Abstract
Background: ADAM15 is a membrane-bound member of the adamalysin family that is up-regulated in areas of tissue remodelling. Previous studies have demonstrated the role of ADAM15 in mesangial cell migration, which is integral in tissue remodelling in pathology and repair. The current study was designed to identify and analyse the genomic regions upstream of ADAM15 that would regulate its transcription. Methods: Using 5′-RACE and RT-PCR, the ADAM15 5′-UTR was extended and luciferase constructs assembled to examine the transcription start site and characterise the promoter region of this gene. Results: A 145-bp proximal promoter construct showed full activity in unstimulated cells. Analysis of this region identified three potential Sp1-binding sites. Electromobility and supershift assays confirmed that Sp1 was constitutively present in MC nuclei. Mutations in each Sp1 site confirmed each was needed for full activity, while mutation of all three sites abrogated luciferase activity demonstrating that Sp1 was involved in the promoter activity of ADAM15. Methylation of this promoter fragment abolished the activity, while the methyltransferase inhibitor 5-aza-3′-deoxycytidine showed no increased activity in transfected cells, implying that the promoter was not methylated in our cells. Conclusion: These results demonstrate the intrinsic promoter activity of ADAM15 in quiescent MC and show the involvement of Sp1 in its regulation.
Journal Section:
Original Paper
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