Low-molecular-weight heparin prevents high glucose- and phorbol ester-induced TGF-beta 1 gene activation.
BACKGROUND
Hyperglycemia-induced overexpression of prosclerotic transforming growth factor-beta 1 (TGF-beta 1) has been implicated in the pathogenesis of diabetic nephropathy. Since previous in vivo studies demonstrated a renoprotective effect of low-molecular-weight (LMW) heparin in experimental animals, and recent in vitro data showed an interaction of this drug with the overactivated TGF-beta 1 cascade in high glucose- and phorbol ester-stimulated mesangial cells, we studied the molecular mechanism of these effects on TGF-beta 1 gene expression.


METHODS
Mesangial cells were stimulated with 30 mmol/L glucose or with 0.5 micromol/L phorbol ester [phorbol myristate acetate (PMA)] in the absence or presence of LMW heparin. TGF-beta 1 promoter activity was determined in promoter-reporter luciferase assays. The effect of LMW heparin on the binding of nuclear proteins to a regulatory activator protein-1 (AP-1) site, which mediates the high glucose and PMA responsiveness of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays.


RESULTS
The presence of LMW heparin completely prevented TGF-beta 1 gene activation in both high glucose- and PMA-stimulated cells. Preincubation of the cells with LMW heparin and subsequent stimulation of the cells with both stimuli yielded the same result. Furthermore, treatment with LMW heparin prevented the enhanced binding of nuclear proteins to the regulatory AP-1 site, while binding to a Sp1 site was unaffected. Basal promoter activity and basal AP-1 binding also was reduced by LMW heparin. The LMW heparin effect on basal promoter activity was abolished by mutation of the regulatory AP-1 box B and by deletion of this AP-1 binding site.


CONCLUSIONS
LMW heparin prevents high glucose- and PMA-mediated TGF-beta 1 expression by inhibiting the activation of the TGF-beta 1 promoter and by preventing the enhanced binding of nuclear proteins to the regulatory AP-1 site.
Weigert
C
C
Department of Internal Medicine, Division of Endocrinology, Metabolism and Pathobio-chemistry, University of Tübingen, Tübingen, Germany.
Brodbeck
K
K
Häring
H U
HU
Gambaro
G
G
Schleicher
E D
ED
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005786:Gene Expression Regulation / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology*; D010703:Phorbol Esters / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D013552:Swine; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D053773:Transforming Growth Factor beta1
D006495:Heparin, Low-Molecular-Weight; D010703:Phorbol Esters; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D005947:Glucose