[Effects of sense and antisense tissue inhibitor metalloproteinase-1 on apoptosis of rat glomerular mesangial cells and its related-gene expressions].
OBJECTIVE
To investigate the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) in apoptosis of rat glomerular mesangial cells (RMC) in vitro.


METHODS
TIMP-1 full length cDNA cloned in previous work was used to construct two recombinant plasmids, eukaryotic expression rector, pcDNA3-TIMP-1 (PTs) encoding sense TIMP-1 and pcDNA3-ATIMP-1 (PTas) encoding antisense TIMP-1. These two vectors were then transfected into RMC with lipofectin DOTAP. Northern blotting was used to detect the expression of sense and antisense TIMP-1. Apoptosis was induced in normal RMC, RMC transfected with pCDNA3, PTs and PTas by serum deprivation. Those cells were monitored for apoptosis over extended periods of time (12 h approximately 4 d) by TUNEL and DNA electrophoressis. Furthermore, the expression of apoptosis related-genes was also investigated by RT-PCR.


RESULTS
Northern blot analysis and RT-PCR demonstrated that cells transfected with exogenous genes effectively expressed sense or antisense TIMP-1 mRNA. RMC transfected with PTas underwent apoptosis after 12 h of serum deprivation, normal RMC and RMC transfected with pcDNA3 underwent apoptosis after 48 hours in serum-free medium, while RMC transfected with PTs did not undergo apoptosis until 4 days after serum deprivation. Furthermore, TIMP-1 upregulation inhibited expression of bax but had no effect on the expression of bcl-2.


CONCLUSIONS
TIMP-1 suppressed apoptosis of RMC, which suggests a novel activity of TIMP-1 in kidney. Antisense TIMP-1 promotes apoptosis in RMC. The effects of TIMP-1 on RMC apoptosis perhaps correlate with inhibition of bax expression.
Lin
H
H
Department of Nephrology, General Hospital and Kidney Center of Chinese People's Liberation Army, Beijing 100853, China.
Chen
X
X
Fu
B
B
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology*; D017209:Apoptosis; D015152:Blotting, Northern; D053938:DNA Fragmentation; D019254:Genes, bcl-2; D005920:Glomerular Mesangium / Q000166:cytology* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000502:physiology*; D014162:Transfection; D051028:bcl-2-Associated X Protein
D016375:Antisense Elements (Genetics); C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019715:Tissue Inhibitor of Metalloproteinase-1; D051028:bcl-2-Associated X Protein