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Frontier Health Science (J.-W.X., K.I.), School of Human Environmental Science, Department of Pathophysiology (T.M.), School of Pharmaceutical Sciences, and Institute for World Health Development (Y.Y.), Mukogawa Women’s University, Nishinomiya 663-8179, Japan; and Section of Cellular Physiological Chemistry (I.M.), Graduate School, Tokyo Medical and Dental University, Tokyo 113-8549, Japan
Address all correspondence and requests for reprints to: Jin-Wen Xu, M.D., Ph.D., Frontier Health Science, School of Human Environmental Science, Mukogawa Women’s University, Nishinomiya, Hyogo 663-8179, Japan. E-mail: jwxu1001{at}yahoo.co.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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phagocytic receptor I (FcRI), but not anti-CD16 (FcRIIIa) or anti-CD32 (FcRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chronic low-grade endothelial inflammation is an important agent in the pathogenesis of insulin resistance and diabetes (12). Type 2 diabetes carries an increased risk of cardiovascular disease and death. One such possible precursor is insulin resistance, which constitutes both a major feature of type 2 diabetes and an independent risk factor for cardiovascular disease. Nystrom and co-workers (13) indicated that persistent endothelial dysfunction is related to elevated CRP levels in type 2 diabetic patients. A recent study demonstrated that one of the serum leptin-interacting proteins is CRP, the expression of which can be stimulated by leptin in human hepatocytes (14).
Spleen tyrosine kinase (Syk) protein-tyrosine kinase has been implicated in a variety of hematopoietic cell responses, in particular immunoreceptor signaling events that mediate diverse cellular responses including proliferation, differentiation, and phagocytosis (15, 16, 17, 18). Syk becomes activated through tandem Src homology 2 interaction with immunoreceptor tyrosine-based activation motifs (ITAMs) in immune response receptors, and the important role of Syk in immunoreceptor signaling has been well documented (19). On the other hand, Syk exhibits a more widespread expression pattern in nonhematopoietic cells such as fibroblasts, epithelial cells, breast tissue, hepatocytes, neuronal cells, and vascular endothelial cells and has been shown to be functionally important in these cell types (20), suggesting that Syk appears to play a general physiological function in a wide variety of cells.
Although few epidemiological studies (21) have demonstrated that CRP is related to insulin resistance, no study to date has examined the molecular mechanism by which CRP inhibits insulin signaling. In this study, we show that CRP attenuates insulin signaling through the regulation of Syk tyrosine kinase and RhoA in the phosphorylation of the insulin receptor substrate-1 (IRS-1), Akt, and endothelial nitric oxide synthase (eNOS) in vascular endothelial cells, leading to an imbalance between NO and endothelin-1 production.
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RESULTS |
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, A and B); however, the boiled CRP did not elevate this phosphorylation (supplemental Fig. I-A published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Moreover, pretreatment with recombinant CRP restrained the insulin-induced phosphorylation of both Akt and eNOS proteins (each n = 3, P < 0.05; Fig. 1C), decreased intracellular phosphatidyl inositol-3,4,5-triphosphate or phosphatidyl inositol-3,4,5-triphosphate [(PIP3), n = 3, P < 0.01, supplemental Fig. I-B], and suppressed insulin-induced NO2 production (insulin group: 2.32 ± 0.25 µmol/liter/106 cells·8 h vs. CRP-treated group: 0.47 ± 0.25 µmol/liter/106 cells·8 h, n = 6, P < 0.01; Fig. 1D).
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). Pretreatment with 10 µmol/liter of Y27632, an inhibitor of Rock, also blocked the inhibitory effect of recombinant CRP on IRS-1 phosphorylation at the Ser307 site (n = 3, P < 0.01; Fig. 2B).
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), suggesting that the small GTP-binding protein RhoA regulates JNK signaling. Moreover, pretreatment with 50 µmol/liter of SP600125, an inhibitor of JNK, blocked recombinant CRP-induced IRS-1 phosphorylation at the Ser307 site (n = 3, P < 0.05; Fig. 2D).
Phagocytic Receptor I (FcRI) (CD64) Mediated the Inhibitory Effect of Recombinant CRP on Insulin Signaling
As shown in Fig. 3A, after coincubation with phagocytic receptor antibodies of anti-CD16 (FcRIIIa), anti-CD32 (FcRII), or anti-CD64 (FcRI) at 1:500 for 30 min, anti-CD64, but not anti-CD32 and anti-CD16 antibodies, blocked the recombinant CRP-induced IRS-1 phosphorylation at the Ser307 site (n = 3, P < 0.05). Similarly, treatment with anti-CD64 antibody, but not anti-CD16 antibody, blocked the recombinant CRP-induced phosphorylation of JNK (n = 3, P < 0.01; Fig. 3B). Coincubation with anti-CD64, but not with anti-CD16, also partially reversed the insulin-stimulated phosphorylation of Akt (n = 3, P = 0.057; supplemental Fig. III published as supplemental data on The Endocrine Society’s Journals Online web site).
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). Moreover, pretreatment with piceatannol at 10 µmol/liter for 30 min or 1 h also suppressed RhoA activity (n = 3, P < 0.05; Fig. 4B). Similarly, preincubation with piceatannol prevented phosphorylation at the Ser307 site of IRS-1 induced by CRP (n = 3, P < 0.01; Fig. 5A). To further confirm the role of Syk in the inhibitory effect of recombinant CRP on insulin signaling, we transfected the control-small interference RNA (siRNA) or Syk-siRNA to mouse kidney vascular endothelial cells, then incubated with or without recombinant CRP for 1 h, and observed recombinant CRP effect on insulin signaling. The results in Fig. 5, B and C, showed that Syk-siRNA, but not control-siRNA, blocked the CRP-induced phosphorylation of IRS-1 and JNK.
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RII), or CD64 (FcRI) was precipitated, and blots were probed with antibody agonist Syk kinase. The data presented in Fig. 6A clearly show that Syk kinase is associated with Pyk2 in CRP-activated cells, and densitometric analyses displayed an increase of 6.8-fold arbitrary units compared with the control (n = 3, P < 0.005). A similar result was also observed in an anti-CD64 immunoprecipitated sample, i.e. up to 2.2-fold arbitrary units of the control, whereas the anti-CD32 precipitated sample did not exhibit a further increase in complex formation (each n = 3; Fig. 6B).
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, CRP elevated insulin-induced endothelin-1 production from 7.70 ± 0.33 to 8.61 ± 0.25 ng/ml/105 cells·24 h (each n = 4; insulin vs. insulin plus CRP, P < 0.05). However, the elevation of endothelin-1 was effectively attenuated by pretreatment with piceatannol (fell to 5.92 ± 0.65 ng/ml/105 cells·24 h; vs. insulin plus CRP group, P < 0.01). On the other hand, a nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 µmol/liter), also assuaged the increase in the level of endothelin-1 production (7.20 ± 0.34 ng/ml/105 cells·24 h; vs. insulin plus CRP group, P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recombinant CRP caused the elevation of Rho GTPase acitivity in a time-dependent manner. Caron and Hall (31) showed in 1998 that different Rho GTPases control the distinct mechanism of phagocytosis in macrophages by FcR. Endothelial cells also express FcR (32). In our experiments, anti-CD64 (FcRI), but not anti-CD32 (FcRII) or anti-CD16 (FcRIIIa) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1, or partially restored the insulin-stimulated phosphorylation of Akt. FcRs contain a specific motif (ITAM), which is located either in the cytoplasmic part of FcRII (CD32) or in -chains associated with FcRI (CD64) and FcRIII (CD16) in hematopoietic cells. These ITAM tyrosine residues are phosphorylated by cross-linking of the receptors with specific antibodies and are associated with Syk tyrosine kinase that activates downstream signals (17, 33).
Syk tyrosine kinase is expressed in all hematopoietic cells and contains a C-terminal kinase domain and tandem N-terminal Src homology 2 domains that bind phosphorylated ITAM and play critical roles in signaling through immune receptors. Syk is essential for lymphocyte development (15, 16) and signal transduction (17, 18, 23, 24) via immune receptors in nonlymphoid cells. Syk is also expressed in vascular endothelial cells (25). Many studies have indicated that Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors, and regulate the activation of Rho GTPase, such as Rac1, Cdc42, and RhoA (34, 35). Moreover, one recent report suggested that Vav proteins are able to bind to the Tyr-348 site of Syk in ß2 integrin-mediated neutrophil migration in vitro (36). In this study, some evidence indicates that the Syk-RhoA-JNK pathway is essential for inhibiting insulin signaling. The Syk-RhoA-JNK pathway brings on the phosphorylation of IRS-1 at the Ser307 site. 1) The Rock inhibitor Y27632 and dominant negative RhoA cDNA blocked the recombinant CRP-induced phosphorylation of IRS-1 at the Ser307 site. 2) Recombinant CRP stimulated the phosphorylation of JNK, which could be blocked by dominant negative RhoA cDNA. 3) The recombinant CRP-induced phosphorylation of IRS-1 at the Ser307 site was also blocked by SP600125, an inhibitor of JNK. 4) Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk siRNA, blocked CRP-induced RhoA activity, phosphorylation of JNK and IRS-1 at Ser307. On the other hand, we showed that CRP stimulated the formation of a complex between Syk and Pyk2. We do not understand the Pyk2 role in CRP-induced RhoA activation. Only one study has shown that M1 mAChR activated small GTPase RhoA through the Pyk2 pathway (37). Our results suggest that the Syk-RhoA-JNK signaling axis is essential for regulating the phosphorylation of IRS-1.
Endothelin-1, a peptide of 21 amino acid residues, is the most potent vasoconstrictive substance known. On the other hand, hyperinsulinemia is a potent inducer of endothelin-1 release (38). In the endothelium, insulin resistance displays decreased IRS-1/PI 3-kinase signaling, leading to an imbalance between decreased NO and increased endothelin-1 production (39). In this study, we found that CRP increased insulin-induced endothelin-1 secretion when inhibiting NO production; in addition, piceatannol, an inhibitor of Syk tyrosine kinase, effectively blocked this increase in endothelin-1 secretion, and the phosphorylation of IRS-1 at Ser307 was stimulated by CRP. Moreover, a nitric oxide donor SNAP assuaged the increase in the level of endothelin-1 production, which agrees with the knowledge of nitric oxide as an antagonist of endothelin-1 secretion (40).
In conclusion, this study demonstrated the molecular mechanism by which recombinant CRP inhibits the insulin signal in endothelial cells and causes endothelial dysfunction involvement by inducing the phosphorylation of IRS-1 at the Ser307 site via the Syk-RhoA axis. Our results provide potential evidence of an epidemiological and clinical association between proinflammatory recombinant CRP and diabetes, obesity, and hypertension.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-tubulin antibody was purchased from Molecular Probes (Eugene, OR). Anti-CD16 (FcRIIIa), anti-CD32 (FcRII), and anti-CD64 (FcRI) mouse monoclonal antibodies were obtained from Caltag Laboratories (Burlingame, CA). Fugene 6 transfection reagent was purchased from Roche Diagnostics (Mannheim, Germany). Opti-MEM I and G418 were purchased from Invitrogen (Carlsbad, CA). NO2/NO3 Assay Kit-C II (Colorimetric) was obtained from Dojindo Laboratories (Kumamoto, Japan). ECL plus Western Blot Detection System was from Amersham Biosciences Corp. (Piscataway, NJ). The endothelin EIA immunoassay kit was purchased from Peninsula Laboratories, Inc. (San Carlos, CA).
Endothelial Cell Culture
BAECs, and mouse kidney vascular endothelial cells were obtained from the Human Science Research Resources Bank (Osaka, Japan). BAECs and mouse kidney vascular endothelial cells were maintained at 37 C in 5% CO2 in DMEM containing 10% fetal bovine serum (FBS). Medium was changed every 2 d and the cells were passaged with trypsin-EDTA.
Immunoprecipitation and Western Blotting
BAECs were washed twice with ice-cold PBS containing 1 mmol/liter sodium orthovanadate, and harvested in ice-cold RIPA buffer (50 mmol/liter Tris/HCl, pH 8.0; 150 mmol/liter NaCl; 2 mmol/liter sodium orthovanadate; 1% Nonidet-P40, 1% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 0.1 mmol/liter dithiothreitol; 0.05 mmol/liter of phenylmethylsulfonylfluoride, 0.002 mg/ml of aprotinin; and 0.002 mg/ml of leupeptin). Lysates were scraped from the dishes and precleared by centrifugation at 12,000 x g for 20 min at 4 C. For immunoprecipitation, antiphosphatase and tensin homolog, anti-Rock, anti-Pyk2 anti-CD32, and anti-CD64 antibody (1:200) were added to the precleared lysates for 1 h at 4 C, and incubation was continued overnight at 4 C with protein A/G Plus agarose (50 µl/ml). Immunoprecipitates were washed five times with 1 ml ice-cold PBS. Immunoprecipitated proteins were eluted from the agarose beads by boiling for 5 min in sodium dodecyl sulfate sample buffer. Aliquots of the cell lysate (100 µg of each sample) were resolved on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in 5% skim milk overnight at 4 C. This was followed by incubation with a primary antibody for 2 h and then exposure to a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Visualization was performed using enhanced chemiluminescence (Amersham). Quantitative analysis of band density was done using NIH Image 1.63 Software. Western blot experiments were performed in duplicate or triplicate.
Rho Activation Assay
Intracellular Rho activity was measured with a Rho activation assay kit (Upstate Biotechnology) according to the manufacturer’s instructions. In brief, approximately 106 serum-starved BAECs were stimulated with recombinant CRP for the period indicated, rinsed twice with ice-cold Tris-buffered saline, and lysed on ice for 15 min in 1x MLB buffer (25 mM HEPES, pH 7.5; 150 mM NaCl; 1% Igepal CA-630; 10 mM MgCl2; 1 mM EDTA; 2% glycerol). The samples were then centrifuged at 14,000 x g at 4 C for 5 min, and the protein concentrations of the supernatants were measured. A 1-ml sample of each cell extract was immediately affinity precipitated at 4 C for 45 min with 30 µl of the Rho assay reagent (one vial containing 650 µg recombinant glutathione-S-transferase-rhotekin-Rho binding domain fusion proteins precoupled to 1-ml glutathione-agarose beads). The precipitates were washed three times with MLB buffer, and the bound Rho-GTP samples were eluted in 40 µl of 2x Laemmli reducing sample buffer and boiled for 5 min. Samples were separated by 12% SDS-PAGE and detected by monoclonal Rho antibody.
RhoA cDNA Transfection
BAECs were seeded at 105 cells per well in six-well plates and maintained in DMEM supplemented with 10% FBS for 16 h. Transfection of wild-type or dominant negative RhoA cDNA was carried out using Fugene 6 transfection reagent according to the manufacturer’s instructions. Briefly, transfection complexes were prepared by adding three parts Fugene 6 reagent to one part cDNA (2 µg) in up to 100 ml Opti-MEM I. After incubation for 30 min at room temperature, the complexes flooded the cells in Opti-MEM I. After 4 h, transfected cells were maintained in DMEM supplemented with 10% FBS and selected in 1 mg/ml of G418 for 5 d. The transfected BAECs were then plated onto 100-mm-diameter dishes for 8 h before use.
siRNA Transfection
siRNA duplexes (siRNAs) were synthesized and purified by TAKARA Bio (Otsu, Japan). The siRNA sequence for targeting mouse Syk (GenBank accession no. NM_011518) was Syk siRNA (5'-UUG AUC CUU GAG AUU AUU CTT-3'). Control siRNA (5'-UUG CUU UUA UCA AUG GUA CTT-3') was used as a negative control. Transfection of siRNAs was performed using the manufacturer’s protocol for siRNA Transfection Reagent (Santa Cruz Biotechnology). Briefly, 4 µl of 20 µM siRNA was mixed with 200 µl of siRNA Transfection Medium, and 4 µl of siRNA Transfection Reagent was diluted in 200 µl of siRNA Transfection Medium and incubated at room temperature for 5 min. After the incubation, the diluted siRNA Transfection Reagent was combined with the diluted siRNA and then incubated for an additional 20 min at room temperature. A total of 400 µl of complexes was applied to each well of cultured mouse kidney vascular endothelial cells at 70% confluence in a six-well plate.
Measurement of NO Release
NO production by BAECs was measured as the nitrite or nitrate concentration in cell culture supernatants according to the manufacturer’s instructions. In brief, BAECs (106 cells/ml) were plated onto six-well tissue culture plates in phenol red-free DMEM. The cells were pretreated with or without Y27632 or SP600125 for 30 min, and then treated with or without recombinant CRP for 2 h as well as stimulated with 10 ng/ml of insulin for 8 h at 37 C. At the end of incubation, 200 µl of each supernatant was carefully transferred to a 96-well plate, with the subsequent addition of 100 µl of Griess reagent (50 µl of 1% sulfanilamide containing 5% phosphoric acid and 50 µl of 0.1% N-1-naphthyl-ethylenediamine). After color development at room temperature for 10 min, the absorbance was measured on a microplate reader at a wavelength of 520 nm. Each sample was assayed in triplicate. A calibration curve was plotted using known amounts of sodium nitrate solution.
PIP3 Content Measurements
PIP3 content was also assessed by immunoblotting. Approximately 106 BAECs were washed three times with large volumes of ice-cold PBS, scraped off the wells with a rubber policeman, and sedimented. The cells were resuspended in 15 vol of methanol-chloroform-concentrated HCl (100:50:1). The mixture was vortexed and 1 vol (equal to the volume of the original cell pellet) of 100 mM EDTA was added. Chloroform (5 vol) and water (5 vol) were added, and the cloudy mixture was swirled and centrifuged at 400 x g for 5 min to induce phase separation. The bottom (organic) phase was transferred to glass tubes and dried. The dried lipid extract was redissolved in 10 µl of 1:1 chloroform-methanol (containing 0.1% HCl) and spotted onto polyvinylidene difluoride membranes. After drying, the membrane was blocked in 5% skim milk overnight at 4 C. Blots were exposed to a 1:500 dilution of monoclonal antibodies against PIP3. Immunoreactive bands were visualized by enhanced chemiluminescence.
Detection of Endothelin-1 Level
Endothelin-1 levels in media were measured with an ELISA immunoassay kit according to the manufacturer’s instructions. In brief, media and the primary antibody, as well as the biotinylated peptide solution, were dispensed into designated wells of a 96-well immunoplate and incubated for 2 h at room temperature. After washing five times, streptavidin-horseradish peroxidase was added to each well and incubated for 1 h. Finally, the microplate was washed and tetra-methylbenzidine solution was added to each well. After 30 min, the reaction was terminated with HCl. Absorbance at 450 nm was measured within 20 min.
Data Presentation
Statistical analysis was performed using ANOVA or t test when appropriate. The data are expressed as the mean ± SD. A value of P < 0.05 was considered significant. Quantitative analysis of band density was done using NIH Image 1.63 Software. Western blot experiments were performed in duplicate or triplicate.
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FOOTNOTES |
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First Published Online November 9, 2006
Abbreviations: BAEC, Bovine artery endothelial cell; CRP, C-reactive protein; eNOS, endothelial nitric oxide synthase; FBS, fetal bovine serum; FcR, Fc phagocytic receptor; IRS-1, insulin receptor substrate-1; ITAM, immunoreceptor tyrosine-based activation motifs; JNK, jun N-terminal kinase; PIP3, phosphatidyl inositol-3,4,5-triphosphate; Rock, RhoA-dependent kinase; siRNA, small interference RNA; SNAP, S-nitroso-N-acetyl-DL-penicillamine; Syk, spleen tyrosine kinase.
Received for publication August 28, 2006. Accepted for publication November 2, 2006.
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REFERENCES |
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receptor signaling in macrophages and neutrophils. Mol Cell Biol 18:4209–4220RI and FcRII crosslinking is determined by the differentiation state of U-937 monocytic cells. Immunol Lett 99:169–179[CrossRef][Medline]R. J Immunol 169:6787–6794 receptors on human aortic endothelial cells mediates biological effects. Arterioscler Thromb Vasc Biol 25:1359–1363 receptor-mediated phagocytosis. FEBS Lett 400:11–14[CrossRef][Medline]This article has been cited by other articles:
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