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AP-1 inhibits expression of MMP-2/9 and its effects on rat smooth muscle cells
Hong-Wei Zhang1, Xin Wang, Zhi-Hong Zong
1Veteran Carder Clinic, the First Affiliated Hospital, China Medical University, Liaoning, China.
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 alpha-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-beta1 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.
