Abstract
Purpose: :
Matrix metalloproteinase–9 (MMP–9) is involved in remodeling of corneal epithelium during the wound healing. We have previously shown that platelet–activating factor (PAF) upregulates the activity of MMP–9 promoter in human corneal epithelial cells, and that this induction is dependent on a PAF–responding segment (PRS) spanning from –670 to –460 relative to the transcription starting point (ARVO 2005). The PRS contains the consensus sequences for several transcription factors including NFΚB (–600) and Sp1 (–558), suggesting a possible role for these transcription factors in regulation of MMP–9 promoter activity during the PAF stimulation. Here we investigated the specific role of the two binding sites by mutational studies.
Methods: :
HCEC were transfected with Firefly luciferase reporter constructs fused to the wild type (–670/+54) or mutated (mut–NFΚB and mut–Sp1) human MMP–9 promoter sequences using FuGene 6 reagent. Transiently transfected cells were either left untreated or stimulated overnight with 100nM PAF and the expression of luciferase was tested using Renila luciferase activity for normalization and a proximal AP–1 site (–79) mutation as a control.
Results: :
The basal activity of MMP–9 promoter decreased by 30% when the NFΚB site was mutated while mutation in proximal AP–1 site decreased the activity by more than 60%. Mutation of Sp1 site, on the other hand, caused an increase of 50% in the basal activity of MMP–9 promoter. However, mutation at either NFΚB or Sp1 site knocked down the PAF–induced activity of MMP–9 promoter as compared to that of wild type promoter sequence by 50% and 75%, respectively.
Conclusions: :
In HCEC the NFΚB consensus sequence located within the PRS of MMP–9 promoter is partly involved in the basal expression of this gene while the Sp1 site is a negative regulator. In PAF–stimulated cells, however, both NFΚB and Sp1 sites play a remarkable role in upregulation of the MMP–9 gene expression. Considering the fact that the DNA binding activity of Sp1 is upregulated by PAF, our findings suggest that at higher levels of DNA binding activities this transcription factor plays a positive regulatory role through the consensus sequence located within the PRS of MMP–9 promoter. This is in contrast with the role of NFΚB site that is always an inducer of MMP–9 gene expression both in untreated and PAF stimulated HCEC. (Supported by NIH EY04928)
Keywords: cornea: epithelium • extracellular matrix • gene/expression