Inhibition of apoptosis in rat mesangial cells by tissue inhibitor of metalloproteinase-1.
BACKGROUND
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important inhibitor of extracellular matrix degradation. Recently, it was reported that TIMP-1 also could inhibit apoptosis in B type lymphocyte. This study was designed to examine the effects of TIMP-1 on mesangial cell apoptosis.


METHODS
The full-length cDNA of TIMP-1 was cloned and used to construct two recombinant vectors, TIMP-1S and TIMP-1AS, encoding sense TIMP-1 and antisense TIMP-1, respectively. The vectors were transfected into rat mesangial cells (RMC) and their expressions detected by Northern and Western blotting. Apoptosis was induced by serum deprivation, and was monitored for DNA fragmentation by TUNEL assay and DNA laddering. In addition, the expression of endogenous TIMP-1, matrix metalloprotein-2 (MMP-2), and MMP-9, as well as apoptosis-related genes Bcl-2 and Bax were investigated.


RESULTS
TIMP-1AS transfection induced a suppression of TIMP-1 expression accompanied by an earlier onset of apoptosis, and TIMP-1S transfection induced TIMP-1 over-expression accompanied by a much later onset of apoptosis. A neutralizing antibody of TIMP-1 restored the sensitivity of TIMP-1S-transfected RMC to serum deprivation, but a synthetic matrix metalloproteinase inhibitor BB-94 did not influence the sensitivity of TIMP-1S-transfected RMC to serum deprivation. Finally, TIMP-1 over-expression inhibited the expression of Bax but with no effect on the expression of Bcl-2.


CONCLUSIONS
TIMP-1 inhibits the serum deprivation-induced apoptosis in RMC, in which Bax might be involved.
Lin
Hongli
H
Department of Nephrology, Kidney Center & Key Lab of Chinese PLA, General Hospital of Chinese PLA, 28 Fu-xing Road, Beijing 100853, People's Republic of China.
Chen
Xiangmei
X
Wang
Jianzhong
J
Yu
Zhiheng
Z
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005920:Glomerular Mesangium / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013876:Thiophenes / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000502:physiology*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013876:Thiophenes; D019715:Tissue Inhibitor of Metalloproteinase-1; D051028:bcl-2-Associated X Protein; D010649:Phenylalanine; C080985:batimastat; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9