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Shinwu Jeong, M. Elizabeth Fini; Clusterin (CLU) Protects Against the MMP-9-Mediated Reduction of Epithelial Barrier Integrity and Its Expression is Regulated in Corneal Epithelial Cells by Factors Associated with Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4395.
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© ARVO (1962-2015); The Authors (2016-present)
CLU is expressed in the corneal epithelium, but its function is unknown. Last year we reported that CLU inhibits MMP-9 proteolytic activity in a biochemical assay. MMP-9 is also expressed in the corneal epithelium, where it plays a causative role in the epithelial pathology associated with dry eye disease (DE). In this study we further analyzed CLU function and studied its expression under normal and stress conditions associated with DE.
Epithelial tight junction integrity in MCF-7 cell culture was quantified using immunofluorescent localization and confocal microscopic image analysis after treatment with recombinant human active MMP-9 in the presence or absence of recombinant human CLU. Cells of a human corneal limbal epithelial line (HCLEC; a gift of Ilene Gipson) were exposed in culture to conditions of hyperosmolarity (increased NaCl concentration), oxidative stress (t-butylhydroxy peroxide (t-BHP)), CLU, TNF-α, dihyrotestosterone (DHT), or 17-β-estradiol (E2). RT-PCR, Western blotting, and/or gelatin zymography was used to measure resulting changes in gene expression.
Treatment of confluent epithelial monolayers with MMP-9 exhibited a loss of epithelial tight junction complexes as assessed by a reduction in the total density of ZO-1 protein and continuity of the interconnecting ZO-1 network. Co-treatment with CLU protected against these changes. CLU expression was up-regulated under hyperosmolar and oxidative stress conditions by 7-fold and 9-fold, respectively. DHT, a known suppressor of DE symptoms, also induced CLU expression up to 9-fold, showing a dose response, whereas E2 showed no effect. Recombinant CLU or TNF-α treatment significantly increased their own expression by up to 40% and 300%, respectively. TNF-α, a proinflammatory cytokine, decreased the basal expression of CLU by more than 70%.
These findings suggest that CLU acts to protect corneal epithelial barrier integrity under conditions (MMP-9 treatment) linked to DE pathology. Up-regulation of CLU expression may be a protective response to conditions associated with DE and protective stimuli may work by up-regulating CLU. CLU may be a potential therapeutic in the treatment of DE disease.
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