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J Mario Wolosin, Quin Liu, Zheng Wang, Celine Portal, Daniel K Scott, Carlo Iomini; K14+ epithelial-targeted Myc ablation induces ΔDNp63 overexpression in corneal epithelium.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2243. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the impact of MYC in the stationary and wound-activated states in mouse corneal epithelium.
Ablation of Myc in the epidermis and corneal epithelium was obtained by crossbreeding floxed MYC and K14-cre mice to obtain the K14cre;Mycfx/fx. MYC gene ablation was monitored by PCR of DNA. Depletion of the Myc mRNA in the corneal epithelium was confirmed using real-time PCR. Health, and survival were monitored for 45 d. Corneal epithelial stratification was then assessed from plastic-embedded sections. Immunohistology of frozen sections was used to determine BrdU incorporation after 4 h administration and Ki67 and keratin distibution. Wound closure rates were determined by the reduction of fluorescein stain following a limbus to limbus epithelial debridement. Western blot and real-time PCR were used to examine p63 isoform expression.
K14cre;Mycfx/fx (mutant) mice were born at a normal weight but, between P5 and P42, postnatal weight gain was only 47 % (n = 10; p<0.01) than that of heterozygous myc littermates. Shortly after weaning (~21 days) mutant mice displayed spontaneous skin ulcerations, in particular in the tail and the lower lip and occasionally die, most likely from malnutrition due to competitive displacement during lactation and subsequent food intake difficulties. In spite of these skin-specific defects, the corneal epithelium displayed a seemingly normal gross morphology. However, the central epithelium was 35 % thicker, the nuclear index for Ki67 was greatly reduced (the suprabasal keratin12 stain was markedly reduced, and surface cell exfoliation profiles were visible lower than in control. Additionally, upon debridement the wound closure rate in the K14cre;Mycfx/fx was more than 4 times slower than control (full closure < 24 h in control vs. >112 h in the knockout), and BrdU incorporation at the periphery at 24 h was reduced by 41 % (n = 3, p <0.05). Finally, Western blot analysis of Dispase-isolated corneal epithelium showed that ablation of MYC resulted in the abnormal, very high overexpression of ΔNp63, a major regulatory factor of the epithelial proliferation/ differentiation equilibrium.
Myc ablation causes specific changes in corneal epithelial features. These changes may be underpinned by a dramatic activation of ΔNp63. The driver for and impact of ΔNp63 overexpression remains to be determined.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Slow healing of the myc-/- corneal epithelium.
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