endothelial cells is upregulated by NF-κB and AP-1 [22]. GA stimulates cardiomyocyte ROS production through a protein kinase C-dependent activation of NADPH oxidase, which results in NF-κB activation and
Fig. 4. Activation of NF-κB by glycated albumin determined by ELISA. NA = native albumin; GA = glycated albumin; Gen = genistein, PTK inhibitor; RO = PKC inhibitor; PD = MEK-1 inhibitor; SB = MAPK inhibitor. MAPK and PKC inhibitors prevented GA stimulated activation of NF-κB. Genistein was ineffective. The results are means of three independent experiments. *p < 0.005 compared with the level of activation by NA, **p < 0.005 compared with the level of activation by GA.
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upregulation of atrial natriuretic factor mRNA. These findings suggest that early glycation may play a role in the pathogenesis of diabetic heart disease [25]. Reduction of Amadori-glycated albumin levels in diabetic animals ameliorates the progression of nephropathy and retinopathy, indicating a causal role of glycated albumin in the pathogenesis of diabetic microangiopathies [5]. Inhibition of GA may be a target for prevention of its cellular interactions and of diabetic complications [5,17]. These and our results show that GA induces different signaling pathways in various cells, which activate the transcription factors NF-κB and AP-1. Further studies are necessary to characterize the receptors for Amadori-modified proteins and the signaling pathways. These experiments have significance for the pathophysiology of diabetic sequelae, because the MM6 cell line is very similar to human monocytes. MM6 constitutively expresses phenotypic and functional characteristics of mature monocytes [26], which also bind GA [11]. Fructoselysine adducts are the principal forms, in which glycated albumin exists in vivo and may be involved in the pathogenesis of diabetic vascular disorders, when formed in excess during chronic hyperglycemia. Acknowledgements We thank Professor Dr. R. Jack for valuable comments and critical reading of the manuscript. This study was financially supported by the Medical Faculty of the Ernst Moritz Arndt University Greifswald.
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
[18]
[19]
References [20] [1] R. Singh, A. Barden, T. Mori, L. Beilin, Advanced glycation end-products: a review, Diabetologia 44 (2001) 129–146. [2] G. Suji, S. Sivakami, Glucose, glycation and aging, Biogerontology 5 (2004) 365–373. [3] Y. Hattori, M. Suzuki, S. Hattori, K. Kasai, Vascular smooth muscle cell activation by glycated albumin (Amadori adducts), Hypertension 39 (2002) 22–28. [4] D. Ruggiero-Lopez, N. Rellier, M. Lecomte, M. Lagarde, N. Wiensperger, Growth modulation of retinal microvascular cells by early and advanced glycation products, Diabetes Res. Clin. Pract. 34 (1997) 135–142. [5] C.G. Schalkwijk, M. Lieuw-a-Fa, V.W.M. van Hinsbergh, C.D.A. Stehouwer, Pathophysiological role of Amadori-glycated proteins in diabetic microangiopathy, Semin. Vasc. Med. 2 (2002) 191–197. [6] M.P. Cohen, F.N. Ziyadeh, Role of Amadori-modified nonenzymatically glycated serum proteins in the pathogenesis of diabetic nephropathy, J. Am. Soc. Nephrol. 7 (1996) 183–190. [7] S. Mandl-Weber, B. Haslinger, C.G. Schalkwijk, T. Sitter, Early glycated albumin, but not advanced glycated albumin, methylglyoxal, or 3-deoxyglucosone increases the expression of PAI-1 in human peritoneal mesothelial cells, Perit. Dial. Int. 21 (2001) 487–494. [8] S. Krantz, R. Brandt, B. Gromoll, Binding sites for short-term glycated albumin on peritoneal cells of the rat, Biochim. Biophys. Acta 1177 (1993) 15–24. [9] R. Salazar, R. Brandt, S. Krantz, Expression of fructosyllysine receptors on
[21]
[22]
[23]
[24]
[25]
[26]
1753
human monocytes and monocyte-like cell lines, Biochim. Biophys. Acta 1266 (1995) 57–63. S. Krantz, R. Salazar, R. Brandt, J. Kellermann, F. Lottspeich, Purification and partial amino acid sequencing of a fructoselysine-specific binding protein from cell membranes of the monocyte-like cell line U937, Biochim. Biophys. Acta 1266 (1995) 109–112. R. Brandt, C. Landmesser, L. Vogt, B. Hehmke, R. Hanschke, J. Kasbohm, K. Hartmann, B. Jäger, S. Krantz, D. Michaelis, Differential expression of fructosyllysine-specific receptors on monocytes and macrophages and possible pathophysiological significance, Diabetologia 39 (1996) 1140–1147. R. Salazar, R. Brandt, J. Kellermann, S. Krantz, Purification and characterization of a 200 kDa fructosyllysine-specific binding protein from cell membranes of U937 cells, Glycoconj. J. 17 (2000) 713–716. R. Salazar, R. Brandt, S. Krantz, Binding of Amadori glucose-modified albumin by the monocytic cell line MonoMac 6 activates protein kinase Cε, protein tyrosine kinases and the transcription factors AP-1 and NF-κB, Glycoconj. J. 18 (2001) 769–777. R. Brandt, M. Nawka, J. Kellermann, R. Salazar, D. Becher, S. Krantz, Nucleophosmin is a component of the fructoselysine-specific receptor in cell membranes of Mono Mac 6 and U937 monocyte-like cells, Biochim. Biophys. Acta 1670 (2004) 132–136. T. Naitoh, M. Kitahara, N. Tsuruzoe, Tumor necrosis factor-α is induced through phorbol ester- and glycated albumin-dependent pathway in THP-1 cells, Cell Signalling 13 (2001) 331–334. M.P. Cohen, F.N. Ziyadeh, Amadori glucose adducts modulate mesangial cell growth and collagen gene expression, Kidney Int. 45 (1994) 475–484. S. Chen, M.P. Cohen, F.N. Ziyadeh, Amadori-glycated albumin in diabetic nephropathy: pathophysiologic connections, Kidney Int. 58 (2000) S40–S44. M.P. Cohen, E. Shea, C.W. Shearman, ERK mediates effects of glycated albumin in mesangial cells, Biochem. Biophys. Res. Commun. 283 (2001) 641–643. M.P. Cohen, E. Shea, S. Chen, C.W. Shearman, Glycated albumin increases oxidative stress, activates NF-κB and extracellular signalregulated kinase (ERK), and stimulates ERK-dependent transforming growth factor-β1 production in macrophage RAW cells, J. Lab. Clin. Med. 141 (2003) 242–249. C. Miele, A. Riboulet, M.A. Maitan, F. Oriente, C. Romano, P. Formisano, J. Giudicelli, F. Beguinot, E. van Obberghen, Human glycated albumin effects glucose metabolism in L6 skeletal muscle cells by impairing insulininduced insulin receptor substrate (IRS) signaling through a protein kinase C α-mediated mechanism, J. Biol. Chem. 278 (2003) 47376–47387. V.Y. Wu, C.W. Sherman, M.P. Cohen, Identification of calnexin as a binding protein for Amadori-modified glycated albumin, Biochem. Biophys. Res. Commun. 284 (2001) 602–606. K. Higai, A. Shimamura, K. Matsumoto, Amadori-modified glycated albumin predominantly induces E-selectin expression on human umbilical vein endothelial cells through NADP oxidase activation, Clin. Chim. Acta 367 (2006) 137–143. T. Ichiki, Y. Funakoshi, K. Ito, A. Takeshita, Expression of monocyte chemoattractant protein-1 by nonenzymatically glycated albumin (Amadori adducts) in vascular smooth muscle cells, Biochem. Biophys. Res. Commun. 269 (2000) 666–670. Y. Hattori, M. Suzuki, S. Hattori, K. Kasai, Vascular smooth muscle cell activation by glycated albumin (Amadori adducts), Hypertension 39 (2002) 22–28. M. Zhang, A.L. Kho, N. Anilkumar, R. Chibber, P.J. Pagano, A.M. Shah, A.C. Cave, Glycated proteins stimulate reactive oxygen species production in cardiac myocytes, Circulation 113 (2006) 1235–1243. H.W.L. Ziegler-Heitbrock, E. Thiel, A. Fütterer, V. Herzog, A. Wirtz, G. Riethmüller, Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes, Int. J. Cancer 41 (1988) 456–461.