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


CONCLUSIONS
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.
Song
Chi Young
CY
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
Kim
Bong Cho
BC
Hong
Hye Kyoung
HK
Lee
Hyun Soon
HS
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005920:Glomerular Mesangium / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008077:Lipoproteins, LDL; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C099252:oxidized low density lipoprotein; D004247:DNA