Abstract
Purpose: :
Matrix metalloproteinase (MMP) family members are key regulators and effectors of tissue remodeling in the cornea and other organs. Expression of one family member, gelatinase B (MMP–9), is induced in corneal epithelial cells (CECs) upon injury where it plays a role in normal re–epithelialization, failure to heal, and dry eye disease. TGF–ß and IL–1 family members are expressed by CECs and are released when cells are damaged. The goal of this study is to determine whether IL–1 and TGF–ß family members interact to induce MMP–9 expression and to investigate the mechanism of induction at the transcriptional promoter level.
Methods: :
Primary cultures of Rabbit CECs were used for all experiments. To ensure a CE phenotype, immunocytochemistry was performed against CEC markers cytokeratins 3/12. Dose–dependent experiments: 24 hours post–plating, replicate cultures were treated with TGF–ß1, TGF–ß2, IL–1α, or IL–1ß separately or in combinations in a dose range of 0.1 to 10 ng/ml. Phorbol myristate acetate (1µM) was used as a positive control. After an additional 24 hours, culture medium was collected for assessment of accumulated MMP–9 by gelatin zymography. Transfection experiments: 24 hours post plating, cells were transfected with a previously characterized construct, Pr22, containing a 541 fragment of the MMP–9 gene (–522 to +19) linked to a ß–Galactosidase (ß–Gal) reporter gene. Co–transfection with the pEGFP vector (Clonetech) was used to determine transfection efficiency. After another 24 hours, cells were treated with TGF–ß1, TGF–ß2, IL–1α, or IL–1ß separately or in combinations. After an additional 24 hours, cells were lysed and ß–Gal activity assessed (Promega, cat #E2000).
Results: :
Individual cytokines could not induce MMP–9 expression, but TGF–ß2 and Il–1ß together stimulated MMP–9 expression in a dose–dependent manner. Concentrations as low as 1 ng/ml of either factor combined with 0.1 ng/ml of the other factor was sufficient for induction. Individual cytokines could only weakly induce Pr22 ∼1.25–1.5 fold above basal in vitro levels; but in combinations, the expression was induced ∼1.75–2 fold, or the sum of the two individual treatments.
Conclusions: :
The findings indicate that TGF–ß and IL–1 proteins act synergistically to induce MMP–9 expression and that some of the transcriptional promoter elements required for this effect are contained in the 541 bp portion of the gene studied. Further studies using deletion constructs of Pr22 will define the specific promoter elements involved.
Keywords: cornea: epithelium • wound healing • gene/expression