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. 2022 Feb 1;63(2):3.
doi: 10.1167/iovs.63.2.3.

The c-Myc Oncogene Maintains Corneal Epithelial Architecture at Homeostasis, Modulates p63 Expression, and Enhances Proliferation During Tissue Repair

Céline Portal  1 , Zheng Wang  2 , Donald K Scott  3 , J Mario Wolosin  2 , Carlo Iomini  1   4
Affiliations

The c-Myc Oncogene Maintains Corneal Epithelial Architecture at Homeostasis, Modulates p63 Expression, and Enhances Proliferation During Tissue Repair

Céline Portal et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The transcription factor c-Myc (Myc) plays central regulatory roles in both self-renewal and differentiation of progenitors of multiple cell lineages. Here, we address its function in corneal epithelium (CE) maintenance and repair.

Methods: Myc ablation in the limbal-corneal epithelium was achieved by crossing a floxed Myc mouse allele (Mycfl/fl) with a mouse line expressing the Cre recombinase gene under the keratin (Krt) 14 promoter. CE stratification and protein localization were assessed by histology of paraffin and plastic sections and by immunohistochemistry of frozen sections, respectively. Protein levels and gene expression were determined by western blot and real-time quantitative PCR, respectively. CE wound closure was tracked by fluorescein staining.

Results: At birth, mutant mice appeared indistinguishable from control littermates; however, their rates of postnatal weight gain were 67% lower than those of controls. After weaning, mutants also exhibited spontaneous skin ulcerations, predominantly in the tail and lower lip, and died 45 to 60 days after birth. The mutant CE displayed an increase in stratal thickness, increased levels of Krt12 in superficial cells, and decreased exfoliation rates. Accordingly, the absence of Myc perturbed protein and mRNA levels of genes modulating differentiation and proliferation processes, including ΔNp63β, Ets1, and two Notch target genes, Hey1 and Maml1. Furthermore, Myc promoted CE wound closure and wound-induced hyperproliferation.

Conclusions: Myc regulates the balance among CE stratification, differentiation, and surface exfoliation and promotes the transition to the hyperproliferative state during wound healing. Its effect on this balance may be exerted through the control of multiple regulators of cell fate, including isoforms of tumor protein p63.

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Conflict of interest statement

Disclosure: C. Portal, None; Z. Wang, None; D.K. Scott, None; J.M. Wolosin, None; C. Iomini None

Figures

Figure 1.
Figure 1.
Main phenotypic manifestations of the K14-cre;Mycfl/fl mouse. (A) Weight gain for male and female K14Cre;Mycfl/ fl versus control littermates (mean ± SD; n = 4 CKO females, 8 control females, 4 CKO males, and 6 control males). (B) Dorsal view of 7-week-old CKO and control littermates. Insets Bi and Bii show disorganization of the hair pattern; insets Biii and Biv show a loss of pigmentation periodicity and spontaneous development of abrasions in the tail. (C) Lip degeneration in black and white mixed-background CKO mice. Pronounced abrasions at the mucocutaneous junctions develop in 100% of these CKO mice by the second or third week of life. The extent of the abrasions is more pronounced in white-background CKO mice, where it extends to the upper lip (bottom micrographs). (D) Histological defects in the tail of adult mice. (Top frames) The desquamation pattern observed in cross-sections of the tail in the epidermis (arrowheads) of the CKO is markedly more abundant than the one observed in the control. (Bottom frame) A histological section at the intersection between the epithelialized and abraded area of the tail in the CKO mouse. Arrows point to the epidermal ends of the abrasion. (E, top frames) neonatal mice heads. There are no evident overt differences in mucocutaneous epithelia between the CKO and control mice. (Center frames) The CKO epithelium is only one or two layers thick, and the Krt layer is very thin compared with that of the control (see insets). (Bottom frames) Skin side of the mucocutaneous zone; epifluorescent visualization of H&E staining. Arrowheads indicate desquamation areas. Arrows point to the epidermal ends of the tail abrasion shown in Biv. EPI, epithelium; ns, not significant (P ≥ 0.05, Student's t-test). *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars: 500 µm.
Figure 2.
Figure 2.
Ablation of Myc results in abnormal CE thickness and strata organization. (A) Myc western blot of isolated CE from control and CKO mice corneas. (B–D) Hematoxylin-only staining of cross-sections of central corneas (B, C) and the limbus (D) from mice at P21. (E) Semi-thin cross-sections of corneas embedded in epoxy resin from mice at P21. Representative micrographs (left) and mean central CE thickness (right side) are shown (mean ± SD, n = 3). (A–E) Images show that, in the CKO mice, the normal flattening of the cells at the surface of the CE was delayed, generating thicker strata incorporating extra superficial nucleated layers. (F) Representative micrographs of the corneal surface of P55 mice following whole eye exposure to 0.4% trypan blue in PBS for 5 minutes. In the CKO corneas, the frequency of staining profiles corresponding to overtly devitalized cells is less than 1/5 of the frequency of control corneas (mean ± SD, n = 3/group). ns, not significant (P ≥ 0.05, Student's t-test). *P < 0.05, **P < 0.001.
Figure 3.
Figure 3.
CE wound-healing response in the absence of Myc. (A) Wound closure of the CE in 2-month-old mice of a circular Algerbrush-generated corneal debridement with a diameter of 1.5 mm. The wound size was visualized by fluorescein staining, and rates of wound closure were quantified over time as indicated (n = 3/group). (B) Representative images of stained flatmounts showing BrdU (green) incorporation at the corneal periphery in intact corneas or 24 hours after debridement (wounded). The proliferation rate was determined by normalizing the number of BrdU-positive nuclei to the total number of DAPI-stained nuclei (mean ± SD, n = 2/group). (C) Ki-67 staining of cross cryosections at the periphery of intact corneas. The ratio between stained basal cells expressing Ki67 (red) and DAPI-stained nuclei (blue) was quantified. Note that in the intact CE there were no differences in the Ki67 stain and BrdU incorporation rates between control and CKO littermates (mean ± SD, n = 3/group). (D) Western blots for Myc and p-(Ser62)-Myc (pMyc) in isolated CE before and 24 hours after 1.5-mm-diameter corneal debridement relative to two similar experiments. Note that, before debridement, the activated Myc, p-(Ser62)-Myc, levels were not detectable in one experiment (lower panels) but were faintly visible in another (higher panels). ns, not significant (P ≥ 0.05, Student's t-test). *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Scale bars: 25 µm.
Figure 4.
Figure 4.
Expression and localization of TP63 isoforms and other selected factors associated with CE homeostasis and stratification in absence of Myc. (A, left panel) CE immunostaining of Krt12 (red) and DAPI (blue) on cross-sections of the paraffin-embedded cornea. The Abs utilized were directed to either the N- or the C-terminus of the Krt12 protein. Krt12 accumulation at the CE surface observed in the mutant using either Ab was severely reduced in the CE of the CKO. Bar graphs represent the percentage of Krt12 apical staining along the CE length of cornea cross-sections (mean ± SD, n = 3). (A, right panel) Line-intensity profile plot of Krt12 fluorescence signal across a CE cross-section. The y-axis represents the average intensity of 15 line-intensity scans (31 µm wide) from the basal membrane (BM) to the apical side of the CE taken every 100 µm along the length of each cornea section. The x-axis represents the length of the line scan from the BM to the apical side of the CE (n = 3). (B) CE immunostaining with Ab directed to the epithelial-specific ΔNp63 (n = 4/group). (C) CE immunostaining with the pan (TP) p63 Abs (n = 2 control and 4 CKO). The bar graphs in B and C describe the mean ± SD values of p63 intensities (green fluorescence) over the whole epithelium, normalized by the corresponding nuclear stain (blue fluorescence). (D, left panel) GAPDH-normalized CKO/control chemiluminescence signal ratios (mean ± SD). Number of independent replicates for each protein are indicated on top of the bars. Myc ablation led to a fourfold increase of ΔNp63 and about twofold increases in ETS1 and Notch 1. Krt14 and Krt 12 displayed no substantial differences in two duplicates and were not quantitated. (E) Level of expression of p63 isoforms relative to Gapdh in the wild-type mouse (mean ± SD, n = 3/group). (F) Expression of p63 isoforms and genes involved in the CE differentiation. Data are represented as the fold change in the CKO relative to the control (n = 3/group). ns, not significant (P ≥ 0.05, Student's t-test). *P < 0.05, **P < 0.001. Scale bars: 25 µm.
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References

    1. Marques-Pereira JP, Leblond CP.. Mitosis and differentiation in the stratified squamous epithelium of the rat esophagus. Am J Anat. 1965; 117: 73–87. - PubMed
    1. Rheinwald JG, Green H.. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975; 6(3): 331–343. - PubMed
    1. Lavker RM, Sun TT.. Epithelial stem cells: the eye provides a vision. Eye (Lond). 2003; 17(8): 937–942. - PubMed
    1. Mackenzie IC, Bickenbach JR.. Label-retaining keratinocytes and Langerhans cells in mouse epithelia. Cell Tissue Res. 1985; 242(3): 551–556. - PubMed
    1. Lehrer MS, Sun TT, Lavker RM.. Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation. J Cell Sci. 1998; 111(pt 1): 2867–2875. - PubMed

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