Abstract
Purpose::
Hypoxic modulation of gene expression leads to reduction in cell-cell adhesion, decreased barrier function, and increase in cell migration. Expression of Pinin (Pnn) positively modulates E-cadherin gene expression. Numerous studies have demonstrated that Pnn functions as a critical mediator of multiprotein complexes involved in regulation of gene expression, pre-mRNA splicing in Human Corneal Epithelial (HCE-T) Cells. Previously, we provided morphological evidence of Pnn's dynamic redistribution in the nucleus due to hypoxia. These data raised the possibility of hypoxia impacting Pnn's function in the basal transcription and mRNA processing machineries. Here we present combination of morphological and biochemical support for hypoxia-induced uncoupling of Pnn from these machineries.
Methods::
Cells were placed in hypoxic conditions for 48 hours and subsequently processed for nuclear extractions or immunostained and immuno-blotted with 143(anti-Pnn), anti-SR proteins, anti-SC35, anti-CtBP1, anti-Magoh, Anti-PolII Ser5, anti-PolII CTD and anti-ASF/SF2 antibodies.
Results::
Although morphological observation showed Pnn redistribution from the nuclear speckle to a diffuse nucleoplasmic pattern, other speckle proteins retained a strong presence at the nuclear speckle in hypoxia. Western blotting of hypoxic samples also revealed a significant shift in Pnn’s solubility without significant changes in other mRNA-processing or transcription proteins. Furthermore, along with this uncoupling event, a significant reduction in both BrU incorporation and activated-PolII were seen in hypoxic samples.
Conclusions::
Pnn is a dynamic protein that resides in the speckle. Pnn has been linked to the maintenance of an epithelial phenotype, likely as a result of its role in transcription and splicing. These data further support a hypoxia-induced uncoupling of Pnn from the mRNA splicing/transcription machinery. These data uncover a novel molecular mechanism by which hypoxic conditions impact gene expression, which in turn may result in decreased barrier function in the Corneal Epithelial Cells.EY07883
Keywords: hypoxia • transcription • cornea: basic science