title: Interferon-gamma-induced MD-2 protein expression and lipopolysaccharide (LPS) responsiveness in corneal epithelial cells is mediated by Janus tyrosine kinase-2 activation and direct binding of STAT1 protein to the MD-2 promoter.
abstract: The inability of epithelial cells from the cornea and other tissues to respond to LPS is reportedly due to low expression of the TLR4 co-receptor MD-2. We generated MD-2(-/-) bone marrow chimeras, and showed that MD-2 expression on non-myeloid cells was sufficient to mediate LPS-induced corneal inflammation. As IFN-γ is produced during Pseudomonas aeruginosa corneal infection, we examined the role of this cytokine on MD-2 expression by primary human corneal epithelial (HCE) cells and HCE cell lines. Exogenous IFN-γ was found to induce MD-2 mRNA, MD-2 cell surface expression, and LPS responsiveness as determined by p65 translocation to the nucleus and production of IL-6, CXCL1, and CXCL8/IL-8. Incubation with either the AG490 JAK2 inhibitor or with STAT1 siRNA blocked STAT1 phosphorylation and MD-2 transcription. Furthermore, EMSA analysis demonstrated that STAT1 binds to the MD-2 promoter, indicating that STAT1 is an MD-2 transcription factor. Together, these findings demonstrate that IFN-γ induces MD-2 expression and LPS responsiveness in HCE cells by JAK-2-dependent STAT1 activation and direct binding to the MD-2 promoter. Furthermore, given our findings on LPS-induced corneal inflammation, it is likely that IFN-γ-induced MD-2 expression by corneal epithelial cells contributes to the host response in vivo, determining the extent of tissue damage and bacterial clearance.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio 44095, USA.
authors.forename: Sanhita
authors.initials: S
authors.lastname: Roy
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.forename: Eric
authors.initials: E
authors.lastname: Pearlman
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism* / Q000382:microbiology; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000382:microbiology; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism*; D007634:Keratitis / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D011552:Pseudomonas Infections / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D020218:Response Elements; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; C495377:LY96 protein, human; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C494080:Stat1 protein, mouse; D007371:Interferon-gamma; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2