Activation of the MAPK Signaling Pathway Following Transient Hyperosmolar Shock

Abstract

Abstract: : Purpose: Rather than centering on global tear film hyperosmolarity as a mechanism in dry eye, we hypothesize that local areas of tear breakup undergo rapid, transient hyperosmolar spikes, which stress underlying cells and lead to inflammation. In this in vitro study, we examined the first step of this process by testing whether short–term hypertonic shock triggered the p38 Mitogen Activated Protein Kinase (MAPK) signaling pathway. Methods: Second passage bovine corneal epithelial cells (BCEC) were cultured in 35mm Petri dishes. Once confluent, cell samples were exposed to a transient hypertonic shock for 30s, 2m, 5m, 10m, 15m, and 30m using DMEM–F12 culture media osmotically adjusted to 600 mOsm by adding sucrose. Following each time point, cells were washed three times with isotonic cold phosphate buffered saline (PBS). Negative controls were identically processed, except they did not receive hypertonic shock. Cellular proteins from each cell sample were extracted and total protein concentration was determined with the Lowry Assay. Activation of the p38 MAPK pathway was detected by monitoring the level of phosphorylated p38 (P–p38) by Western Blot using a phospho–specific p38 antibody (Cell Signaling Technology). To verify equal loading, films were stripped and reprobed for total P38 and then scanned for analysis by densitometry. The ratio of p–p38 to overall p38 expression was used to calculate fold–induction of the response. Results: P38 phosphorylation appeared after all time periods of hypertonic shock. After 30 seconds, the P–P38/P38 ratio averaged 1.43 times higher than controls. Then at the 2m, 5m, 10m, 15m and 30m time periods, the ratio steadily increased, averaging 1.97, 2.84, 3.38, 4.12, and 5.74 times higher than that the control, respectively. Conclusions: Short term hyperosmolar shock, which may occur locally in areas of tear breakup, can stimulate the p38 MAPK signaling pathway. Activation of this signaling pathway is known to be a first step in inflammatory reactions, which may contribute to the etiology of dry eye.

Keywords: cornea: tears/tear film/dry eye • cornea: epithelium • inflammation