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Priscilla Hiu-Mei Too, Tsz Ying (Judy) Lee, Connie Tam; Bacterial Antigen Challenge Promotes Solubilization of Keratin 6A Filaments And Generation of Keratin-Derived Antimicrobial Peptides (KDAMPs) in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):853.
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© ARVO (1962-2015); The Authors (2016-present)
KDAMPs, a novel family of antimicrobial peptides derived from keratin 6A (K6A), are constitutively expressed in human corneal epithelia cells. K6A is a major intermediate filament protein of stratified epithelial cell cytoskeleton. Phosphorylation-induced solubilization of keratin filaments is an important cellular response to combat external stress stimuli. Pseudomonas aeruginosa has been shown to trigger protective innate immune responses in corneal epithelium via activation of p38/JNK signaling. To begin deciphering the molecular mechanisms of KDAMP generation, we tested the hypothesis that bacterial antigens increase solubilization of K6A and generation of KDAMPs.
Human corneal epithelia cells were grown on plates or transwell filters respectively to form single- or stratified multi-layered cells, followed by 16-h exposure to culture supernatant of P. aeruginosa strain PAO1 or Salmonella flagellin (500 ng/ml), or by 1-h exposure to serine phosphatase inhibitor Calyculin A (0.2 uM) as positive control for keratin solubilization. Cells were lysed by freeze and thaw cycles to release soluble lysate fractions. KDAMPs/total soluble K6A, and phosphorylated K6A, were detected by Western blotting using anti-K6A 19mer and anti-phosphoserine antibodies respectively. Band intensities were quantified by Licor Image Solution and normalized to GAPDH.
K6A solubilization in untreated human corneal epithelial cells (both single- and multi-layered) was not detectable unless the cells were treated with P. aeruginosa culture supernatant, purified Salmonella flagellin or Calyculin A. An increase of 240% and 30% above baseline were found for KDAMPs production in supernatant-treated or flagellin-treated cells respectively. Soluble K6A in all challenged cells was found to be serine-phosphorylated.
The data suggest that bacterial antigen challenge triggers serine phosphorylation of keratin 6A, which in turns promotes solubilization of its filament network, and ultimately upregulates generation of KDAMPs. This could potentially represent a novel innate defense mechanism for the cornea and other mucosal surfaces. Further studies will determine the signaling pathway(s) involved in bacteria-induced phosphorylation of K6A, and if increased amount of soluble K6A is sufficient to upregulate KDAMPs production.
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