Abstract
Purpose: :
The barrier function of the corneal epithelium is highly dependent on cell–cell and cell matrix adhesion. Hypoxia compromises the corneal epithelium barrier by altering gene expression resulting in a reduction of cell–cell adhesion and an increase in cell migration. Over–expression of Pnn has been shown to increase levels of E–cadherin expression and drive cells to a more epithelial phenotype. Furthermore, decreasing Pnn by shRNA results in a reduction of E–cadherin expression and weakened cell–cell adhesion. As a first step in examining the impact of hypoxia on Pnn’s function, we address a dynamic redistribution of Pnn in the nucleus due to hypoxia.
Methods: :
Human corneal epithelial cells (HCE–T, RCB1384, K. Sasaki) were placed in a hypoxia chamber (0.5% O2, 5% CO2) for 48hrs or alternatively treated with hypoxia mimicking agents; dimethyloxalylglycine (DMOG) and CoCl2, which have been shown to inhibiting prolyl hydroxylase – mediated regulation of HIF–alpha. Cells were immunostained and Western–blotted with 143(anti–Pnn), anti–SRm300, anti–SR proteins and anti–SC35.
Results: :
Immunostaining of HCE–T cells demonstrated a dramatic redistribution of Pnn from the nuclear speckle, resulting in a more diffuse nucleoplasmic staining. SR and SRm300 showed some increase in the nucleoplasmic fill yet retained intense speckle presence. Interestingly, SC35, a well–established speckle protein required for mRNA splicing, did not demonstrate any significant redistribution due to hypoxic conditions.
Conclusions: :
Pnn is a dynamic protein that resides in the speckle. Pnn expression has been linked to the maintenance of an epithelial phenotype exhibited by corneal epithelial cells. These data suggest a possible uncoupling of Pnn to a subset of members of the mRNA splicing/transcription machinery during hypoxia. These data may indicate a novel molecular mechanism by which hypoxic conditions impact gene expression and in turn epithelial barrier integrity of the cornea. (Supported by NIH grant EY07883)
Keywords: hypoxia • cell adhesions/cell junctions • cornea: epithelium