Glycogene expression in conjunctiva of patients with dry eye: downregulation of Notch signaling

Abstract

Purpose: Glycoconjugates regulate a variety of biological events in mucosal surfaces, such as differentiation of postmitotic epithelial cells and maintenance of the wet-surfaced phenotype. This study aimed to identify the repertoire of genes (glycogenes) involved in biosynthesis of glycoconjugates in conjunctiva of normal subjects and patients with dry eye.

Methods: RNA from conjunctival impression cytology samples was amplified and hybridized to a custom-designed glycogene microarray. Intensity data were converted to expression values and analyzed by ANOVA. Microarray data for selected Notch glycogenes were confirmed by quantitative real-time PCR. Notch receptors and ligands were immunolocalized on conjunctival biopsies by confocal microscopy.

Results: By microarray, 424 glycogenes were identified in normal conjunctival epithelium; galectins, glycosyltransferases, mucins, Notch signaling molecules, and proteoglycans were among the most highly expressed. In dry eye, 46 glycogenes were significantly downregulated, including five members of the Notch signaling pathway (Notch1, Notch 2, Notch 3, Jagged1, Delta1), four Wnt signaling molecules (Wnt4, -5A, Frizzled6, -7), and three heparan sulfate glycotransferases (HS2ST1, HS3ST6, EXTL2). Only interferon-induced transmembrane protein 1 was upregulated. By real-time PCR, expression ratios of Notch1, Notch 3, and Jagged1 in dry eye were 0.43, 0.56, and 0.50, respectively, compared to controls (P < 0.05). Notch1, Notch3, and Jagged1 were immunolocalized throughout the conjunctival epithelium, whereas Notch2 and Delta1 were distributed apically.

Conclusions: This study revealed the differential glycogene expression profiles in normal subjects and patients with dry eye. Downregulation of Notch signaling in dry eye may result in abnormal differentiation of the conjunctival epithelium and have implications in the pathogenesis of the disease.

Figure 1

Distribution of gene ontology groups in normal conjunctiva. Glycogenes (n=424) were categorized according to gene function as defined in the GLYCOv3 microarray database.

Figure 2

Real-time PCR analysis of Notch1, Notch3 and Jagged1. (A) By real-time PCR, Notch1, Notch3, and their ligand Jagged1 were significantly downregulated in the conjunctiva of patients with dry eye (p<0.05). (B) Comparison of microarray and real-time PCR showed downward trends in the results, with real-time PCR analyses resulting in more pronounced differences in expression of Notch signaling genes between normal subjects and dry eye patients. (C) Conventional PCR after 30 cycles of cDNA amplification produced a unique band corresponding to the predicted size for Notch1 (75 bp), Notch3 (105 bp) and Jagged1 (76 bp).

Figure 3

Immunolocalization of Notch receptors and ligands in normal conjunctiva. By confocal microscopy, Notch1, -3, and Jagged1 were present throughout conjunctival epithelium. Antibodies to Notch2 and Delta1 bound primarily to apical cells. Propidium iodide was included in the mounting medium to localize the position of the nuclei in all sections (red). In control experiments, antibodies to Notch1, -2, -3, Delta1, and Jagged1 bound to epithelial cells in normal colon (data not shown). Negative control corresponds to secondary antibody only. Scale bar = 75 μm.