Hyperosmolarity-induced cornification of human corneal epithelial cells is regulated by JNK MAPK

Abstract

Purpose: To evaluate the effects of hyperosmolar stress on expression of cornified envelope (CE) precursors and transglutaminases (TGs) by primary cultured human corneal epithelial (PCHCE) cells and the regulatory effects of JNK MAPK on this process.

Methods: Expression of CE precursors and TGs were evaluated in PCHCE cells exposed to media of increasing osmolarity (350-450 mOsM) for 24, 48, and 72 hours. JNK1 and -2 MAPKs were inhibited by addition of short interfering (si)RNA. Relative levels of mRNA transcripts and proteins were evaluated. TG activity, cell viability, and apoptosis were detected in PCHCE cells, with or without siRNA-JNKs.

Results: Exposure of PCHCE cells to hyperosmolar medium increased TG activity at 3 hours, levels of the CE precursors SPRR1b and -2a and membrane-associated TG1 mRNA at 6 hours, and tissue-type TG2 mRNA at 24 hours. Osmotic stress decreased corneal epithelial cell viability, which was due in part to stimulation of apoptosis and cornification death. Inhibiting JNK2 production by siRNA in osmotically stressed PCHCE cells prevented the stimulation of SPRR and membrane-associated TG1 production and TG activity, and improved cell viability, whereas inhibition of JNK1 prevented early apoptosis.

Conclusions: Osmotic stress promotes production of certain CE proteins and cross-linking membrane-associated TG1 and decreases cell viability via JNK MAPK-mediated pathways. Strategies that inhibit JNK production downregulate the cornification response of PCHCE cells to osmotic stress. These findings have potential therapeutic implications for preventing cornification of the corneal epithelium in response to the hyperosmolar tear film in dry eye disease.

Figures

Figure 1.

Relative levels of CE precursor (A) and TG (B) mRNA transcripts evaluated by real-time PCR. PCHCE cells were treated with NaCl medium (osmolarity 450 mOsM) for 6 to 48 hours. All data (mean ± SEM) were compared to the control: *P < 0.05; **P < 0.01.

Figure 2.

Effects of osmotic stress on levels of CE precursor and TG proteins in PCHCE cells by Western blot, using β-actin as a control (A). The ratio of band intensity (y-axis) of CE precursors and membrane-associated TG1 compared with β-actin (mean ± SD in three experiments) in osmotically stressed PCHCE cells at 24 and 48 hours (B). TG activity measured by F-CDV incorporation at 3 and 24 hours in osmotically stressed PCHCE cells (C).

Figure 3.

Effects of osmotic stress (exposure to 450-mOsM medium) on corneal epithelial viability: MTT (A) and TUNEL-positive cells (B). All data were compared to the control: *P < 0.05; **P < 0.01. (C) Representative results of flow cytometry analysis of Annexin V and PI staining on osmotically stressed PCHCE cells for 24 hours (right) compared with controls (left). Cells undergoing early apoptosis are in the bottom right quadrant and those with late apoptosis are in the top right quadrant.

Figure 4.

Effects of osmotic stress in PCHCE cells in 450-mOsM medium on JNK mRNA level as shown by real-time PCR (A) and total JNK protein measured by immunobead assay (B). Data are the mean ± SEM. All data were compared with the control: *P < 0.05; **P < 0.01.

Figure 5.

Effects of RNA silencing on the level of JNK. JNK mRNA measured by real-time PCR (JNK1: A; JNK2: B) and total JNK protein measured by immunobead assay (C). (A, B) Mean ± SEM; (C) mean. Results from siRNA-JNKs (siRNA-JNK1, -2, and -1+2) were compared to that of siRNA-F. Data from the siRNA-F and siRNA-JNK groups with NaCl added were compared to the corresponding siRNA-F and siRNA-JNK groups; *P < 0.05; **P < 0.01.

Figure 6.

Effects of JNK-specific siRNAs on levels of SPRR2a (A) and SPRR2b (B) mRNA transcripts in control and osmotically stressed PCHCE cells (450 mOsM) measured by real-time PCR. Data are the mean ± SEM. All data were compared to siRNA-F; *P < 0.05; **P < 0.01. siRNA-JNKs with osmotic stress groups were compared to corresponding groups without osmotic stress; ¥¥P < 0.01; ¥P < 0.05.

Figure 7.

Effect of JNK-specific siRNAs on levels of expression of SPRR2 and membrane-associated TG1 protein in PCHCE cells grown in hyperosmolar medium (450 mOsM) for 24 hours. β-Actin was used as the control (A). Ratio of SPRR or membrane-associated TG1 with β-actin (y-axis); mean ± SD detected by Western blot (B).

Figure 8.

Immunofluorescence staining for SPRR2 (top) and membrane-associated TG1 (bottom) in siRNA (siRNA-F- or JNK-specific) treated PCHCE cells for 24 hours grown in control or hyperosmolar sodium chloride (+NaCl) medium (450 mOsM for 24 hours).

Figure 9.

TG activity measured by F-CDV incorporation in siRNA-treated PCHCE cells grown in control or hyperosmolar medium (+NaCl) for 3, 24, and 48 hours (A). Levels of membrane-associated TG1 mRNA transcripts measured by real-time PCR (B) in PCHCE cells grown in control or hyperosmolar sodium chloride (+NaCl) medium with JNK1-, -2-, or -1+2-specific siRNA for 6 to 48 hours. Data are the mean ± SEM. All siRNA-JNK groups (siRNA-JNK1, -2, and -1+2) were compared to siRNA-F (*P < 0.05; **P < 0.01) and all siRNA-JNKs+NaCl groups (siRNA-JNK1, -2, and -1+2 +NaCl) were compared to the corresponding siRNA-JNK groups (siRNA-JNK1, -2, and -1+2; ¥¥P < 0.01; ¥P < 0.05), respectively.

Figure 10.

Number of viable cells measured by MTT in control (siRNA-F) and JNK-specific siRNA-treated PCHCE cells exposed to 450-mOsM sodium chloride (+NaCl) medium for up to 72 hours.

Figure 11.

A summary diagram of the signaling pathways controlling apoptosis and cornification that are mediated by JNK MAPK pathways in osmotically stressed cells.