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发表于 2009-4-22 08:10 |只看该作者 |倒序浏览 |打印

作者：Hyun Soon Lee, Kyung Chul Moon, Chi Young Song, Bong Cho Kim, Suxia Wang, and Hye Kyoung Hong作者单位：Department of Pathology, Seoul National University College of Medicine, Seoul 110-79 Korea

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      【摘要】/ s) `  K. P7 s1 Q! S
   Amadori-modified glycated albumin stimulates extracellular matrix and transforming growth factor- (TGF- ) expression in cultured mesangial cells. Smad proteins transduce the TGF- -mediated signal, and Smad-binding CAGA sequences are present in the plasminogen activator inhibitor-1 (PAI-1) promoter. This study examined whether glycated albumin induces PAI-1 transcription in human mesangial cells (HMC) through Smad-binding sites in the PAI-1 promoter. Quiescent HMC were exposed to 200 µg/ml bovine serum albumin (BSA) or glycated BSA (Gly-BSA) for 12-72 h. At 24 h, Gly-BSA stimulated TGF- 1 and PAI-1 mRNA expression in HMC to 1.8 and 3.2 times that in the BSA-treated control cells. Gly-BSA also activated the PAI-1 promoter luciferase activity 2.3-fold. Gly-BSA-treated cells enhanced Smad2 and Smad3 protein levels 2.5 times the control levels in the nuclei. An electrophoretic mobility shift assay performed using CAGA sequences as a probe showed that Gly-BSA increased DNA/protein complexes. When nuclear extracts were preincubated with 100-fold molar excess of unlabeled CAGA oligonucleotide, the formation of complex was prevented. The DNA-binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotide, a CAGA antisense analog, inhibited Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked Gly-BSA-induced PAI-1 promoter luciferase activity. These results indicate that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA sequences in the PAI-1 promoter in HMC. Thus progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.   S5 s0 y: s' |$ q+ D5 d) Q
      【关键词】 Amadori adducts extracellular matrix CAGA boxes diabetic nephropathy
   GLUCOSE CAN REACT NONENZYMATICALLY with the amino groups of proteins to form reversible Schiff bases and, then, stable Amadori constructs. Albumin modified by Amadori glucose adducts is the predominant form of circulating glycated protein in vivo ( 3, 7 ), and its concentration is significantly increased in diabetes ( 31 ). In addition, glycated proteins are localized in the glomeruli of patients with diabetic nephropathy ( 30 ). Glycated albumin stimulates type IV collagen and fibronectin expression and upregulates the transforming growth factor- (TGF- ) system in cultured mesangial cells ( 2, 4 - 6, 46 ).
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The ECM can be degraded, and a distortion of the balance between ECM synthesis and turnover may result in an abnormal ECM accumulation in the mesangium in diabetic nephropathy. Plasminogen activator inhibitor-1 (PAI-1) is a component of the ECM and plays an important role in regulating blood coagulation and ECM accumulation. High media glucose concentration increases PAI-1 secretion ( 10 ) or activates the PAI-1 gene promoter in mesangial cells ( 11 ). However, the effects of glycated albumin on mesangial cell PAI-1 gene expression are unknown.
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PAI-1 gene transcription is activated by TGF- ( 9, 35, 39 ). TGF- signals through sequential activation of two cell-surface receptor serine-threonine kinases, which phosphorylate Smad2 and/or Smad3 ( 19, 22, 25, 40, 44 ). The phosphorylated Smads form heteromeric complexes with Smad4, which then translocate to the nucleus. In the nucleus, the Smad3-Smad4 complex can activate transcription through direct binding to DNA ( 9, 43 ).

Two different consensus sequences for Smad binding, such as GTCTAGAC (Smad-binding element) ( 43 ) and AG(C/A)CAGACAC (CAGA box) ( 9 ), have been described. Both sequences contain the core motif AGAC, which represents the optimal binding sequence for Smad3 and Smad4 ( 33, 43 ). CAGA boxes appear to be necessary and sufficient to mediate TGF- transcriptional effects ( 9 ), and human PAI-1 promoter contains three CAGA boxes ( 9, 12, 36 ).4 ~4 ^5 s4 R  M

We raised the possibility that glycated albumin influences PAI-1 gene expression in human mesangial cells (HMC) and that glycated albumin-induced PAI-1 expression is mediated by the TGF- /Smad signaling pathway. To address this question, we examined the effects of glycated albumin on TGF- 1 and PAI-1 mRNA expression, PAI-1 promoter activity, and DNA binding activity of Smad proteins in HMC.

MATERIALS AND METHODS; S/ y; p3 v% a( G" a" p5 ^

Reagents. Rabbit anti-Smad3 and anti-Smad4 were obtained from Zymed Laboratories (San Francisco, CA). Rabbit anti-Smad2/3, anti-actin, and anti-CREB-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies against Smad3 and Smad4 were kindly donated by Prof. Eui Yul Choi (Hallim Univ., Choon Chun, Korea). Monoclonal antibody against PAI-1 was obtained from York Biologicals (Stony Brook, NY). pGL3-basic vector, the luciferase assay system, and a -galactosidase enzyme assay kit were purchased from Promega (Madison, WI). Lipofectamine reagent and pCMV- -galactosidase vector (pCMV- gal) were obtained from Invitrogen (Carlsbad, CA). Nonidet P-40 was purchased from Sigma (St. Louis, MO). Other reagents were from sources as previously reported ( 14, 34 ).4 b8 T0 u. _7 L8 [' C

Culture of HMC and preparation of glycated albumin. HMC were obtained from adult nephrectomy specimens, as previously described ( 14, 15 ). The culture medium was made of DMEM supplemented with 20% fetal calf serum, 200 mM L -glutamine, and antibiotics. For the present experiments, cells from between passages 5 and 7 were used.
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Glycated BSA (Gly-BSA) was prepared by incubating 10 mg/ml BSA (fraction V) in PBS containing protease inhibitors and antibiotics for 7 days at 37°C in the presence of 0.3 mol/l D -glucose. Native BSA was processed under the same conditions in the absence of glucose. At the end of the incubation period, the solutions were dialyzed against PBS at 4°C for 24 h to remove unincorporated sugars and/or antibiotics and then sterile filtered. The extent of glycation of Gly-BSA, as measured by m -aminophenyl-boronate affinity chromatography ( 38 ), reached 76%. We measured bacterial endotoxin in BSA preparations using the Limulus amebocyte lysate test gel-clot method ( 1 ). No endotoxin was detected.- x0 [! A6 Q# G8 i: N

Experimental conditions. HMC were grown to confluency. The cells were synchronized to quiescence in serum-free DMEM containing 5 µg/ml insulin-transferrin-selenite for 48 h. After synchronization, experiments were performed by the addition of 200-1,000 µg/ml of Gly-BSA or BSA to the HMC for 12-72 h at 37°C. In given experiments, simultaneous control monolayers were treated with DMEM alone. In nondiabetic individuals, 1% of serum albumin is in the glycated form, which is equivalent to concentrations of 300-400 µg/ml of glycated albumin ( 46 ). Plasma Amadori albumin levels in patients with diabetic nephropathy are 2.5-fold higher than in healthy control subjects ( 31 ).8 V  O0 L( |5 N, q9 i
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Northern blot analysis. Isolated RNA samples were transferred onto nylon filters. The blotted membranes were incubated with the specific 32 P-labeled cDNA probes for human TGF- 1, PAI-1, and -actin as we previously described ( 14, 34 ). The filters were dried and exposed at -70°C using Agfa film (Agfa-Gevaert, Hortsel, Belgium). The mRNA levels for TGF- 1 and PAI-1 were expressed as a ratio of the optical density units for TGF- 1 or PAI-1 to -actin.# f9 N1 d# @# t& h2 a
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Western blot analysis. Cell lysates or nuclear extracts were electrophoretically resolved using a 10% polyacrylamide gel in an SDS buffer and then transferred onto nitrocellulose membranes as previously described ( 16 ). The blots were incubated in blocking solution for 1 h and incubated with rabbit anti-Smad3 and anti-Smad2/3 or mononclonal antibody against human PAI-1. Bound primary antibody was visualized after incubation with horseradish peroxidase-conjugated secondary antibody using an enhanced chemiluminescence system kit (Amersham, Arlington Heights, IL). To assess the equality of protein loading, the membrane was reprobed with anti-actin or anti-CREB-1 antibody (1:1,000 dilution).0 j2 `, m. c" f% E% L& s

Plasmid constructs. PAI-1 reporter vectors (740PAI-1-LUC) were generated using pGL3-basic plasmid (Promega) containing the firefly luciferase coding sequence. The oligonucleotides containing the sequence from -740 to  44 of the human PAI-1 promoter were prepared by PCR from genomic DNA and inserted between BgI II and Sac 1 cloning sites of pGL3-basic vector. The sequences of the oligonucleotide cloned were 5'-TTG-AGC-TGC-CCA-GAC-AAG-GTT-GTT-GAC-3' and its complementary strand 5'-CGA-GAT-CTG-TCT-TCT-TGA-CAG-CGC-TCT-TGG-3' ( 24 ).

Preparation of CAGA, mutant CAGA, and phosphorothioate CAGA oligonucleotides. The sequence of the double-strand CAGA oligonucleotides was 5'-TCG-AGA-GCC-AGA-CAA-AAA-GCC-AGA-CAT-TTA-GCC-AGA-CAC-3' and its complementary strand ( 9 ). The sequence of the mutant CAGA oligonucleotides was 5'-TCG-AGA-GCT-ACA-TAA-AAA-GCT-ACA-TAT-TTA-GCT-ACA-TAC-3'and its complementary strand ( 9 ).
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Oligonucleotides with modified phosphodiester bonds, such as phosphorothioate, are relatively resistant to nucleases and have been used as antisense agents ( 20 ). In this respect, CAGA antisense analog was prepared by attaching phosphorothioate to each end of CAGA oligonucleotides.; t, I# d" |7 J9 P* m+ |

Transfection and luciferase assays. Cells were grown in six-well plates to 80% confluence. The cells were transfected with 2 µg of DNA (1.5 µg of PGL3-PAI-1 construct and 0.5 µg of pCMV- gal) mixed with Lipofectamine according to the manufacturer's instructions. After 6 h of transfection, cells were serum starved for 8 h before incubation with BSA or Gly-BSA for 24 h. Then, cells were lysed on ice in 100 mM KH 2 PO4, pH 7.9, and 0.5% Triton X-100 and centrifuged. Luciferase and -galactosidase assays were performed with reagents from Promega. Luciferase activity was normalized to -galactosidase activity.2 t6 Y4 l3 U: t+ S( E$ v9 \+ q

Preparation of nuclear extracts. Nuclear extracts were prepared as previously described by Schreiber et al. ( 32 ). Briefly, the cells were washed with Tris-buffered saline (TBS), centrifuged, and resuspended in 400 µl cold buffer A [(in mM) 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol, 0.5 PMSF]. After a 15-min incubation on ice, 25 µl of a 10% solution of nonionic detergent Nonidet P-40 were added. After centrifugation, the nuclear pellet was resuspended in 50 µl ice-cold buffer B (20 HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and the tube was rocked at 4°C for 15 min on a shaking platform. The nuclear extracts were centrifuged, and the supernatant was frozen in aliquots. Protein content was measured using bicinchoninic acid with BSA as a standard.

EMSA. The human PAI-1 promoter construct and CAGA oligonucleotides described above were end-labeled with [ - 32 P]ATP using the T4 polynucleotide kinase. Five micrograms of nuclear extracts were incubated for 30 min at 4°C with 40,000 cpm of 32 P-labeled probes in 10 mM HEPES, pH 7.9, 5% glycerol, 0.1 mM EDTA, 1 mM DTT, and 1 µg of poly(deoxyinosin-deoxycytosin). The DNA-protein complexes were separated on a 5% polyacrylamide gel in 0.5 x TBE buffer and visualized by autoradiography.% {4 b1 T% i4 E# q& R% g7 J: C
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For competition and supershift assays, nuclear extracts were preincubated with a 100-fold molar excess of unlabeled CAGA oligonucleotides or 10 µl of mouse monoclonal anti-Smad3 or anti-Smad4 for 30 min or overnight before addition of the labeled probe./ S! r% ]: l3 D& j

Statistics. Results were expressed as means ± SD of three separate experiments. Results were analyzed by the two-way ANOVA for three groups or by Wilcoxon's rank sum test between two groups. A P value of

RESULTS9 m1 G; Q' V( h8 c5 D: q

Gly-BSA stimulates TGF- 1 mRNA expression. Twelve to twenty-four hours after incubation with HMC, Gly-BSA at a concentration of 200 µg/ml significantly increased TGF- 1 mRNA synthesis to 1.8 times that in the BSA-treated control cells ( Fig. 1 ).
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Fig. 1. Northern blot analysis of transforming growth factor- 1 (TGF- 1 ) mRNA in human mesangial cells (HMC). Cells were incubated with 200 µg/ml of BSA ( lane 2 ) or glycated BSA (Gly-BSA; lane 3 ) or DMEM alone ( lane 1 ) for 12 or 24 h. The blots were hybridized with 32 P-labeled cDNAs for TGF- 1 ( A ) and -actin ( B ). C : quantitative expression of TGF- 1 mRNA abundance after correction for -actin signal. Values are means ± SD of 3 separate experiments expressed as percent increases above mRNA levels of DMEM-treated controls. * P . R" I6 K$ z* P# e8 L) U* V
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Gly-BSA increases PAI-1 expression and PAI-1 gene promoter activity. Gly-BSA at a concentration of 200 µg/ml significantly increased PAI-1 mRNA transcripts to 3.2 times the levels in the BSA-treated cells at 24 h, but this effect disappeared at 48 and 72 h ( Fig. 2 ). When a dose-response of Gly-BSA was tested at 24 h, no significant difference was observed between 200 and 1,000 µg/ml Gly-BSA in PAI-1 mRNA expression (data not shown).& X+ g% @1 u+ V6 O1 ~8 r. o
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Fig. 2. Northern blot analysis of plasminogen activator inhibitor-1 (PAI-1) mRNA in HMC. Cells were incubated with 200 µg/ml of BSA (-) or Gly-BSA ( ) for 24, 48, or 72 h. The blots were hybridized with 32 P-labeled cDNAs for PAI-1 ( A ) and -actin ( B ). C : quantitative expression of PAI-1 mRNA abundance after correction for -actin signal. Values are means ± SD expressed as perecent increases of mRNA levels of Gly-BSA-treated HMC above those of BSA-treated controls. * P   m' j/ E3 U; \( ?  w* S
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Incubation of HMC with 200 µg/ml of Gly-BSA for 24 h increased PAI-1 protein expression 1.5 times the control levels, as assessed by Western blotting ( Fig. 3 ).
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Fig. 3. Western blot analysis of PAI-1 from cells treated with 200 µg/ml BSA ( lane 1 ) or Gly-BSA ( lane 2 ) for 24 h. Membranes were probed with anti-PAI-1 ( A ) and anti-actin ( B ). C : quantitative expression of PAI-1 after correction for actin signal. Values are means ± SD of 3 comparable experiments expressed as the relative ratio of PAI-1 density in Gly-BSA-treated cells to that in cells incubated with BSA, assigned an arbitrary value of 1. * P
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In cells transfected with the 740PAI-1-LUC construct, containing the sequence from -740 to  44 of the human PAI-1 promoter in front of the luciferase reporter gene, Gly-BSA treatment for 48 h increased luciferase activity 2.3 times compared with that in BSA-treated cells ( Fig. 4 ).
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Fig. 4. Stimulation of PAI-1 promoter activity by Gly-BSA. Cells were transiently transfected with a luciferase reporter gene containing the PAI-1 promoter (740PAI-1-LUC; -740 to  44) and were treated for 48 h with 50-200 µg/ml of BSA or Gly-BSA. Values are means ± SD of 3 separate experiments expressed as fold-stimulation by Gly-BSA compared with BSA-treated cells. * P
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Gly-BSA increases nuclear Smad2/3 and Smad3 expression in HMC. Because activated Smads are translocated into the nucleus, we measured the expression of Smads using nuclear extracts by Western blotting. Incubation of HMC with 200 µg/ml of Gly-BSA for 24 h increased Smad2/3 or Smad3 expression 1.6-2.5 times the control levels in the nuclei ( Fig. 5 ). When we tested a dose-response of Gly-BSA at 24 h, no significant difference was found between 200 and 1,000 µg/ml Gly-BSA in nuclear Smad3 expression (data not shown).

Fig. 5. Western blot analysis of Smad2/Smad3 and Smad3 in nuclear extracts from cells treated for 24 h with 200 µg/ml BSA ( lane 1 ) or Gly-BSA ( lane 2 ). Membranes were probed with anti-Smad2/Smad3 ( A ), anti-Smad3 ( B ), and anti-CREB-1 ( C ). D : quantitative expression of Smad2/Smad3 and Smad3 after correction for CREB-1 signal. Values are means ± SD of 3 comparable experiments expressed as relative ratio of Smad2/Smad3 or Smad3 density in cells incubated with Gly-BSA to that in cells incubated with BSA, assigned an arbitrary value of 1. * P

Anti-TGF- attenuates Gly-BSA-induced increases in nuclear Smad3 expression. When cells were incubated with pan-specific anti-TGF- antibody (25 µg/ml) along with Gly-BSA for 24 h, the expected increase in Smad3 expression was significantly reduced. However, the same concentration of control rabbit IgG had no effect on the Gly-BSA-induced increase in Smad3 levels ( Fig. 6 ).

Fig. 6. Western blot analysis of Smad3 in nuclear extracts from cells treated for 24 h with 200 µg/ml BSA alone ( lane 1 ), with addition of 200 µg/ml Gly-BSA (GA; lane 2 ), both Gly-BSA and anti-TGF- (GA anti-TGF-; lane 3 ), or both Gly-BSA and IgG (GA IgG; lane 4 ). Membranes were probed with anti-Smad3 ( A ) and anti-CREB-1 ( B ). C : quantitative expression of Smad3 after correction for CREB-1 signal. Values are means ± SD of 3 separate experiments expressed as relative ratio of Smad3 density in cells incubated with Gly-BSA to that in cells incubated with BSA, assigned an arbitrary value of 1. * P
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Gly-BSA increases the DNA/protein-binding complexes. An EMSA was performed using a -740 to  44 PAI-1 promoter construct or an oligonucleotide containing three CAGA boxes as a probe. Nuclear extracts were prepared from HMC exposed to 200 µg/ml of BSA or Gly-BSA for 24 h. DNA/protein complexes were identified, and their intensity significantly increased in Gly-BSA-treated cells vs.BSA-treated cells ( Fig. 7 A, top, lanes 1 and 2 ).% g1 a( i8 {, c1 K, H

Fig. 7. EMSA using a labeled CAGA sequence probe and nuclear extracts from cells treated with 200 µg/ml BSA ( lane 1, A ) or Gly-BSA ( lane 2, A; all lanes, B and C ) for 24 h. DNA/protein complexes are identified (arrows). For competition experiments, nuclear extracts were preincubated with a 100-fold molar excess of unlabeled CAGA oligonucleotides ( lane 2, B ). Anti-Smad3 ( lane 3, B; lane 2, C ) or anti-Smad4 ( lane 4, B ) antibody was preincubated with nuclear extracts before being mixed with labeled probe. The supershifted complex is indicated (arrowheads). Addition of normal mouse IgG did not affect formation of these complexes ( lane 3, C )." @, s) A" J0 p* j6 S

Smad3 is present in the DNA/protein complexes and binds to the CAGA box in the PAI-1 promoter. To demonstrate the presence of Smad3 or Smad4 in the DNA/protein complexes, monoclonal antibody against Smad3 or Smad4 was incubated with nuclear extracts from Gly-BSA-treated cells before addition of the radiolabeled probe. Addition of anti-Smad3 resulted in a slower migrating complex, whereas formation of DNA/protein complex was completely inhibited [ Fig. 7, B ( lane 3 ) and C ( lane 2 )]. With anti-Smad4, DNA/protein complex formation was also inhibited, although no supershifted complex was detected ( Fig. 7 B, lane 4 ). As expected, normal mouse IgG did not affect formation of these complexes ( Fig. 7 C, lane 3 ).- K0 f' R0 y% L$ _8 E
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To prove that the Smad3-binding sequences within the PAI-1 promoter are CAGA boxes, CAGA oligonucleotides were used as cold competitors in the EMSA. When nuclear extracts were preincubated with 100-fold molar excess of unlabeled CAGA oligonucleotides before addition of radiolabeled probe, the complex formation was prevented ( Fig. 7 B, lane 2 ). These results suggest that Gly-BSA increases the binding activity of Smad3 to the CAGA box within the PAI-1 promoter.3 U" K/ d8 B  z4 V, M8 T
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Gly-BSA increases PAI-1 transcription mediated by the CAGA sequences. To examine whether Gly-BSA-induced PAI-1 transcription is mediated through Smad-binding CAGA sequences, phosphorothioate CAGA oligonucleotides and mutant CAGA oligonucleotides were transfected into HMC. Transfection of cells using 32 P-labeled phosphorothioate CAGA oligonucleotides resulted in strong radioactivity, confirming their successful transfection. On Gly-BSA treatment, phosphorothioate CAGA oligonucleotide-transfected cells exhibited significantly reduced PAI-1 mRNA expression compared with nontransfected cells or mutant CAGA oligonucleotide-transfected cells ( Fig. 8 ). Luciferase activity on Gly-BSA treatment was significantly decreased in cells cotransfected with phosphorothioate CAGA oligonucleotide and 740PAI-1-LUC compared with those transfected with 740PAI-1-LUC alone ( Fig. 9 ).
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Fig. 8. Northern blot analysis of PAI-1 mRNA in HMC without ( lanes 1 and 2 ) or with transfection of phosphorothioate CAGA oligonucleotide (P-CAGA; lane 3 ) or mutant CAGA oligonucleotide (M-CAGA; lane 4 ). Cells were incubated with 200 µg/ml of BSA ( lane 1 ) or Gly-BSA (GBSA; lanes 2-4 ) for 24 h. Blots were hybridized with 32 P-labeled cDNAs for PAI-1 ( A ) and -actin ( B ). C : quantitative expression of PAI-1 mRNA abundance after correction for the -actin signal. Values are means ± SD of 3 separate experiments expressed as in Fig. 2. * P 9 t; q% c. {4 |! [6 ]

Fig. 9. Luciferase activity in HMC transfected with 740PAI-1-LUC alone (BSA or GBSA) or cotransfected with phosphorothioate CAGA oligonucleotide (GBSA P-CAGA), or phosphorothioate CAGA oligonucleotide alone (P-CAGA), which were then treated for 24 h with 200 µg/ml BSA (BSA) or GBSA (GBSA or GBSA P-CAGA) or DMEM (P-CAGA). Values are means ± SD of 3 separate experiments. * P

DISCUSSION1 |# q/ n5 i+ }' `; ]: d8 d: w

This study demonstrates, to the best of our knowledge for the first time, that Gly-BSA increases PAI-1 mRNA expression, PAI-1 promoter activity, and DNA-binding activity of Smad3 in HMC. Furthermore, it shows that the Gly-BSA-induced PAI-1 gene upregulation is mediated through Smad3-binding CAGA sequences in the PAI-1 promoter.

We found that treatment of mesangial cells with Gly-BSA at a concentration of 200 µg/ml increased steady-state PAI-1 mRNA levels at 24 h. We also observed that Gly-BSA increased transiently transfected PAI-1 promoter activity compared with that in BSA-treated cells. These findings are similar to results with mesangial cells incubated with high ambient glucose ( 11 ). However, we could not observe a dose-response effect in PAI-1 mRNA and Smad3 expression between 200 and 1,000 µg/ml Gly-BSA, suggesting that concentrations of glycated albumin to activate the TGF- /signaling pathway in cultured mesangial cells might be lower than those found in clinical specimens. Ziyadeh et al. ( 46 ) also observed a minimal or negligible difference between 200 and 600 µg/ml glycated albumin in inducing fibronectin and TGF- 1 mRNA expression.: }& ?% C: t8 x* `5 e5 G
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In agreement with the report of Ziyadeh et al. ( 46 ), we found that Gly-BSA increased TGF- 1 mRNA expression. Increased glomerular TGF- 1, which can be induced by either elevated glucose or glycated albumin in kidneys of diabetic animals ( 42 ), stimulates the production of fibronectin and type IV collagen ( 46 ). Similarly, Gly-BSA-induced TGF- 1 upregulation observed in this study could increase mesangial PAI-1 overproduction, which can be linked to the development of mesangial matrix accumulation.
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In the present study, incubation of Gly-BSA with HMC increased nuclear Smad2 and Smad3 protein levels compared with those in BSA-treated cells. Although high-glucose-induced phosphorylated Smad2 expression was demonstrated in the nuclei of mesangial cells by immunohistochemistry ( 18 ), no study has shown that glycated albumin can induce enhanced nuclear Smad2 and Smad3 expression in mesangial cells. Our results with increased nuclear Smad2 and Smad3 expression by Gly-BSA suggest that glycated albumin can activate Smad signaling in mesangial cells with translocation of Smad complexes into nuclei. Furthermore, anti-TGF- significantly attenuated the Gly-BSA-induced increases in nuclear Smad3 expression in this study, suggesting an intermediate role for TGF- in the effects of the Gly-BSA-induced Smad pathway.

Our EMSA results demonstrate that Gly-BSA significantly increased the intensity of DNA/protein (binding) complexes within the -740 to  44 region of the PAI-1 promoter compared with BSA. We also found that the PAI-1 promoter- or CAGA sequence-binding protein was Smad3 by antibody supershift, confirming the report of Dennler et al. ( 9 ). Furthermore, we observed that formation of the DNA/protein complex was also inhibited with anti-Smad4, suggesting that Smad4 can also bind to the CAGA boxes. However, a supershifted complex was not detected with anti-Smad4, possibly because the amounts of Smad4 bound to the complex are too small to be detected.
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Smad2 cannot bind directly to DNA compared with Smad3 ( 23, 41 ). Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice ( 45 ). In cultured mesangial cells, Smad3 overexpression increases fibronectin promoter activity ( 13 ), and Smad3 has been found to be involved in TGF- -induced stimulation of the 2 (I) collagen ( 26, 27 ). Although Smad4 is essential for TGF- -induced 1 (I) collagen expression in mesangial cells, TGF- -induced fibronectin expression and sustained TGF- -induced PAI-1 expression are independent of Smad4 ( 37 ). These reports together with our observations showing Gly-BSA-induced increased Smad3/DNA interactions suggest that Smad3 plays a key role in intracellular Smad signaling and ECM accumulation.

In our study, transfection of phosphorothioate CAGA oligonucleotides, a CAGA antisense analog, inhibited Gly-BSA-induced PAI-1 mRNA expression. Cotransfection of the phosphorothioate CAGA oligonucleotide with the PAI-1 promoter construct also blocked Gly-BSA-induced luciferase activity. These results suggest that CAGA boxes in the PAI-1 promoter could mediate Gly-BSA-induced PAI-1 promoter activation and PAI-1 gene transcription in HMC.

In the present study, the increase in Smad/DNA binding could explain the increased PAI-1 promoter activity induced by Gly-BSA. CAGA boxes themselves might be sufficient to mediate TGF- transcriptional effects ( 9 ), although Smads must cooperate with other transcription factors to effect the transcription of a target gene ( 8, 21 ).

Increased levels of glycated albumin seem to be related to multiple diabetic complications ( 31 ). Upregulation of PAI-1 gene expression by glycated albumin may be relevant to diabetic nephropathy. Not only high glucose ( 10, 11 ) but also low-density lipoprotein ( 34 ), mediators of diabetic nephropathy, stimulate expression of PAI-1 in mesangial cells. PAI-1 has been implicated in renal disease as being a mediator of ECM accumulation ( 28 ) and as a feedback mechanism to limit vascular fibrinolysis ( 17, 29 ). Our results describing the Gly-BSA-induced transcriptional activation of PAI-1 suggest that increased circulating glycated albumin levels in diabetic patients could lead to a mesangial ECM accumulation and to eventual renal fibrosis partly linked to impaired matrix degradation.5 S5 d0 ~7 L3 _

In summary, our results show that Gly-BSA increases DNA binding activity of Smad3 and that it stimulates PAI-1 transcription through Smad-binding CAGA boxes in the PAI-1 promoter in HMC. Thus progression of diabetic nephropathy may be promoted by PAI-1 upregulation mediated by the glycated albumin-induced Smad/DNA interactions.
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ACKNOWLEDGMENTS: R) P7 e: ?& x/ m( N" K& l! i

Present address of S. Wang: Dept. of Pathology, Peking University, Beijing, China.
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