Abstract
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. 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. 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. 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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
