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Mingwu Wang, Yang Zhao, Jing Li, Fayez Ghishan, Hua Xu; TNF-α Inhibits NHE8 Expression in Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2761.
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NHE8 is a membrane protein in the sodium/hydrogen exchanger (NHE) family. It is highly expressed in the conjuntival epithelial cells in both human and mouse. Our previous study demonstrated that mice deficient of NHE8 displayed dry eye phenotype, suggesting that NHE8 plays an important role in the ocular surface homeostasis. Since TNF-α is implicated to be one important pro-inflammatory cytokine in dry eye disease, this current study investigated the role of TNF-α on NHE8 expression in human conjunctival epithelial cells.
Localization of NHE8 was determined by immunochemical staining in the human conjunctival epithelial cell line (CCL-20.2). The cell culture was treated with various concentrations of human recombinant TNF-α to investigate the effect of TNF-α on NHE8 expression. RNA and protein were purified from treated and non-treated cells for comparison. NHE8 gene expression was assessed by Real time PCR and Western blot. NHE8 gene promoter activity was measured using promoter reporter assay system.
Immunohistochemistry confirmed that NHE8 is expressed on the plasma membrane of human conjunctival epithelial cells. In a dose higher than 25 ng/ml, TNF-α down regulates the expression of NHE8 at both mRNA and protein levels in human conjunctival epithelial cells. TNF-α mediated NHE8 mRNA regulation could be blocked by actinomycin D, a transcription inhibitor. NHE8 promoter activity was reduced in the presence of TNF-α as demonstrated in the promoter reporter assay.
NHE8 is a membrane protein in human conjunctival epithelial cells. Inflammatory cytokine TNF-α is capable of impairing NHE8 function by inhibiting NHE8 expression at both mRNA and protein levels in conjunctival epithelial cells. The mechanism of TNF-α regulation on NHE8 involves gene transcriptional inhibition.
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