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Published on: September 27, 2016
Hypoxia-induced downregulation of ΔNp63α in the corneal epithelium
Danielle M Robertson1, Meifang Zhu, Yu-Chieh Wu
1Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA. danielle.robertson@utsouthwestern.edu
Purpose:
The aim of this study was to establish a relationship between hypoxic stress and the expression of ΔNp63α in an established rabbit contact lens model and in cultured corneal epithelial cells.
Methods:
New Zealand white rabbits were fit in one eye with either a non-oxygen transmissible or hyper-oxygen-permeable rigid contact lens for 24 hrs of wear; the contralateral eye was used as a control. All the rabbits underwent a bilateral nictitating membranectomy to facilitate lens retention. ΔNp63α expression was analyzed by immunofluorescence and western blot. Telomerase-immortalized human corneal epithelial cells (hTCEpi) were grown in serum-free media and treated with the hypoxia-mimetic cobalt chloride to simulate hypoxia for 6 hrs (short term) or 24 hrs (prolonged). Transcriptional activity and protein levels were assessed using luciferase reporter assays, reverse transcription polymerase chain reaction, and western blot. Cell viability was assessed by live/dead assay.
Results:
Compared with the non-lens wearing eye, 24 hrs of non-oxygen transmissible lens wear in vivo decreased ΔNp63α protein levels in both the limbal and central corneal epithelium; this decrease was not found in the hyper-oxygen transmissible lens group. In hTCEpi cells in vitro, hypoxia increased the activity of the ΔN promoter but reduced the levels of ΔNp63α mRNA after 24 hrs of prolonged culture. Similarly, ΔNp63α expression levels were unaffected from short-term exposure but decreased after 24 hrs. Live/dead assay confirmed the presence of viable cells after CoCl2 treatment at 6- and 24-hr time points. Cells treated for 24 hrs were viable but were smaller and rounded with signs of membrane blebbing, consistent with early stages of apoptosis.
Conclusions:
Hypoxic stress induced by either prolonged wear of a nonoxygen transmissible lens in vivo or hypoxic-mimic conditions by cobalt chloride in vitro downregulates ΔNp63α in the corneal epithelium. The loss of ΔNp63α in response to hypoxic stress may contribute to the disruption of normal renewal mechanisms reported with low oxygen transmissible contact lens wear and prolonged eyelid closure.
