Abstract
Purpose: :
Extracellular PAI–2 is a potent inhibitor of u–PA and, although the intracellular targets and the molecular mechanism of PAI–2 remain undefined, different studies have indicated that PAI–2 acts as a multifunctional protein. The significance of the different expression pattern of PAI–2 in corneal and conjunctival epithelia is not yet clear, but may suggest a particular role of this inhibitor in regulating cell proliferation and cell turnover in the cornea and conjunctiva. Recently, we showed that pRb2/p130 and Rb1/p105, but not p107, interact with PAI–2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cell cultures and we suggested a model of how pRb2/p130 and PAI–2 may cooperate in modulating PAI–2 gene expression by chromatin remodeling.
Methods: :
Here, we assessed the level of PAI–2 basal transcription in normal primary corneal and conjunctiva cells by multiplex semiquantitative RT–PCR. We also investigated if the presence of different pRb2/130–multimolecular complexes on the PAI–2 promoter in normal corneal and conjunctival epithelial cells may stem from unrelated differences between these two cell types in expressing specific proteins by Western blotting analysis. Lastly, we investigated if the presence of these complexes may dictate different levels of local H3 and H4 histone acetylation by performing XChIP assays.
Results: :
We found that the protein expression levels of p300, SUV39H1, E2F4 and E2F1 are similar in the normal primary human corneal and conjunctival cell cultures investigated as well as the protein expression levels of pRb family members. We reported that the level of PAI–2 mRNA is higher in conjunctival cells than in corneal cells. Finally we revealed a different pattern of H3 and H4 acetylation on the PAI–2 promoter fragment bound by specific pRb2/p130–multimolecular complexes.
Conclusions: :
Our data suggest the intriguing hypothesis that in normal human corneal and conjunctival epithelial cell cultures, the binding of pRb2/p130–PAI–2 complexes on a specific region of PAI–2 promoter may modulate the PAI–2 basal transcription by inducing local changes in chromatin structure, maybe by altering the activity of transcription regulators bound nearby.
Keywords: cornea: epithelium • conjunctiva • gene/expression