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A. P. Surguchov, K. Cain, I. Surgucheva; New Structural and Regulatory Properties of Matrix Metalloproteinase 9. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1649. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recently we found that: 1. γ-Synuclein is highly expressed in retinal ganglion cells (RGC) and 2. γ-Synuclein is a potent upregulator of matrix metalloproteinase 9 (MMP-9) in several types of neurons. To study the molecular mechanisms of this phenomenon we investigated rat MMP-9 and found some new structural and regulatory properties of this enzyme.
RNA was isolated from immortalized RGC culture (RGC-5), astrocyte culture (A7), rat brain and retina, converted to cDNA by reverse transcriptase and used for PCR and qRT-PCR. Cultured media (CM) from RGC-5 and A7 was collected, concentrated and analyzed for MMP-9 by Western blotting or by in-gel activity with gelatin as a substrate. A fragment of 3’-untranslated region (3'-UTR) of MMP-9 was generated by PCR, inserted in the pGL3-control vector and the effect of this fragment on the efficiency of the luciferase gene transcription was analyzed by Dual-Luciferase Reporter Assay System (Promega).
We found that the fluorescent oligonucleotide probe designed for qRT-PCR to the junction of exon 12 and exon 13 produces normal amplification with cDNA from A7, but does not amplify cDNA from RGC-5, rat brain and retina. Similar results were obtained when we used an unlabeled probe and regular PCR. The sequencing of different fragments from the 3’-end of MMP-9 cDNA showed the presence of a part of the intron 12 sequence in cDNA from RGC-5 and brain, but not in astrocytes. These results point to a differential splicing of the MMP-9 in glial and neuronal cells. Further analysis of CM showed the presence of MMP9 of different molecular size in RGC-5 and A7. While the size of MMP9 from A7 coincides with the size of human MMP9, the size of MMP9 from RGC-5 is different. This data is supported with the analysis of gelatinase activity of the enzyme. We also found an in frame insertion of a fragment of 24 nucleotides in RGC-5 cDNA corresponding to eight amino acids in exon 13 of MMP-9. In addition, MMP-9's 3’-UTR from RGC-5 contains additional regulatory CA repeats absent in 3'-UTR of cDNA from A7. The analysis of luciferase activity shows that the 3’-UTR of MMP-9 possesses a position-dependent effect on the level of transcription.
This data supports the view that neuronal cells contain a new form of MMP-9 different in some structural and regulatory properties. These results are discussed in connection with the upregulation of MMP-9 by γ-synuclein, possible implication of MMP-9 in diseases and approaches to modulate its expression.
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