Expression of matrix metalloproteinase-9 associated with ets-1 proto-oncogene in rat tubulointerstitial cells.
BACKGROUND
Ets-1 proto-oncogene exhibits multiple activities in the transcriptional regulation of numerous genes including metalloproteinase (MMP)-1, -3 and -9. MMPs play an important role in the remodelling of extracellular matrix in various renal diseases. However, the role of the Ets-1-MMP axis in advanced renal diseases is uncertain. In the present study, we investigated whether Ets-1 is involved in interleukin (IL)-1-mediated expression of MMPs in tubulointerstitial cells.


METHODS
Rat renal fibroblasts (NRK-49F) and tubular epithelial cells (NRK-52E) were cultured and allocated to an IL-1beta-treated group (10 ng/ml), a platelet-derived growth factor (PDGF)-BB-treated group (25 ng/ml) and a control group. Protein and mRNA were extracted after 1, 6, 12 and 24 h of treatment. Parallel flasks were treated with 2 muM ets-1 antisense oligodeoxynucleotides (ODNs) before exposure to IL-1beta. The expression of Ets-1 protein was evaluated by western blotting. The activities of MMPs were evaluated by gelatin zymography. The expression of ets-1 and/or MMP-9 mRNA was evaluated semiquantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
In NRK-49F cells, Ets-1 protein increased significantly by 6.8-fold at 6 h, and MMP-9 activity increased significantly by 9.9-fold at 12 h in the IL-1beta-treated group compared with controls. MMP-2 and -3 activities also increased significantly in the IL-1beta-treated group. In NRK-52E cells, Ets-1 protein was 3.1 times higher at 1 h, and the latent form of MMP-9 activity increased 3.4-fold at 6 h in the IL-1beta group compared with controls. However, MMP-2 or MMP-3 activities were not markedly altered by IL-1beta treatment compared with controls. When the cells were treated with ets-1 antisense ODNs before IL-1beta treatment, Ets-1 protein expression decreased at least 50%, and MMP-9 activity was clearly inhibited in both cells. We also confirmed that MMP-9 activity was upregulated on days 21 and 28 in renal cortex of rat crescentic glomerulonephritis.


CONCLUSIONS
The Ets-1 transcriptional factor may participate in IL-1beta-mediated MMP-9 expression in tubulointerstitial cells.
Naito
Takashi
T
Department of Medicine, Tokyo Women's Medical University Daini Hospital, Arakawaku, Tokyo, Japan. takashi020304@yahoo.co.jp
Tanihata
Yoko
Y
Nishimura
Hideki
H
Tanaka
Toshihisa
T
Higuchi
Chieko
C
Taguchi
Takashi
T
Sanaka
Tsutomu
T
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004195:Disease Models, Animal; D015870:Gene Expression; D005921:Glomerulonephritis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007684:Kidney Tubules / Q000378:metabolism* / Q000473:pathology; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis / Q000235:genetics*; D050527:Mesangial Cells / Q000378:metabolism* / Q000473:pathology; D050784:Proto-Oncogene Protein c-ets-1 / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
C494043:Ets1 protein, rat; D050784:Proto-Oncogene Protein c-ets-1; D012333:RNA, Messenger; D020780:Matrix Metalloproteinase 9