Abstract
Purpose::
Squamous metaplasia occurs in severe ocular surface diseases (e.g., Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP) and Sjögren’s syndrome (SS)) and represents an important clinical problem because it causes pathological keratinization of the ocular surface. Squamous metaplasia is phenotypic change whereby epithelial cells initiate the synthesis of specialized, squamous-specific proteins like small proline-rich protein 1B (SPRR1B) to form the cornified envelope. Very little is known about the molecular mechanisms triggering squamous metaplasia although it presence has been extensively correlated with proinflammatory activity of the ocular surface. Based on this, we examined the hypothesis that inflammatory mediators are key regulators of pathological keratinization by assessing the regulatory effects of several inflammatory mediators on SPRR1B expression in human corneal epithelial cells (HCECs).
Methods::
Quantitative real-time PCR was used to examine the endogenous effects of IFN-γ, IL-1α, IL-1ß and IL-4 on SPRR1B transcription in HCECs. Transient transfection of HCECs with the SPRR1B 5’-flanking region was used to characterize the cis-acting elements that respond to stimulation with the same mediators.
Results::
Endogenous expression of SPRR1B was increased between 1.8- and 6-fold depending on the inflammatory mediator. Using a -620 bp fragment of the SPRR1B promoter, we found that IFN-γ was able to increase reporter gene expression up to 5-fold. In contrast, this region did not contain response elements for stimulations by IL-1α, IL-1ß or IL-4. Through deletion and mutation of the -620 bp promoter, we observed that the stimulatory effect of IFN-γ was significantly reduced using a -150 bp promoter that contains two AP1 sites and was completely lost with a -96 bp promoter. Signal transduction studies suggest the involvement of p38 in the stimulatory effects of IFN-γ on SPRR1B expression.
Conclusions::
Inflammatory mediators induce the expression of squamous metaplasia biomarker, SPRR1B. Studies to define the molecular events that mediate SPRR1B regulation may suggest possible therapeutic targets to prevent pathological keratinization of the ocular surface in human patients.
Keywords: cornea: epithelium • gene/expression • signal transduction