NDT Advance Access originally published online on July 26, 2005
Nephrology Dialysis Transplantation 2005 20(11):2333-2348; doi:10.1093/ndt/gfi013
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Expression of matrix metalloproteinase-9 associated with ets-1 proto-oncogene in rat tubulointerstitial cells
1 Department of Medicine, Tokyo Women's Medical University Daini Hospital, Tokyo and 2 Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan
Correspondence and offprint requests to: Takashi Naito, MD, Department of Medicine, Tokyo Women's Medical University Daini Hospital, 2-1-10, Nishiogu, Arakawaku, Tokyo, 116-8567, Japan. Email: takashi020304{at}yahoo.co.jp
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-1ß-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 µM ets-1 antisense oligodeoxynucleotides (ODNs) before exposure to IL-1ß. 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-1ß-treated group compared with controls. MMP-2 and -3 activities also increased significantly in the IL-1ß-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-1ß group compared with controls. However, MMP-2 or MMP-3 activities were not markedly altered by IL-1ß treatment compared with controls. When the cells were treated with ets-1 antisense ODNs before IL-1ß 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-1ß-mediated MMP-9 expression in tubulointerstitial cells.
Keywords: ets-1 proto-oncogene; interleukin-1 (IL-1); matrix metalloproteinase-9 (MMP-9); MMP-2; MMP-3