Molecular Mechanism of Proinflammatory Cytokine-Mediated Squamous Metaplasia in Human Corneal Epithelial Cells

Abstract

Purpose.: The cornified envelope protein small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia. Proinflammatory cytokines IL-1β and IFN-γ are potent inducers of ocular surface keratinization and SPRR1B expression. Here the molecular mechanisms controlling SPRR1B gene expression in response to IL-1β and IFN-γ are elucidated. Methods.: A 3-kb fragment of the SPRR1B gene 5′-flanking region was amplified from human chromosome 1, sequentially deleted, and cloned into a luciferase vector. Constructs were transiently transfected into human corneal epithelial cells, and activity was assessed in response to IL-1β, IFN-γ, or basal medium. Functional cis-elements responding to IL-1β and IFN-γ were characterized by site-directed mutagenesis and gel mobility shift assay. Effects of mitogen-activated protein kinases p38, ERK, and JNK were assessed using inhibitors and dominant-negative mutants. Results were validated by real-time RT-PCR. Results.: The first 620 bp of the SPRR1B 5′-flanking region regulated constitutive expression and increased promoter activity in response to IL-1β and IFN-γ. Corresponding cis-elements for IL-1β and IFN-γ were bound by cAMP response element binding protein (CREB) and zinc-finger E-box binding homeobox 1 (ZEB1), respectively. Inhibition of p38 abolished the stimulatory effects of IL-1β and IFN-γ on SPRR1B, whereas inhibition of JNK and ERK had no effect. Dominant-negative mutants targeting p38α and p38β2 blocked cytokine-induced SPRR1B promoter activity and mRNA expression. Conclusions.: SPRR1B is upregulated by the proinflammatory cytokines IL-1β and IFN-γ via p38 MAPK-mediated signaling pathways that lead to the activation of transcription factors CREB and ZEB1, respectively. These results identify key intracellular signaling intermediates involved in the pathogenesis of immune-mediated ocular surface squamous metaplasia.