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Matrix metalloproteinase-12 gene expression in human vascular smooth muscle cells
Lihua Wu1, Akihide Tanimoto, Yoshitaka Murata
1Department of Pathology, Institute of Basical Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Background:
Matrix metalloproteinases (MMPs) play an important role in smooth muscle cell (SMC) migration and proliferation during vascular remodelling. To investigate the expression of MMP-12 by SMCs, we examined the protein secretion and mRNA expression of MMP-12 by cultured medial SMCs and intimal SMCs derived from human aortic atherosclerotic lesions. To further elucidate the molecular mechanism for MMP-12 expression in SMCs, we determined the sequence requirements for MMP-12 gene transcriptional activity.
Results:
Cultured medial SMCs and intimal SMCs showed substantial MMP-12 expression at both the protein and mRNA levels. A series of 5'-deletion and site-directed mutants of the human MMP-12 promoter demonstrated that an AP-1 site spanning -81 to -75 bp was critical for the MMP-12 promoter activity in SMCs. An electrophoretic mobility shift assay confirmed the AP-1 binding activity in SMCs and showed that the protein bound to the AP-1 site consisted predominantly of c-Jun, JunD and Fra-1. Two structurally different inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002, inhibited MMP-12 transcriptional activity and AP-1 binding.
Conclusion:
These results indicated the expression of MMP-12 in vascular SMCs and showed that the MMP-12 gene expression was dependent on the AP-1 binding activity. Phosphatidylinositol 3-kinase signalling may be involved in MMP-12 transcriptional activation through AP-1 binding activity.
Insights
Vascular smooth muscle cells express matrix metalloproteinase-12 (MMP-12), with its gene expression regulated by AP-1 binding activity. Phosphatidylinositol 3-kinase signaling pathways may influence MMP-12 transcription in these cells.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Cell Biology
Background:
- Matrix metalloproteinases (MMPs) are crucial in vascular remodeling, influencing smooth muscle cell (SMC) migration and proliferation.
- Investigating the role and regulation of MMP-12 in human aortic atherosclerotic lesions is important for understanding vascular disease.
Purpose of the Study:
- To examine MMP-12 protein secretion and mRNA expression in cultured medial and intimal SMCs from human atherosclerotic lesions.
- To elucidate the molecular mechanisms governing MMP-12 gene transcription in SMCs.
Main Methods:
- Analysis of MMP-12 protein and mRNA levels in cultured SMCs.
- Utilized 5'-deletion and site-directed mutants of the human MMP-12 promoter to identify critical regulatory elements.
- Performed electrophoretic mobility shift assays (EMSA) to assess AP-1 binding activity.
- Investigated the effect of phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin, LY294002) on MMP-12 transcription and AP-1 binding.
Main Results:
- Cultured medial and intimal SMCs demonstrated significant MMP-12 expression at both protein and mRNA levels.
- A specific AP-1 binding site (-81 to -75 bp) within the MMP-12 promoter was identified as critical for transcriptional activity in SMCs.
- EMSA confirmed AP-1 binding in SMCs, with c-Jun, JunD, and Fra-1 identified as the predominant proteins involved.
- Inhibition of PI3K with wortmannin and LY294002 suppressed MMP-12 transcriptional activity and AP-1 binding.
Conclusions:
- MMP-12 is expressed in vascular SMCs, and its gene expression is dependent on AP-1 binding activity.
- The findings suggest that PI3K signaling pathways are involved in the transcriptional activation of MMP-12 through AP-1 binding.