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M. Massaro-Giordano, M. Montanari, C. M. Marshall, M. Macaluso; TGF B1 and PMA Treatments Indirectly Regulate PAI-2 Expression in Corneal and Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4311.
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Several studies have reported that plaminogen activator inhibitor type 2 (PAI-2) is a multifunctional protein. It plays a role in cell differentiation, prevention of programmed cell death, regulation of cell proliferation, inhibition of microbial proteinases and in protection against stromal degradation.Previously we observed a different expression pattern of PAI-2 protein in the epithelium of the cornea and conjunctiva. We reported an interesting nuclear interaction between PAI-2 and the pRb2/130 proteins and suggested a model of how pRb2/p130 and PAI-2 may cooperate with specific chromatin remodeling enzymes in modulating PAI-2 transcription. We investigated the effects of transforming growth factor Î²-1 (TGF-Î²1) and phorbol 12-myristate 13-acetate (PMA) on the PAI-2 gene and protein expression and found that in both corneal and conjunctival cells TGF-Î²1 treatment down-regulates PAI-2 gene expression and decreases PAI-2 protein levels, while PMA treatment up-regulates the expression. Here we investigated the mechanisms by which TGF-Î²1 and PMA induce changes in both PAI-2 gene and protein expression in primary corneal and conjunctival cells. Our hypothesis is that following the treatments, specific signal transduction pathways may be turned on, which may affect pRb2/p130 and PAI-2 interaction and the binding on PAI-2 promoter.
We assessed the binding of pRb2/p130 and PAI-2 protein to PAI-2 promoter by performing XChIP assay in the presence or absence of TGF-Î²1 and PMA. We assessed the interaction of pRb2/p130 and PAI-2 proteins by co-immunoprecipitation assay in the presence or absence of TGF-Î²1 and PMA.
We reported that TGF-Î²1 or PMA treatments do not affect the interaction of PAI-2 and pRb2/p130 on the PAI-2 proximal promoter. After the treatments these proteins still bind the PAI-2 promoter. We confirmed that after TGF-Î²1 treatment pRb2/p130 and PAI-2 proteins co-immunoprecipitate only in the nucleus of both corneal and conjunctival cells.
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