AP-1 Inhibits Expression of MMP-2/9 and Its Effects on Rat Smooth Muscle Cells
Received 5 December 2008 published online 16 March 2009.
Background
We designed AP-1 small interfering RNA (siRNA) to study the relationship between AP-1 expression and matrix metalloproteinase (MMP)-2 activation.
Methods
To suppress the expression of AP-1 gene, we used RNA interference to silence the AP-1 gene post-transcriptionally in rat smooth muscle cells. Effects of AP-1 siRNA on mRNA expression of AP-1 were examined using reverse transcriptase polymerase chain reaction (RT-PCR). Phosphorylation levels of c-Jun, c-Fos, and the activity of AP-1 were determined by Western blotting and AP-1 DNA-binding activity assay. To observe the expression of SM α-actin and downstream genes of AP-1, we simultaneously examined the activity of cell matrix metal proteinases and the migration ability of smooth muscle cells using a modified Boyden-chamber assay. Effects of AP-1 siRNA on rat vascular smooth muscle cells (VSMC) in vitro were evaluated using cell cycle analysis, MTT-tests, BrdU-ELISA and immunofluorescence.
Results
AP-1 siRNA decreased not only AP-1 mRNA and AP-1 binding activity, but also c-Jun and c-Fos phosphorylation levels and the expression levels of urokinase-type plasminogen (u-PA), MMPs, TGF-β1 and bFGF in VSMCs. The AP-1 siRNA also significantly inhibited PDGF/IL-1-induced MMP-2 and MMP-9 gelatinolytic activity in VSMCs. The invasion and migration of VSMC were also induced greatly.
Conclusions
AP-1 inhibited expression of MMP-2/9 and AP-1 siRNA was able to effectively inhibit the proliferation, migration and de-differentiation of rat smooth muscle cells.
∗Veteran Carder Clinic, the First Affiliated Hospital, China Medical University, Liaoning, China
†Department of Biochemistry, China Medical University, Liaoning, China
To whom correspondence and reprint requests should be addressed at Veteran Carder Clinic, The First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China.
ABSTRACT