High Glucose Increases Angiopoietin-2 Transcription in Microvascular Endothelial Cells through Methylglyoxal Modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor α. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease. Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor α. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease. Methylglyoxal (MG) 3The abbreviations used are: MG, methylglyoxal; Ang-2, angiopoietin-2; GLO1, glyoxalase 1; ICAM-1, intracellular adhesion molecule-1; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc-transferase; SOD2, manganese superoxide dismutase; TNF-α, tumor necrosis factor α; UCP-1, uncoupling protein-1; VCAM-1, vascular cell adhesion molecule-1; MKEC, murine kidney endothelial cell; IP, immunoprecipitation; aa, amino acid; WT, wild type.3The abbreviations used are: MG, methylglyoxal; Ang-2, angiopoietin-2; GLO1, glyoxalase 1; ICAM-1, intracellular adhesion molecule-1; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc-transferase; SOD2, manganese superoxide dismutase; TNF-α, tumor necrosis factor α; UCP-1, uncoupling protein-1; VCAM-1, vascular cell adhesion molecule-1; MKEC, murine kidney endothelial cell; IP, immunoprecipitation; aa, amino acid; WT, wild type. is a highly reactive α-oxoaldehyde formed in cells primarily from the triose phosphate intermediates of glycolysis, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (1Phillips S.A. Thornalley P.J. Eur. J. Biochem. 1993; 212: 101-105Crossref PubMed Scopus (449) Google Scholar, 2Richard J.P. Biochem. Soc. Trans. 1993; 21: 549-553Crossref PubMed Scopus (207) Google Scholar). It is the major physiologic substrate for the enzyme glyoxalase I, which is encoded by the GLOI gene. Together with glyoxalase II and a catalytic amount of glutathione, glyoxalase I reduces methylglyoxal to d-lactate (3Thornalley P.J. Biochem. Soc. Trans. 2003; 31: 1343-1348Crossref PubMed Google Scholar). In cells, methylglyoxal reacts almost exclusively with arginine residues to form the major methylglyoxal-derived epitope hydroimidazolone MG-H1 (Nα-acetyl-Nδ (5-hydro-5-methyl)-4-imidazolone) (4Ahmed N. Thornalley P.J. Biochem. Soc. Trans. 2003; 31: 1417-1422Crossref PubMed Google Scholar). Changes in methylglyoxal concentration have been implicated in the pathobiology of a variety of important diseases, including diabetic vascular disease. Diabetes increases levels of the methylglyoxal arginine-derived hydroimidazolone adduct MG-H1 in retina, renal glomerulus, and sciatic nerve of rats (5Hammes H.P. Du X. Edelstein D. Taguchi T. Matsumura T. Ju Q. Lin J. Bierhaus A. Nawroth P. Hannak D. Neumaier M. Bergfeld R. Giardino I. Brownlee M. Nat. Med. 2003; 9: 294-299Crossref PubMed Scopus (667) Google Scholar, 6Karachalias N. Babaei-Jadidi R. Ahmed N. Thornalley P.J. Biochem. Soc. Trans. 2003; 31: 1423-1425Crossref PubMed Scopus (135) Google Scholar), and MG-H1 is also increased in aortic endothelial cells cultured in high glucose (7Du X. Matsumura T. Edelstein D. Rossetti L. Zsengeller Z. Szabo C. Brownlee M. J. Clin. Investig. 2003; 112: 1049-1057Crossref PubMed Scopus (621) Google Scholar). Diabetes also induces a significant increase in retinal and glomerular expression of angiopoietin-2 (Ang-2) in rats (8Hammes H.P. Lin J. Wagner P. Feng Y. Vom Hagen F. Krzizok T. Renner O. Breier G. Brownlee M. Deutsch U. Diabetes. 2004; 53: 1104-1110Crossref PubMed Scopus (264) Google Scholar, 9Yuan H.T. Tipping P.G. Li X.Z. Long D.A. Woolf A.S. Kidney Int. 2002; 61: 2078-2089Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Singh A.K. Gudehithlu K.P. Pegoraro A.A. Singh G.K. Basheerudin K. Robey R.B. Arruda J.A. Dunea G. Lab. Investig. 2004; 84: 597-606Crossref PubMed Scopus (24) Google Scholar, 11Rizkalla B. Forbes J.M. Cao Z. Boner G. Cooper M.E. J. Hypertens. 2005; 23: 153-164Crossref PubMed Scopus (54) Google Scholar). In diabetic retinal capillaries, increased Ang-2 is associated with pericyte loss and acellular capillary formation, while in kidney it is associated with glomerular capillary loss in anti-glomerular basement membrane glomerulonephritis (9Yuan H.T. Tipping P.G. Li X.Z. Long D.A. Woolf A.S. Kidney Int. 2002; 61: 2078-2089Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). A mechanistic link between elevated angiopoietin-2 levels and vascular pathology is suggested by the finding that Ang-2 can function as an autocrine regulator of endothelial inflammatory responses (12Fiedler U. Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. Witzenrath M. Rosseau S. Suttorp N. Sobke A. Herrmann M. Preissner K.T. Vajkoczy P. Augustin H.G. Nat. Med. 2006; 12: 235-239Crossref PubMed Scopus (713) Google Scholar). These observations led us to hypothesize that hyperglycemia-induced methylglyoxal formation might directly regulate transcription of genes involved in diabetic vascular disease, such as Ang-2, by covalently modifying proteins that bind to the Ang-2 promoter. In the present study, we demonstrate that in mouse kidney microvascular endothelial cells increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC-box in the Ang-2 promoter, resulting in increased Ang-2 expression. High glucose-induced Ang-2 increased expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) both in cultured cells and in diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor α (TNF-α). This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease. Materials and Methods—Mouse Ang-2 reporter plasmid (pGL3-mANG-2 (short)-Luc) was kindly provided by Drs. G. D. Yancopoulos and P. C. Maisonpierre. The related deletion promoter constructs were generated by PCR methods, and the indicated mutations were generated using the site-directed mutagenesis kit from Promega (Madison, WI). Rat UCP-1 cDNA was provided by D. Ricquier (CNRS-Unite Propre 1511, Meudon, France), human SOD2 cDNA was provided by L. Oberly (University of Iowa, Iowa City, IA), and human glyoxalase-I (GLO1) cDNA was provided by Dr. K. D. Tew (University of South Carolina, Charleston, S.C.). These cDNAs were cloned into the shuttle vector pAd5/CMV/K-NpA, and adenoviral vectors were prepared by the Gene Transfer Vector Core (University of Iowa). Mouse Gal4mSin3A was obtained from Dr. R. M. Evans. Rat O-GlcNAc-transferase (OGT) cDNA was obtained from G. W. Hart. pcDNA3 Gal4-AD-msin3A (mouse) and pVP16-mSin3A (888-955)/925 + 938(Q) double mutants were digested and ligated into Mfe1/XbaI sites for construction of Gal4-msin3A/925 + 938(Q) full-length double mutants. Detailed information regarding each construct is available upon request. Murine Ang-2 small interfering RNA (number 162193) was from Ambion. Ang-2 antibody (ab8452) was from Abcam (Cambridge, MA). GLO1 rabbit polyclonal antibody was produced and characterized by P. J. T. A monoclonal antibody to the major intracellular methylglyoxal-derived epitope, Nα-acetyl-Nδ (5-hydro-5-methyl)-4-imidazolone (M. G.), was generated and characterized by M. B., I. G., and P. J. T. Antibodies for Sp1, Sp3, mSin3A, and Gal4 were obtained from Santa Cruz Biotechnology. OGT rabbit polyclonal antibody (AL28) was kindly provided by Dr. G. W. Hart. O-GlcNAc monoclonal antibody (MA1-072) was purchased from Affinity BioReagents, Golden, CO. The murine conditionally transformed kidney endothelial cell (MKEC) line was obtained from H-2Kb-tsA58 mice (13Langley R.R. Ramirez K.M. Tsan R.Z. Van Arsdall M. Nilsson M.B. Fidler I.J. Cancer Res. 2003; 63: 2971-2976PubMed Google Scholar) and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics supplemented with essential amino acids and vitamins. Cells were grown at 33 °C, but experiments and treatment were performed at the non-permissive temperature of 37 °C. Conditionally transformed human aortic endothelial cells were obtained from Dr. Anita Sumaga, Albert Einstein College of Medicine. Plasmid DNA and small interfering RNA were transfected by Lipofectamine™ reagent (Invitrogen). Luciferase activity assays were carried out using the Dual-Luciferase™ Assay System (Promega), and transfection efficiencies were normalized using a cotransfected Renilla plasmid. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce Biotechnology). Protein concentration was measured by Coomassie Protein Assay kit (Pierce) using bovine serum albumin as a standard. Reverse Transcription Reaction and Real-time Quantitative PCR—Total RNA from treated cells was extracted using the RNeasy Mini kit (Qiagen), and the RNA was reverse transcribed by SuperScript™ III First Strand Synthesis System (Invitrogen). Real-time quantitative PCR (qPCR) was run on a Light-Cycler Roche 480 (Roche Molecular Systems) with the Light-Cycler Roche 480 master kit. PCR was performed by denaturing at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C, annealing at 60 °C, and extension at 72 °C for 10 s. Results were normalized by β-actin. Immunoprecipitation (IP) and Western Blotting—Cell lysates or nuclear extracts were precleared by preimmune IgG plus Protein A-agarose beads for 2 h, and the supernatants were immunoprecipitated by the indicated antibodies and a 50% slurry of Protein A-agarose beads overnight at 4 °C (14Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). After washing with buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and 0.5% deoxycholate with protease inhibitors, proteins were released and separated on 10% SDS-PAGE gels. The membranes were blotted by primary antibodies and then simultaneously incubated with the differentially labeled species-specific secondary antibodies anti-rabbit IRDye™ 800CW (green) and anti-mouse (or goat) Alexa Fluor 680 (red). Membranes were scanned and quantitated by the Odyssey Infrared Imaging System (LI-COR Biosciences). Chromatin Immunoprecipitation—Treated cells were cross-linked by 1% formaldehyde for 20 min and terminated by addition of 0.1 m glycine. Cell lysates were sonicated and centrifuged. 500 μg of protein were precleared by bovine serum albumin/salmon sperm DNA plus preimmune IgG and a slurry of Protein A-agarose beads as previously described (14Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). Immunoprecipitations were performed with the indicated antibodies, bovine serum albumin/salmon sperm DNA, and a 50% slurry of Protein A-agarose beads. Input and immunoprecipitated DNA were washed and eluted and then incubated for 2 h at 42 °C in the presence of Proteinase K followed by 6 h at 65 °C to reverse the formaldehyde cross-linking. DNA fragments were recovered by phenol/chloroform extraction and ethanol precipitation. A 196-bp fragment from mice Ang-2 promoter (forward primer 5′-ccccctacaggaagatagtgg-3′ and reverse primer 5′-agctgtcctgagaggaaggag-3′) was amplified by real-time quantitative PCR. of mouse mSin3A was amplified by PCR and into the vector mutations for of the sites in the mSin3A were prepared using the site-directed mutagenesis kit from mutants were prepared by I and of indicated fragments from pVP16-mSin3A mutants. The of OGT was amplified by PCR and into the vector These and the reporter vector were cotransfected into cells, and activity was measured by the Dual-Luciferase™ Assay System In diabetic mice were induced by of 50 m pH for 5 an with glucose mice The mice were by to The kidney or were for of and protein or the in were by the and as experiments were performed at in was and for were by of and the using Ang-2 Transcription by High in high glucose increases intracellular glucose flux and methylglyoxal concentration in cells by M. PubMed Scopus Google Scholar), we the effects of cells in mm This treatment increased Ang-2 levels with 5 mm glucose and increased Ang-2 protein levels by in mm an not not of superoxide by is the major mechanism by which high glucose increases intracellular levels of the glyoxalase I substrate methylglyoxal M. PubMed Scopus Google Scholar), we also the of uncoupling a protein of of the or manganese superoxide the form of this of the high glucose-induced increase of Ang-2 and A and of GLO1, UCP-1 and SOD2, the high glucose-induced increase of Ang-2 and protein in of a in the Ang-2 the for of the Ang-2 gene by high deletion constructs were generated containing of the murine Ang-2 promoter. In 5 mm the reporter were not the and deletion constructs to In mm were increased with in 5 mm glucose in constructs not a significant of activity was in the construct with the construct in 5 mm and activity was not increased by high These indicated that promoter between and for high glucose-induced of the Ang-2 promoter. of with transcription factor sites and an an and an the of on the high glucose-induced increase in activity of the Ang-2 promoter using a of or Ang-2 in deletion of the not high glucose-induced of the + the or deletion of of also In of the between the and + and + and deletion of of both of high glucose-induced These indicated that the between the and sites is for glucose of the Ang-2 promoter. Sp3 and to the Ang-2 Changes in to High which of nuclear proteins to the GC-box and the effects of high glucose and glyoxalase I in the of was performed using antibodies for the indicated proteins in After and of the the Ang-2 promoter was by real-time PCR using for the Ang-2 GC-box The PCR product from was increased by mm glucose with 5 mm and this increase was by of GLO1, as as by of UCP-1 or SOD2 In the PCR product from Sp3 was decreased in mm glucose with 5 mm This was by of GLO1, as as by of UCP-1 or binding was not by high glucose These indicated that Sp1, Sp3, and bind to the Ang-2 promoter in the of and suggested that the in and Sp3 binding induced by cells in mm glucose might high glucose-induced Ang-2 expression. Sp3 with but Sp3 by GLO1 high glucose-induced in and Sp3 binding to the glucose-responsive in the Ang-2 promoter we that high glucose induced by modifying Sp1, Sp3, or both with and Sp3 were immunoprecipitated and then with protein was modified by not In nuclear extracts from were immunoprecipitated with antibody and for and Sp3, high glucose increased the of the Sp3 and GLO1 this The high glucose-induced increase in Sp3 was also by of UCP-1 and SOD2 high glucose GLO1 These results suggested that an protein was modified by Sp3 and that this modification might Sp3 binding to the Ang-2 promoter. Methylglyoxal mSin3A, with a variety of proteins have been to with Sp3, we performed Western for OGT not and mSin3A. the corepressor mSin3A was modified by A and Cells incubated in high glucose a increase in modification of mSin3A. of GLO1, as as UCP-1 and SOD2, this mSin3A been to the enzyme OGT X. F. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). immunoprecipitated mSin3A was for OGT, cells incubated in high glucose OGT associated with mSin3A of GLO1, as as UCP-1 and SOD2, this This was by OGT and then for mSin3A which residues in mSin3A were for this increased binding of OGT, we that the of mSin3A was both and for this recruitment by mSin3A and into the for assays with not These with by X. F. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of mSin3A was and for binding methylglyoxal reacts primarily with residues in and residues in proteins can also modified F. J. J. K. J. K. U. S. A. 2002; PubMed Scopus Google Scholar, U. S. A. PubMed Scopus Google Scholar). the role of each and in the of mSin3A by mutants in the that each of the residues to assays that of GLO1 the increased of mSin3A with OGT induced by high glucose not mutations of and the increased of mSin3A and OGT, and double of and the increased of and was of and double of and was of not These results with binding from the not that 6 residues as sites for and The of the double on high glucose-induced modification of mSin3A by methylglyoxal was directly by of or mSin3A in high glucose a increase in modification of full-length mSin3A. In the double + 938(Q) increase in modification induced by in high a mSin3A and were to of of the mSin3A double was by experiments in which the of high glucose on Ang-2 expression was High glucose of cells mSin3A increased Ang-2 expression by high glucose of cells + on Ang-2 expression. for and in this mechanism is to cell diabetic we the experiments in and in human aortic endothelial cells and in retinal cells not In both cell to high glucose increased Ang-2 methylglyoxal modification of mSin3A. of Sp3 with OGT Sp3 directly demonstrate that increased methylglyoxal induced by high glucose increased of Sp3 with OGT, nuclear extracts were immunoprecipitated with and then for OGT and O-GlcNAc High glucose increased of Sp3 with OGT and also increased modification of Sp3 by of GLO1, as as UCP-1 and SOD2, both of These results were by with and for Sp3 for A and in High Ang-2 and in Cells and in the levels of Ang-2 induced in cells by high glucose were to expression of proinflammatory adhesion in an autocrine (12Fiedler U. Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. Witzenrath M. Rosseau S. Suttorp N. Sobke A. Herrmann M. Preissner K.T. Vajkoczy P. Augustin H.G. Nat. Med. 2006; 12: 235-239Crossref PubMed Scopus (713) Google Scholar), we measured levels of and and in high glucose increased and levels and and protein levels and in of mice on and expression were incubated in 5 mm the concentration of increased and expression in incubated in mm as in A and for levels and and protein levels and in The effects of high both and in with TNF-α, were by with Ang-2 small interfering These that high glucose-induced Ang-2 is to microvascular endothelial cells to the proinflammatory effects of implicated in an in of diabetic kidneys were obtained from diabetic and in levels of Ang-2, ICAM-1, and increased and protein levels increased and mSin3A modification by was increased in kidneys from diabetic mice with for and in In the present study, we a novel mechanism for of gene expression by high protein modification by the dicarbonyl demonstrate that in mouse kidney endothelial cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. High glucose-induced Ang-2 increased expression of and in both cultured cells and kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of In human aortic endothelial cells and in retinal cells, the major cell angiopoietin-2 in the K. Investig. Google Scholar), high glucose-induced Ang-2 expression is also by this mechanism mSin3A been to the enzyme OGT to in tumor cell which then in with to gene X. F. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In in mouse kidney endothelial cells and in retinal cells that recruitment of OGT to mSin3A gene expression the mSin3A is modified by methylglyoxal from high glucose flux and reactive formation by the mSin3A binding with the transcription factor Sp3 not been Sp3 and for in promoter G. PubMed Scopus Google Scholar, L. S. J.M. Biochem. Cell 2004; PubMed Scopus Google Scholar). both Sp3 and may as or of gene Sp3 been to in a of cell G. S. M. G. J. PubMed Scopus Google Scholar, C. C. L. G. J.P. P. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of may or transcription K. Cell. PubMed Scopus Google Scholar, X. K. Q. U. S. A. PubMed Scopus Google Scholar, H.T. Ju J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Edelstein D. Rossetti L. H. F. J. Brownlee M. U. S. A. PubMed Scopus Google Scholar), on which residues In cells and cells, Sp3 was not modified by O-GlcNAc, as by A. F. G. T. G. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the of increased glucose flux on O-GlcNAc modification of Sp3 was not of gene expression complex transcription and is that and corepressor proteins may primary of physiologic J. J. S. L. M. Z. Cooper S. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins have been described Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. V. G. J. Cell. Biochem. PubMed Scopus Google Scholar) that to regulate demonstrate for the that methylglyoxal causes modification of a protein and that this modification gene expression. The of this modification the of a variety of intracellular including flux and reactive formation, and may function as a to regulate of gene expression. The increase in Ang-2 expression by this mechanism in to high glucose important for the of diabetic High glucose-induced Ang-2 increased expression of and both in cultured cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of is elevated in both kidney and of diabetic and both diabetic and in mice C. C. J. J. 2006; PubMed Scopus Google Scholar, J. Soc. 2005; PubMed Scopus Google Scholar, A. Y. Diabetes. 2005; PubMed Scopus Google Scholar, V. K. C. H. U. N. S. B. J. 2004; PubMed Scopus Google Scholar). that methylglyoxal concentration in cells to diabetic may have important with