AP-1 inhibits expression of MMP-2/9 and its effects on rat smooth muscle cells.
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.
Zhang
Hong-Wei
HW
Veteran Carder Clinic, the First Affiliated Hospital, China Medical University, Liaoning, China.
Wang
Xin
X
Zong
Zhi-Hong
ZH
Huo
Xin
X
Zhang
Qiang
Q
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000201:enzymology*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D017208:Rats, Wistar; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D016760:Proto-Oncogene Proteins c-fos; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9