Abstract
Hypoxic conditions in the cornea affect epithelial function by activating Polo-like kinase 3 (Plk3) signaling and the c-Jun·AP-1 transcription complex, resulting in apoptosis of corneal epithelial cells. Hypoxic stress in the culture conditions also regulates limbal stem cell growth and fate. In this study, we demonstrate that there is a differential response of Plk3 in hypoxic stress-induced primary human limbal stem (HLS) and corneal epithelial (HCE) cells, resulting in different pathways of cell fate. We found that hypoxic stress induced HLS cell differentiation by down-regulating Plk3 activity at the transcription level, which was opposite to the effect of hypoxic stress on Plk3 activation to elicit HCE cell apoptosis, detected by DNA fragmentation and TUNEL assays. Hypoxic stress-induced increases in c-Jun phosphorylation/activation were not observed in HLS cells because Plk3 expression and activity were suppressed in hypoxia-induced HLS cells. Instead, hypoxic stress-induced HLS cell differentiation was monitored by cell cycle analysis and measured by the decrease and increase in p63 and keratin 12 expression, respectively. Hypoxic stress-induced Plk3 signaling to regulate c-Jun activity, resulting in limbal stem cell differentiation and center epithelial apoptosis, was also found in the corneas of wild-type and Plk3(-/-)-deficient mice. Our results, for the first time, reveal the differential effects of hypoxic stress on Plk3 activity in HLS and HCE cells. Instead of apoptosis, hypoxic stress suppresses Plk3 activity to protect limbal stem cells from death and to allow the process of HLS cell differentiation.
Highlights
The corneal epithelial layer forms the first defense barrier to prevent environmental and biological assaults to damage the eye structures behind it
We have reported previously that hypoxia induces the activation of a specific Polo-like kinase 3 (Plk3) pathway in corneal epithelial and MEF2 cells to phosphorylate downstream hypoxia-regulated PTEN, Hif-1, AP-1, p53, and H2XA, resulting in altered cell cycle progression and switching on of apoptotic responses (16 –20)
Application of hypoxic stress (1% oxygen) to human corneal epithelial (HCE) cells induced apoptosis detected by DNA fragmentation, which is consistent with the previous observation
Summary
Effects of Hypoxic Stresses on Corneal Limbal Stem and Epithelial Cell Variability—Plk[3] activity is one of the major regulators to mediate cellular responses to various stresses, including hypoxic stimulation pressure, during the wound healing process (17). Hypoxic Stress-induced Changes in HLS and HCE Cell Markers—p63 and keratin 12 (K12) proteins have significantly higher expression in human limbal stem and center corneal epithelial cells, respectively. We performed further experiments to substantiate the evidence that Plk[3] plays an important role in hypoxic stress-induced limbal stem and corneal epithelial cell fates by studying Plk[3] activity-deficient cells and corneas from wild-type and Plk3Ϫ/Ϫ knockout mice. Immunostaining study results were further verified by Western blotting analysis using primary cultured corneal epithelial cells from wild-type and Plk3Ϫ/Ϫ knockout mice 1 and 4 h after hypoxic stimulation, respectively (Fig. 5E). The results indicate that hypoxia-induced differentiation in human limbal stem cells requires suppression of Plk[3] activity to apoptosis under hypoxic stress conditions. Results obtained from Plk3Ϫ/Ϫ corneas and MEF cells provided more evidence showing that Plk[3] plays a significant role in the mediation of hypoxic stress in various cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
