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S. Jeong, D. R. Ledee, M. E. Fini; Evidence for a Novel Role of Clusterin (CLU) in Ocular Surface Inflammatory Processes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6243.
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Recent findings indicate that matrix metalloproteinase-9 (MMP-9) plays a key role in ocular surface maintenance and pathologies, particularly those involving inflammation. The purpose of the present study was to identify novel protein binding partners for MMP-9 that might be involved in corneal function.
We performed yeast two hybrid experiments to identify proteins that interact with MMP-9, and further proved in vitro binding assay and cell culture experiments. Fluorescence resonance energy transfer (FRET) method was adopted for MMP inhibition tests. Gelatin zymography and Western blotting were used for studying the interaction and secretion of MMP-9.
We found CLU, which has anti-inflammatory function, interacts with MMP-9 when we used the active form of MMP-9 as a bait and cDNA library from mouse corneal cells in yeast two hybrid assays. The significance of their interaction was demonstrated by the observation that CLU inhibited the enzymatic activity of MMP-9. Truncation of C-terminal hemopexin-like domain of MMP-9 did not affect CLU inhibition of MMP-9. Consistently, CLU significantly inhibited the enzymatic activity of MMP-7 that lacks a hemopexin-like domain. CLU also inhibited MMP-2 activity. CLU binds to the unprocessed form of MMP-9 more preferentially than the processed forms, by interacting with the N-terminal propeptide domain of pro-MMP-9. CLU also rendered MMP-2 and MMP -9 soluble in a non-ionic detergent condition. We also observed that exogenous CLU inhibits TNF-α-induced MMP-9 secretion from human corneal limbal epithelial cell line.
As MMP-9 and TNF-α are involved in inflammatory events, we propose a model for the role of CLU in the pathology of inflammatory disorders of the ocular surface such as dry eye disease.
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