Oxidized LDL activates PAI-1 transcription through autocrine activation of TGF-beta signaling in mesangial cells

Kidney Int. 2005 May;67(5):1743-52. doi: 10.1111/j.1523-1755.2005.00271.x.

Abstract

Background: Lipid abnormalities and oxidative stress may be involved in the development of glomerulosclerosis. Plasminogen activator inhibitor-1 (PAI-1) is a component of extracellular matrix (ECM) and target gene of transforming growth factor-beta (TGF-beta). Smad proteins play a key role in TGF-beta signaling, and Smad binding CAGA boxes are present in the PAI-1 promoter. This study examined whether oxidized low-density lipoprotein (Ox-LDL) activates PAI-1 transcription in human mesangial cells, mediated by increased Smad/DNA interactions.

Methods: Quiescent HMC were incubated with 50 microg/mL of Cu(++)-catalyzed Ox-LDL for 15 minutes to 4 hours, and the effects of Ox-LDL on TGF-beta1 and PAI-1 mRNA expression, PAI-1 promoter activity, and DNA binding activity of Smad proteins were examined.

Results: Ox-LDL induced TGF-beta1 and PAI-1 mRNA expression. Ox-LDL increased the transiently transfected PAI-1 promoter activity as compared with controls to 3.9-fold. Ox-LDL-treated cells increased Smad3 protein levels two times the control levels in the nuclei. Electrophoretic mobility shift assay (EMSA) performed using a CAGA sequence probe and nuclear extracts showed that Ox-LDL increased DNA/protein complexes. When nuclear extracts were preincubated with 100 molar excess of unlabeled CAGA oligonucleotide or SB-431542, an inhibitor of the TGF-beta type I receptor, the formation of complex was prevented. The DNA binding protein was shown to be Smad3 by antibody supershift. Transfection of phosphorothioate CAGA oligonucleotides, which compete with the CAGA-containing PAI-1 promoter for Smad3 binding, inhibited the Ox-LDL-induced PAI-1 mRNA expression. Cotransfection of phosphorothioate CAGA oligonucleotides with PAI-1 reporter vector also blocked the Ox-LDL-induced PAI-1 promoter activity.

Conclusion: These results suggest that Ox-LDL activates TGF-beta/Smad signaling to stimulate PAI-1 transcription in human mesangial cells. Thus, progression of glomerular disease may be promoted by PAI-1 up-regulation in human mesangial cells mediated by the Ox-LDL-induced TGF-beta/Smad signaling pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites / genetics
  • Cells, Cultured
  • DNA / genetics
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism
  • Glomerular Mesangium / drug effects*
  • Glomerular Mesangium / metabolism*
  • Humans
  • Lipoproteins, LDL / pharmacology*
  • Plasminogen Activator Inhibitor 1 / genetics*
  • Promoter Regions, Genetic
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Signal Transduction / drug effects
  • Smad3 Protein
  • Trans-Activators / metabolism
  • Transcriptional Activation / drug effects
  • Transfection
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism*

Substances

  • DNA-Binding Proteins
  • Lipoproteins, LDL
  • Plasminogen Activator Inhibitor 1
  • RNA, Messenger
  • SMAD3 protein, human
  • Smad3 Protein
  • Trans-Activators
  • Transforming Growth Factor beta
  • oxidized low density lipoprotein
  • DNA