Essential role for c-Jun N-terminal kinase 2 in corneal epithelial response to desiccating stress

Abstract

Objective: To investigate the protective effects of c-Jun N-terminal kinase (JNK)-1 and -2 gene knockout (KO) on the corneal epithelial response to desiccating stress.

Methods: The C57BL/6, JNK1KO, and JNK2KO mice were subjected to desiccating stress (DS) for 5 days. The effects of DS on the corneal epithelium were evaluated by measuring corneal smoothness and permeability. Expression of matrix metalloproteinases (MMP)-1, MMP-9, and cornified envelope protein precursors (small proline-rich protein [SPRR]-1a, SPRR-2a, and involucrin) in the corneal epithelia was evaluated by immunostaining and real-time polymerase chain reaction. Collagenase and gelatinase activity in corneal sections as measured with in situ fluorescent assays.

Results: The JNK2KO mice had smoother corneal surfaces and less corneal barrier disruption in response to DS than JNK1KO mice and C57BL/6 wild-type control mice. The DS increased levels of MMP-1, MMP-9, SPRR-1a, SPRR-2a, involucrin immunoreactivity, and mRNA transcripts in the corneal epithelium of JNK1KO and C57BL/6 mice, but not in JNK2KO mice. Knockout of JNK2 prevented DS-induced increase in gelatinase and collagenase activity in the cornea.

Conclusion: The JNK2 protein appears to have an essential role in desiccation-induced corneal epithelial disease by stimulating production of MMP-1, MMP-9, and cornified envelope precursors. Clinical Relevance The JNK2 protein could be a novel therapeutic target in dry eye disease.

Figure 1

Corneal smoothness and permeability evaluation of the 3 strains of mice. A, Representative digital images of corneas in C57BL/6, c-Jun N-terminal kinase 1 knockout (JNK1KO), and JNK2KO mice (A) were used to score Oregon green dextran–488 permeability (B) after 5 days of desiccating stress (DS5). Gray levels of fluorescent intensity are shown on the y-axis (B). C, Representative digital images of the corneal surface in C57BL/6, JNK1KO, and JNK2KO mice were used to score corneal smoothness (D) after 5 days of desiccating stress. Data in graphs are mean(standard deviation). NS indicates nonstressed control; *P<.05 compared with NS in the same strain. Scale bars, 500 μm.

Figure 2

Evaluation of metalloproteinase (MMP) expression and activity in corneal sections in the 3 strains of mice. Laser scanning immunofluorescent confocal microscopy of corneal sections stained for MMP-9 (A, green) or MMP-1 (E), with propidium iodide nuclear counterstaining (red) in nonstressed control conditions (NS) and after 5 days of desiccating stress (DS5) in C57BL/6, c-Jun N-terminal kinase knockout 1 (JNK1KO) and JNK2KO mice. B and F, Representative digital images of in situ zymography (B) and in situ collagenase assay (F). The fluorescence intensity is proportional to gelatinase or collagenase activity, respectively, within the tissue. Graphical data are the mean (standard deviation) of measured fluorescence levels of MMP-9 and MMP-1 immunofluorescent staining (C and G) and in situ assays (D and H). *P<.05 compared with NS in the same strain; **P<.001 compared with NS in the same strain. Scale bars, 50 μm (A, E, and F) and 100 μm (B).

Figure 3

Evaluation of cornified envelope protein precursors in the corneal sections of the 3 strains of mice. Laser scanning immunofluorescent confocal microscopy of corneal sections stained for small proline-rich protein (SPRR)–1a (A, green), SPRR-2a (B, green), or involucrin (C, green) with propidium iodide nuclear counterstaining (red) in nonstressed control conditions (NS) and after 5 days of desiccating stress (DS5) in C57BL/6, c-Jun N-terminal kinase knockout 1 (JNK1KO), and JNK2KO mice. Graphical data are the mean (standard deviation) of measured fluorescence levels for each protein. *P<.05 compared with NS in the same strain; **P<.01 compared with NS in the same strain; and ***P<.001 compared with NS in the same strain. Scale bars=50 μm.

Figure 4

Schematic showing the proposed sequence of events after exposure of ocular surface tissue to desiccating stress. JNK indicates c-Jun N-terminal kinase. Smaller font of JNK1 indicates its possible smaller role.