Synergistic effect of hypoxia and TNF-alpha on production of PAI-1 in human proximal renal tubular cells.
BACKGROUND
Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-alpha (TNF-alpha) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs).


METHODS
Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O(2) without TNF-alpha). Hypoxia-inducible factor-1alpha (HIF-1alpha) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay.


RESULTS
In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-alpha, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-alpha. Luciferase reporter gene assay showed that hypoxia, TNF-alpha, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1alpha significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-kappaB (NF-kappaB) caused a moderate decrease in PAI-1 production under hypoxia.


CONCLUSIONS
Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha and partially via NF-kappaB activation in HPTECs. TNF-alpha can synergistically enhance this hypoxia-induced PAI-1 expression.
Li
Xuan
X
Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, Fukui University, Fukui, Japan.
Kimura
Hideki
H
Hirota
Kiichi
K
Kasuno
Kenji
K
Torii
Kunio
K
Okada
Toshiharu
T
Kurooka
Hisanori
H
Yokota
Yoshifumi
Y
Yoshida
Haruyoshi
H
D000970:Antineoplastic Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004357:Drug Synergism; D005786:Gene Expression Regulation; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D010100:Oxygen / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects; D013859:Thiocarbamates / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000494:pharmacology*
D000970:Antineoplastic Agents; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D017395:Plasminogen Activator Inhibitor 1; D013859:Thiocarbamates; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C066229:prolinedithiocarbamate; D011392:Proline; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010100:Oxygen