February 2016
Volume 57, Issue 2
Open Access
Cornea  |   February 2016
APR-246/PRIMA-1Met Inhibits and Reverses Squamous Metaplasia in Human Conjunctival Epithelium
Author Affiliations & Notes
  • Jing Li
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Cheng Li
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Guoliang Wang
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Zhen Liu
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Pei Chen
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Qichen Yang
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
  • Nuo Dong
    Xiamen University Affiliated Xiamen Eye Center, Xiamen, Fujian, China
  • Huping Wu
    Xiamen University Affiliated Xiamen Eye Center, Xiamen, Fujian, China
  • Zuguo Liu
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
    Xiamen University Affiliated Xiamen Eye Center, Xiamen, Fujian, China
  • Wei Li
    Eye Institute of Xiamen University Xiamen, Fujian, China
    Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China
    Xiamen University Affiliated Xiamen Eye Center, Xiamen, Fujian, China
  • Correspondence: Zuguo Liu, Eye Institute of Xiamen University, 4th Floor, Chengyi Building, Xiang'an Campus of Xiamen University, Xiang'an South Road, Xiamen, Fujian, 361102, China; zuguol@xmu.edu.cn
  • Wei Li, Eye Institute of Xiamen University, 4th Floor, Chengyi Building, Xiang'an Campus of Xiamen University, Xiang'an South Road, Xiamen, Fujian, 361102, China; wei1018@xmu.edu.cn
  • Footnotes
     JL and CL contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science February 2016, Vol.57, 444-452. doi:10.1167/iovs.15-17519
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      Jing Li, Cheng Li, Guoliang Wang, Zhen Liu, Pei Chen, Qichen Yang, Nuo Dong, Huping Wu, Zuguo Liu, Wei Li; APR-246/PRIMA-1Met Inhibits and Reverses Squamous Metaplasia in Human Conjunctival Epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(2):444-452. doi: 10.1167/iovs.15-17519.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Squamous metaplasia is a common pathologic condition in ocular surface diseases for which there is no therapeutic medication in clinic. In this study, we investigated the effect of a small molecule, APR-246/PRIMA-1Met, on squamous metaplasia in human conjunctival epithelium.

Methods: Human conjunctival explants were cultured for up to 12 days under airlifting conditions. Epithelial cell differentiation and proliferation were assessed by Cytokeratin 10 (K10), K14, K19, Pax6, MUC5AC, and p63 immunostaining patterns. β-catenin and TCF-4 immunofluorescent staining and real-time PCR characterized Wnt signaling pathway involvement. Pterygium clinical samples were cultured under airlifting conditions with or without APR-246 for 4 days. p63, K10, β-catenin, and TCF-4 expression in pterygial epithelium was determined by immunofluorescent staining and real-time PCR.

Results: Airlift conjunctival explants resulted in increased stratification and intrastromal epithelial invagination. Such pathology was accompanied by increases in K10, K14, and p63 expression, whereas K19 and Pax6 levels declined when compared to those in freshly isolated tissue. On the other hand, APR-246 reversed all of these declines in K10, K14, and p63 expression. Furthermore, K19 and Pax6 increased along with rises in goblet cell density. These effects of APR-246 were accompanied by near restoration of normal conjunctival epithelial histology. APR-246 also reversed squamous metaplasia in pterygial epithelium that had developed after 4 days in ex vivo culture.

Conclusions: Reductions in squamous metaplasia induced by APR-246 suggest it may provide a novel therapeutic approach in different squamous metaplasia–associated ocular surface diseases.

Squamous metaplasia is a common pathologic process induced by injury and inflammation in which a nonkeratinized, mucous-secreting stratified epithelium is gradually replaced by a nonsecretory, keratinized epithelium.1,2 This pathologic process occurs in respiratory tract epithelium,3 urothelium,4 nonneoplastic breast,5 salivary tissues,6 and ocular surface epithelial cells. In the corneal and conjunctival epithelium, such pathology can develop as a consequence of a number of ocular surface diseases such as Sjögren syndrome, ocular cicatricial pemphigoid, Stevens-Johnson syndrome, pterygium, chemical/thermal burns, and vitamin A deficiency,1,2,7,8 which can lead to visual impairment and even blindness. The severity of squamous metaplasia and these ocular diseases correlate well with one another. Our recent studies have gained insights into the mechanisms underlying pathogenesis of squamous metaplasia.7,9 Moreover, we have also found that either coculturing conjunctival epithelium with amniotic membrane or exposing it instead to an amniotic membrane–soluble extract prevents and reverses squamous metaplasia.9 However, it is still unclear which of the numerous amniotic membrane components contribute to this process. Till now, no single molecule has been identified that has therapeutic effects on squamous metaplasia. 
APR-246/PRIMA-1Met (methylated form of p53-dependent reactivation of massive apoptosis) is a small molecule that is capable of restoring the sequence-specific DNA-binding and transcriptional transactivation by mutant p53 in tumor cells.1012 It can induce several p53 target genes and mutant p53-dependent apoptosis in human tumor cells10,13 and can inhibit human xenograft tumor growth in mice.10 It has been tested in a phase I/II clinical trial in patients with hematologic malignancies or hormone-refractory prostate cancer.14 Recent study has shown that APR-246 could rescue mutant p63 in primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations. Keratinocytes from these patients show impaired epidermal differentiation. Treatment with APR-246 partially rescues morphology and gene expression during epidermal stratification and restores expression of p63-regulated genes.15 In addition, APR-246 reverses corneal epithelial lineage commitment and restores a normal p63-related signaling pathway in induced pluripotent stem cells (iPSCs) from EEC patients carrying two different point mutations in the DNA-binding domain of p63.16 
These findings prompted us to hypothesize that APR-246 may have an effect on squamous metaplasia of the ocular surface epithelium, which occurs as a consequence of normal epithelial cells undergoing transdifferentiation. Here we used a well-established ex vivo tissue culture squamous metaplasia model to test this hypothesis. Indeed we found that APR-246 prevented and reversed squamous metaplasia in airlifted conjunctival tissue and restored the normal pterygial conjunctival epithelial phenotype in clinical samples. 
Materials and Methods
Materials and Reagents
PRIMA-1MET/APR-246 (2,2-Bishydroxymethyl-1-aza-bicyclo[2.2.2]octan-3-one) was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), Ham's/F12 medium, HEPES buffer, fetal bovine serum (FBS), amphotericin B, mouse epidermal growth factor (EGF), TRIzoland Alexa Fluor 488 goat anti-mouse, goat IgGs were from Life Technologies (Carlsbad, CA, USA). Hydrocortisone, dimethyl sulfoxide, cholera toxin, 0.3% hydrogen peroxide, insulin-transferrin-sodium selenite media supplement, acetone, Triton X-100, bovine serum albumin (BSA), and FITC-conjugated anti-rabbit IgG were from Sigma-Aldrich Corp. Mouse anti-cytokeratin 10 (K10), cytokeratin 14 (K14), cytokeratin 19 (K19) antibodies, and diaminobenzidine (DAB) were from Dako Cytomation (Carpinteria, CA, USA). Mouse anti-Pax6 and β-catenin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-MUC5AC antibody and rabbit anti-p63 antibody were from Abcam (Cambridge, MA, USA). Immunofluorescence mounting medium with DAPI (4,6-diamino-2-phenyl indole) was from Vector Laboratories (Burlingame, CA, USA). Six-well and 12-well plate inserts were from Merck Millipore (Bedford, MA, USA). Plastic cell culture dishes were from BD Biosciences (Lincoln Park, NJ, USA). 
Human Conjunctival Explant Culture
This study was approved by the Ethics Committee of Xiamen Eye Center (Xiamen, Fujian, China). Human conjunctival tissues were handled in accordance with the Declaration of Helsinki. Bulbar conjunctival tissues from human donor eyes without ocular surface disease history were obtained from the Eye Bank of Xiamen Eye Center. These tissues were harvested within 8 hours postmortem and stored in Optisol medium for less than 24 hours before experimentation. The tissues were rinsed three times with PBS containing 1.25 mg/mL amphotericin B and 50 mg/mL gentamicin. The conjunctival tissues with epithelium and stroma were cut into pieces approximately 3 × 3 mm after carefully removing excessive Tenon's capsule. Some pieces were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Miles, Inc., Elkhart, IN, USA) and frozen at −80°C, and others were immediately used for RNA isolation. We defined these tissues as normal conjunctival control. The rest of the conjunctival tissue pieces were used as explants and placed epithelial side up on inserts and cultured in supplemented hormonal epithelial medium (SHEM) in the presence or absence of 3 μM and 15 μM APR-246. The SHEM contained an equal volume of HEPES-buffered DMEM, bicarbonate, and Ham/F12, supplemented with 5% FBS, 0.5% dimethyl sulfoxide, 2 ng/mL mouse EGF, 5 mg/mL insulin, 5 mg/mL transferrin, 5 ng/mL selenium, 0.5 mg/mL hydrocortisone, 1 nM cholera toxin, 50 mg/mL gentamicin, and 1.25 mg/mL amphotericin B. Explants were airlifted and incubated under a humidified atmosphere of 5% CO2 and 95% air at 37°C (Fig. 1A). The medium was changed every day for up to 12 days.7 
Figure 1
 
Histology of conjunctival tissue. (A) Schematic drawing of airlift culture. Conjunctival tissue was cut into pieces and placed on the inserts with epithelial side up and cultured in SHEM with or without APR-246 for different durations. (B) Histology of conjunctival tissues cultured under the airlifting condition without APR-246 exhibited hyperproliferation of epithelial cells, undulation, and digital invasion of epithelium. In contrast, incubation with either 3 or 15 μM APR-246 prevented hyperproliferation. In the presence of 15 μM APR-246, the epithelial surface maintained near smooth surface after 12 days in culture. Scale bar: 100 μm. (C) Conjunctival epithelial cell layer counting showed dramatic increase of cell layers in control group at day 12 in airlift culture, while it significantly decreased in APR-246–treated groups (***P < 0.001).
Figure 1
 
Histology of conjunctival tissue. (A) Schematic drawing of airlift culture. Conjunctival tissue was cut into pieces and placed on the inserts with epithelial side up and cultured in SHEM with or without APR-246 for different durations. (B) Histology of conjunctival tissues cultured under the airlifting condition without APR-246 exhibited hyperproliferation of epithelial cells, undulation, and digital invasion of epithelium. In contrast, incubation with either 3 or 15 μM APR-246 prevented hyperproliferation. In the presence of 15 μM APR-246, the epithelial surface maintained near smooth surface after 12 days in culture. Scale bar: 100 μm. (C) Conjunctival epithelial cell layer counting showed dramatic increase of cell layers in control group at day 12 in airlift culture, while it significantly decreased in APR-246–treated groups (***P < 0.001).
Human Pterygial Explant Culture
Six cases of primary pterygia from the Xiamen Eye Center were enrolled in this study. All pterygia were surgically removed by one surgeon (HW). The tissues were first rinsed three times with PBS containing 1.25 mg/mL amphotericin B and 50 mg/mL gentamicin and then cut into pieces approximately 3 × 3 mm after removing the excessive pterygial stroma. Next, the tissues were placed on the inserts, with epithelial side facing up and cultured in the presence or absence of 15 μM APR-246 in SHEM. Cultures were incubated under the same conditions as those used for the conjunctival explants. The medium was changed daily during 4 days of culture. 
Histology and Immunostaining
Tissue samples were embedded in OCT compound and frozen at −80°C. Cryostat sections (6 μm) were fixed in acetone for 10 minutes at −20°C and washed three times in PBS and then incubated in 0.2% Triton X-100 for 15 minutes. Subsequently, they were rinsed with PBS for 5 minutes each followed by exposure for 1 hour to 2% BSA to block nonspecific binding, and then overlaid with primary antibodies overnight at 4°C with different dilutions (K10 and K19 at 1:50, β-catenin and K14 at 1:100, MUC5AC at 1:200). Identical dilutions of homologous serum of different primary antibodies were added as negative control. On the next day, the sections were repeatedly washed three times for 30 minutes each with PBS and incubated with an FITC-conjugated secondary antibody (donkey anti-rabbit or anti-mouse IgG at 1:200) for 1 hour at 37°C. After three additional PBS washes for 30 minutes each, the sections were then mounted with mounting medium containing DAPI and photographed under a Nikon TE-2000 U Eclipse epifluorescence microscope (Nikon Instruments, Tokyo, Japan). 
In β-catenin immunostaining, we counted β-catenin–positive epithelial cells in three different high-power (×400) fields in triplicate sections of each group. Among the β-catenin–positive epithelial cells, cells with nuclear β-catenin translocation were also counted in the same field. The percentage was then calculated of nuclear β-catenin–positive cells among the total number of β-catenin–positive epithelial cells. 
For immunohistochemical staining, the endogenous peroxidase activity was blocked by exposure to 0.3% hydrogen peroxide for 10 minutes after fixation, while nonspecific staining was blocked by 2% BSA for 30 minutes. After that, sections were incubated with antibodies overnight at 4°C with varying dilutions (p63 at 1:500, Pax6 at 1:100). Identical dilutions of homologous serum of primary antibodies were added as negative control. After consecutively washing with PBS three times for 30 minutes each, the sections were incubated with biotinylated anti-mouse or anti-rabbit IgG (1:50) at 37°C for 1 hour. The reaction product was then developed with DAB for 2 minutes. Sections were mounted and examined with a light microscope (Eclipse 50i; Nikon Instruments). 
RNA Isolation and Real-Time Polymerase Chain Reaction
RNA was isolated from different explants at the beginning and end of the culture by using TRIzol (Invitrogen, Carlsbad, CA, USA). RNA sample parameters and concentrations were detected by NanoDrop 1000TM (Thermo Fisher Scientific, Waltham, MA, USA). Equal amount of RNA was reverse transcribed to cDNA by using the ExScript RT Reagent kit. Real-time polymerase chain reaction (PCR) was performed with a StepOne Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) using a SYBR Premix Ex Taq Kit (Takara Bio, Otsu, Shiga, Japan), according to the manufacturer's instructions. For each experiment, RT-minus controls (i.e., RNA samples that are treated similarly but without addition of reverse transcriptase) and template-minus controls were included to provide negative controls for subsequent PCR reactions. The amplification program included an initial denaturation step at 95°C for 10 seconds, followed by denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 35 seconds, for 40 cycles. SYBR Green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by melting curve analysis. The primers used to amplify specific gene products from cDNA are shown in the Table. The results of the relative quantitative real-time PCR were analyzed by the comparative CT method and normalized to β-actin as an internal control. 
Table
 
Primer Sequence Pairs Used for Quantitative Real-Time PCR
Table
 
Primer Sequence Pairs Used for Quantitative Real-Time PCR
Image Analysis
To analyze the relative mean density, we first measured the integrated optical density (IOD) expression of positive immunostaining in the conjunctival epithelium. Cytokeratin 19-, K10-, MUC5AC-, Pax6-, p63-, and K14-immunostained images from sections were processed (Image Pro Plus V6.0; Media Cybernetics, Silver Spring, MD, USA) as previously reported.8 In brief, the exposure time was the same when capturing immunostaining images from multiple samples stained by one specific antibody in different groups. After that, correction of unequal illumination (shading correction) and calibration of the measurement system were performed with a reference slide after the images were recorded. The images of immunostained slides were converted to a gray scale, and the IOD was measured as a linear combination between the average gray intensity and the relative area occupied by positive cells. The IOD scores were obtained from the epithelium of the conjunctival tissues, and then the epithelial area of each image was calculated. Finally, the relative epithelial to IOD ratio was calculated, which represents the relative mean density. For each sample, different areas of four to seven sections were scored and subjected to statistical analysis. 
Statistical Analysis
All experiments were repeated at least three times. Quantitative data are presented as means ± SD. Group mean data were compared by using 2-way ANOVA, where P < 0.05 was considered statistically significant. The statistical analysis was conducted with Prism 5.0 software (GraphPad Prism for Windows, version 5.00; GraphPad Software, Inc., La Jolla, CA, USA). 
Results
APR-246 Reverses Histologic Changes Resulting From Airlifting Human Conjunctival Tissue
Freshly isolated conjunctival epithelium appeared intact and smooth with three to six layers. On the other hand, after 12 days of airlifting, the epithelium had undergone hyperproliferation and stratification reaching approximately 15 cell layers. In addition, finger-like epithelial invagination into the underlying stromal occurred. APR-246 at a final concentration of 3 μM and 15 μM was used in this study, based on previous reports.15,16 With the 3 μM APR-246–treated group, conjunctival epithelial stratification decreased from day 4 to day 12. There was some invagination at 4 days, but at 8 and 12 days it was much less evident than in the untreated control group. Meanwhile, epithelial stratification was further reduced in the 15 μM APR-246 group, and there was no stromal invagination (Fig. 1B). Statistical analysis showed that the number of epithelial cell layers in the control group dramatically increased as compared with that in the freshly isolated tissue, while it significantly decreased in APR-246–treated groups compared with the control group (Fig. 1C; ***P < 0.001). 
APR-246 Suppresses Conjunctival Epithelial Transdifferentiation
Cytokeratin 19 and K10 keratin expression profiles were evaluated during airlifting to determine if APR-246 exposure maintained the conjunctival epithelial phenotype. As reported, K19, the major conjunctival epithelial cytokeratin, was uniformly expressed in all freshly isolated conjunctival epithelial cells.17 However, its expression gradually decreased from days 4 to 12 in airlift culture, whereas K19 was nearly maintained in the full-thickness epithelial cells throughout the culture duration with 15 μM APR-246 (Fig. 2A). Similarly, relative IOD analysis showed that there was a significant decrease in K19 expression in the conjunctival tissue cultured in the airlifting condition without APR-246 for 12 days, compared with that in normal freshly isolated conjunctiva. There was no statistical difference between K19 expression in the normal freshly isolated human conjunctival epithelium and the APR-246–treated group (Fig. 2B; ***P < 0.001). Quantitative real-time PCR K19 expression also dramatically decreased in airlift culture, while it was rescued by culturing conjunctival tissue with APR-246 (Fig. 2C; *P < 0.05, ***P < 0.001). 
Figure 2
 
Cytokeratin 19 expression in conjunctival tissue. (A) Cytokeratin 19 was expressed in the full thickness of normal conjunctival epithelium, while its expression gradually decreased from days 4 to 12 in airlift culture. With 3 μM or 15 μM APR-246, K19 expression was well maintained in the conjunctival epithelium at day 12. Scale bar: 100 μm. Both relative IOD analysis (B) and real-time PCR (C) showed that there was significant decrease of K19 expression in the conjunctival tissue cultured in the airlift condition without APR-246 for 12 days as compared with the normal conjunctiva (*P < 0.05, ***P < 0.001).
Figure 2
 
Cytokeratin 19 expression in conjunctival tissue. (A) Cytokeratin 19 was expressed in the full thickness of normal conjunctival epithelium, while its expression gradually decreased from days 4 to 12 in airlift culture. With 3 μM or 15 μM APR-246, K19 expression was well maintained in the conjunctival epithelium at day 12. Scale bar: 100 μm. Both relative IOD analysis (B) and real-time PCR (C) showed that there was significant decrease of K19 expression in the conjunctival tissue cultured in the airlift condition without APR-246 for 12 days as compared with the normal conjunctiva (*P < 0.05, ***P < 0.001).
Cytokeratin 10 is an epidermal keratinocyte-specific intermediate filament. Its expression was negative in normal conjunctival epithelium. In airlift cultures, K10-positive cells gradually increased from day 8 to 12 in the superficial cell layers. In contrast, there were fewer K10-positive epithelial cells in cultures with 3 μM APR-246 at days 8 and 12. Furthermore, with 15 μM APR-246 there were no K10-positive cells throughout the entire culture period (Fig. 3A). The IOD analysis confirmed that after airlifting for 12 days, K10 expression increased in the control group, while it was downregulated in the APR-246–treated group (Fig. 3B; ***P < 0.001). Quantitative real-time PCR also showed that K10 was upregulated under this condition, while it was dramatically downregulated at day 12 in the APR-246–treated group (Fig. 3C; ***P < 0.001). These results indicate that APR-246 inhibited abnormal epidermal transdifferentiation and maintained the normal conjunctival epithelial phenotype. 
Figure 3
 
Cytokeratin 10 expression in conjunctival tissue. (A) Cytokeratin 10 expression was negative in normal conjunctival epithelium. In airlift cultures, K10-positive cells gradually increased from days 8 to 12 in the superficial cell layers. There were fewer K10-positive epithelial cells in cultures with 3 μM APR-246 at days 8 and 12. Furthermore, with 15 μM APR-246, there were no K10-positive cells throughout the culture duration. Scale bar: 100 μm. The IOD analysis (B) and quantitative real-time PCR (C) confirmed a significant increase of K10 expression level in airlift culture, while it was downregulated dramatically at day 12 in the APR-246 treated groups (***P < 0.001).
Figure 3
 
Cytokeratin 10 expression in conjunctival tissue. (A) Cytokeratin 10 expression was negative in normal conjunctival epithelium. In airlift cultures, K10-positive cells gradually increased from days 8 to 12 in the superficial cell layers. There were fewer K10-positive epithelial cells in cultures with 3 μM APR-246 at days 8 and 12. Furthermore, with 15 μM APR-246, there were no K10-positive cells throughout the culture duration. Scale bar: 100 μm. The IOD analysis (B) and quantitative real-time PCR (C) confirmed a significant increase of K10 expression level in airlift culture, while it was downregulated dramatically at day 12 in the APR-246 treated groups (***P < 0.001).
To investigate whether APR-246 had an effect on goblet cell differentiation in airlift cultures, we examined the expression of the most abundant ocular surface mucin, MUC5AC.18 In the normal conjunctival epithelium, MUC5AC-positive cells were sporadically distributed. At day 4 after culturing airlifted conjunctiva without APR-246, MUC5AC staining significantly declined and was totally absent by day 8. With 3 μM APR-246, the number of MUC5AC-positive cells also declined at day 4 but showed sporadic expression from days 8 to 12. However, with 15 μM APR-246, there was no decrease in the number of MUC5AC-positive cells on day 4. Nevertheless, MUC5AC-positive cells became prominent between days 8 to 12 (Fig. 4A). The IOD analysis showed that MUC5AC expression was restored to a certain extent in cultures with APR-246 at day 12 (Fig. 4B; ***P < 0.001). Real-time PCR also showed that MUC5AC gene expression was at a higher level in the 15 μM APR-246 group than in the airlift group without APR-246 at day 12 (Fig. 4C; ***P < 0.001). However, the MUC5AC mRNA level was still much lower than that in the normal control (Fig. 4C; ***P < 0.001). 
Figure 4
 
MUC5AC expression in conjunctival tissue. (A) MUC5AC became negative after 4 days of airlift culture without APR-246. In the 3 μM drug-treated group, MUC5AC was undetectable at day 4, but reappeared at day 8 and had increased by day 12. MUC5AC expression also decreased in conjunctival epithelium at day 4 with 15 μM APR-246, but it was rescued from days 8 to 12. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that MUC5AC expression was significantly reduced in the control group, whereas it was higher in APR-246 treated groups on day 12 in airlift cultures (***P < 0.001).
Figure 4
 
MUC5AC expression in conjunctival tissue. (A) MUC5AC became negative after 4 days of airlift culture without APR-246. In the 3 μM drug-treated group, MUC5AC was undetectable at day 4, but reappeared at day 8 and had increased by day 12. MUC5AC expression also decreased in conjunctival epithelium at day 4 with 15 μM APR-246, but it was rescued from days 8 to 12. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that MUC5AC expression was significantly reduced in the control group, whereas it was higher in APR-246 treated groups on day 12 in airlift cultures (***P < 0.001).
Pax6 (paired-box protein 6) expression evaluation helped determine whether transdifferentiation preceded squamous metaplasia development. Pax6 is a master regulator of ocular mucosal lineage commitment that is necessary for normal epithelial cell growth, directing corneal epithelial cell migration and activation of limbal stem cells.19 Pax6 was uniformly expressed in nearly all the nuclei throughout the full thickness of normal conjunctival epithelium before culture. The nuclear expression of Pax6 decreased in some basal and suprabasal epithelial cells at day 8, and in the full-epithelial thickness it was almost undetectable at day 12 when cultured without APR-246. On the contrary, in conjunctival explants cultured with APR-246, Pax6-positive nuclei remained in the full-thickness conjunctival epithelium from days 4 to 12 in both 3 μM and 15 μM APR-246–treated groups (Fig. 5A). Both IOD analysis and real-time PCR showed significant declines in Pax6 expression in conjunctiva cultured without APR-246. However, irrespective of APR-246 concentration, Pax6 expression levels were maintained (Figs. 5B, 5C; ***P < 0.001). All of these findings are in agreement with an earlier study describing the molecular events, which underlie the development of squamous metaplasia in this model.20 We showed that APR-246 significantly prevented these changes and preserved normal conjunctival epithelial histology. 
Figure 5
 
Pax6 expression in conjunctival tissue. (A) Normal conjunctival epithelial cells exhibit widespread positive Pax6 cells throughout the whole epithelial thickness. Under the airlifting condition, Pax6 expression underwent dramatic decrease in the superficial and basal layers from days 4 to 12, and become almost undetectable at day 12 in the airlift culture without APR-246. However, Pax6 expression in culture with 3 or 15 μM APR-246 remained at a level similar to that in the normal freshly isolated conjunctival epithelial cells. Scale bar: 100 μm. This protective effect of 3 or 15 μM APR-246 against declines in Pax6 expression was also confirmed by relative IOD analysis (B) and real-time PCR (C) (***P < 0.001).
Figure 5
 
Pax6 expression in conjunctival tissue. (A) Normal conjunctival epithelial cells exhibit widespread positive Pax6 cells throughout the whole epithelial thickness. Under the airlifting condition, Pax6 expression underwent dramatic decrease in the superficial and basal layers from days 4 to 12, and become almost undetectable at day 12 in the airlift culture without APR-246. However, Pax6 expression in culture with 3 or 15 μM APR-246 remained at a level similar to that in the normal freshly isolated conjunctival epithelial cells. Scale bar: 100 μm. This protective effect of 3 or 15 μM APR-246 against declines in Pax6 expression was also confirmed by relative IOD analysis (B) and real-time PCR (C) (***P < 0.001).
APR-246 Suppresses Airlift-Induced Conjunctival Epithelial Hyperproliferation
Hyperproliferation in airlifted limbal explants was accompanied by squamous metaplasia.7 To determine whether this change occurs in airlifted conjunctival tissue, p63 and K14 immunostaining was performed. As reported, p63 is expressed in the basal epithelial and all stratified epithelial cell layers and thought to be a biomarker of progenitor cells.21 Its expression was evident in most of the basal and some suprabasal layers in freshly isolated conjunctiva. However, after 4 days of airlift culture, p63 expression dramatically increased in the basal epithelial cells and this trend remained continuous up to 12 days. In contrast, with APR-246 the increase after 4 days was much more modest. Furthermore, p63-positive nuclei were mostly confined to the basal and only some suprabasal epithelial cells at day 12, similarly to normal freshly isolated conjunctival epithelium (Fig. 6A). The IOD analysis and real-time PCR both underwent a dramatic increase in p63 expression in airlift culture, while p63 was significantly downregulated in cultures with APR-246 at day 12 (Figs. 6B, 6C; ***P < 0.001). 
Figure 6
 
p63 expression in conjunctival tissue. (A) p63 was expressed in basal and some suprabasal epithelial cell nuclei. p63 expression increased from days 4 to 12 in the airlifting condition without APR-246, while its expression was relatively stable during exposure to either 3 or 15 μM APR-246. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that APR exposure suppressed increases and spreading of p63 expression at day 12 (***P < 0.001).
Figure 6
 
p63 expression in conjunctival tissue. (A) p63 was expressed in basal and some suprabasal epithelial cell nuclei. p63 expression increased from days 4 to 12 in the airlifting condition without APR-246, while its expression was relatively stable during exposure to either 3 or 15 μM APR-246. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that APR exposure suppressed increases and spreading of p63 expression at day 12 (***P < 0.001).
Cytokeratin 14 keratin, a basal epithelial progenitor cell biomarker of all stratified epithelia, was noted in basal and suprabasal epithelial cells in the normal human conjunctival epithelium before culturing. There was a dramatic increase in K14 expression under the airlifting conditions from days 4 to 12 in the control group. Meanwhile, K14 expression in 3 μM and 15 μM APR-treated groups increased at day 4, while it decreased at days 8 and 12 to a level similar to that in normal conjunctival epithelium before culturing (Fig. 7A). Both IOD analysis and real-time PCR confirmed a significant difference in K14 expression between airlifted human conjunctival tissue cultured with and without APR-246 on day 12 (Figs. 7B, 7C; *P < 0.05, ***P < 0.001). These results indicate that APR-246 suppressed conjunctival epithelial hyperproliferation induced by airlifting conditions. 
Figure 7
 
Cytokeratin 14 expression in conjunctival tissue. (A) There was a slight increase in K14 expression from days 4 to 12 under airlifting conditions, while K14 expression in 3 and 15 μM APR-246–treated groups was stable and similar to the expression level in normal freshly isolated conjunctival epithelium. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that K14 expression level was significantly higher in conjunctiva cultured without APR-246 than in normal conjunctiva and explants cultured with either 3 or 15 μM APR-246 (*P < 0.05, ***P < 0.001).
Figure 7
 
Cytokeratin 14 expression in conjunctival tissue. (A) There was a slight increase in K14 expression from days 4 to 12 under airlifting conditions, while K14 expression in 3 and 15 μM APR-246–treated groups was stable and similar to the expression level in normal freshly isolated conjunctival epithelium. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that K14 expression level was significantly higher in conjunctiva cultured without APR-246 than in normal conjunctiva and explants cultured with either 3 or 15 μM APR-246 (*P < 0.05, ***P < 0.001).
APR-246 Suppresses Activation of Wnt Signaling Pathway in Airlift Cultures
Wnt signaling plays an essential role in regulating many cellular processes, including cell proliferation, inflammation, cell differentiation, migration, and cell survival.22,23 Our recent study has demonstrated that in airlifted conjunctival epithelial cultures the Wnt signaling pathway is transiently activated.20 To determine whether APR-246 inhibits such signaling, time-dependent changes were determined in β-catenin expression localization. β-catenin was expressed in the cell membrane and cytoplasm in the full-thickness freshly isolated normal conjunctival epithelium. In airlifted conjunctiva cultured without APR-246, β-catenin underwent membrane upregulation and was also translocated into the nuclei and cytoplasm of some epithelial cells at day 4. By day 12, its expression became prominent in the nuclei of basal epithelial cells and was weak in the superficial layers of conjunctival epithelium. However, in the conjunctiva cultured with APR-246, there was little β-catenin nuclear translocation from days 8 to 12. From days 4 to 8, β-catenin expression decreased by different degrees in the superficial layers in the APR-246–treated groups (Fig. 8A). In the control group, the percentage of β-catenin–nuclear positive cells was more than that in the APR-246 group whose localization and expression level was nearly the same as that in the normal conjunctival epithelium (Fig. 8B; *P < 0.05, ***P < 0.001). 
Figure 8
 
Wnt signaling pathway activation in conjunctival tissue. (A) In normal freshly isolated conjunctival epithelium, β-catenin expression was essentially cell membrane delimited throughout all tissue layers. During culturing from days 4 to 12 under airlifting conditions, there is some β-catenin translocation from the cell membrane to cytoplasmic and nuclear domains. The number of cells that show β-catenin nuclear translocation gradually increased. Meanwhile, there was also sporadic nuclear translocation of β-catenin from days 4 to 12 when cultured with 3 μM APR-246, which was slightly less with 15 μM APR-246. Scale bar: 100 μm. Inset: High-magnification picture represents nuclear translocation of β-catenin. (B) Percentage of cells with nuclear-positive β-catenin expression in the control group was greater than that in the APR-246 group, which was nearly the same as in normal conjunctival epithelium (*P < 0.05, ***P < 0.001). (C) Real-time PCR analysis of the TCF-4 gene expression changes also showed that APR-246 protects the tissue against Wnt signaling activation induced by tissue airlifting (***P < 0.001).
Figure 8
 
Wnt signaling pathway activation in conjunctival tissue. (A) In normal freshly isolated conjunctival epithelium, β-catenin expression was essentially cell membrane delimited throughout all tissue layers. During culturing from days 4 to 12 under airlifting conditions, there is some β-catenin translocation from the cell membrane to cytoplasmic and nuclear domains. The number of cells that show β-catenin nuclear translocation gradually increased. Meanwhile, there was also sporadic nuclear translocation of β-catenin from days 4 to 12 when cultured with 3 μM APR-246, which was slightly less with 15 μM APR-246. Scale bar: 100 μm. Inset: High-magnification picture represents nuclear translocation of β-catenin. (B) Percentage of cells with nuclear-positive β-catenin expression in the control group was greater than that in the APR-246 group, which was nearly the same as in normal conjunctival epithelium (*P < 0.05, ***P < 0.001). (C) Real-time PCR analysis of the TCF-4 gene expression changes also showed that APR-246 protects the tissue against Wnt signaling activation induced by tissue airlifting (***P < 0.001).
We also measured TCF-4 gene expression, which serves as a downstream gene target in the Wnt pathway. Real-time PCR showed that TCF-4 gene expression was at a higher level in the airlifting condition without APR-246 than in the cultured cells with APR-246 (Fig. 8C; ***P < 0.001). These results indicate that the Wnt signaling pathway was indeed activated from the early stages in the conjunctival airlifting condition and that it was downregulated to varying degrees when conjunctiva was cultured instead with APR-246. 
APR-246 Reversed Squamous Metaplasia of Pterygium
Squamous metaplasia is one of the characteristics of pterygial tissue.24 We determined whether culturing pterygial tissues with 15 μM APR-246 can reverse this condition. Hematoxylin and eosin (H&E) staining showed that there was a mild increase in epithelial thickness after 4 days in airlift culture without APR-246. However, culturing pterygial tissue with APR-246 inhibited this thickening process, as the number of cell layers was significantly less than that in the control group (Figs. 9A, 9B; *P < 0.05). As found previously, p63 was strongly expressed in pterygial epithelium before culture, while it dramatically decreased in pterygial tissues cultured with APR-246. Cytokeratin 10 was mainly expressed in superficial epithelial cell layers in pterygial samples. However, in pterygial tissues cultured with APR-246, K10-positive cells were instead sparse. β-catenin membrane expression and nuclear translocation in pterygial epithelium were detected after 4 days. However, in explants cultured instead with APR-246, there was little detectable β-catenin nuclear translocation on day 4 (Fig. 9A). Real-time PCR detected a significant difference in p63, K10, and TCF-4 expression between pterygial tissue cultured with and without APR-246 on day 4 (Fig. 9C; ***P < 0.001). 
Figure 9
 
Pterygium tissues treated with APR-246. (A) The H&E staining showed a mild increase of epithelial thickness after 4 days airlift culture of pterygium without APR-246, while there was an obvious decrease of epithelial thickness in pterygial tissues cultured with APR-246. p63 was strongly expressed in pterygium epithelium before culture, while it dramatically decreased in pterygial tissues cultured with APR-246. K10 was expressed in superficial epithelial cell layers in pterygial samples. However, in pterygial tissues cultured with APR-246, K10-positive cells were instead sparse. β-catenin membrane expression and nuclear translocation in pterygial epithelium were detected after 4 days. However, in explants cultured with APR-246, there was little detectable β-catenin nuclear translocation on day 4. Scale bar: 100 μm. (B) The number of epithelial cell layers in pterygium cultured with APR-246 was significantly less than that in the control group (*P < 0.05). (C) Real-time PCR analysis detected a significant difference in p63, K10, and TCF-4 gene expression between APR-246–cultured pterygial tissue and that without APR-246 on day 4 (***P < 0.001).
Figure 9
 
Pterygium tissues treated with APR-246. (A) The H&E staining showed a mild increase of epithelial thickness after 4 days airlift culture of pterygium without APR-246, while there was an obvious decrease of epithelial thickness in pterygial tissues cultured with APR-246. p63 was strongly expressed in pterygium epithelium before culture, while it dramatically decreased in pterygial tissues cultured with APR-246. K10 was expressed in superficial epithelial cell layers in pterygial samples. However, in pterygial tissues cultured with APR-246, K10-positive cells were instead sparse. β-catenin membrane expression and nuclear translocation in pterygial epithelium were detected after 4 days. However, in explants cultured with APR-246, there was little detectable β-catenin nuclear translocation on day 4. Scale bar: 100 μm. (B) The number of epithelial cell layers in pterygium cultured with APR-246 was significantly less than that in the control group (*P < 0.05). (C) Real-time PCR analysis detected a significant difference in p63, K10, and TCF-4 gene expression between APR-246–cultured pterygial tissue and that without APR-246 on day 4 (***P < 0.001).
Discussion
In this study, for the first time, we found APR-246 very markedly suppressed squamous metaplasia development in airlifted human conjunctival tissue explants. As we have previously reported,9 human conjunctival culture exposure to air for 4 days was sufficient to induce squamous metaplasia, which was accompanied by upregulation of K10, K14, and p63 expression and downregulation of K19, MUC5AC, and Pax6 expression. In the presence of APR-246, K10, K14, and p63 expression was downregulated, while K19, MUC5AC, and Pax6 expression levels were partially reversed to levels in freshly isolated conjunctival tissue. As a consequence of these restorative effects, conjunctival epithelial cells maintained their normal phenotype despite being airlifted for 12 days. 
One of the distinguishing characteristics of squamous metaplasia is hyperproliferation. p63, a member of the p53 family of transcription factors, affects stem cell proliferative capacity in stratified epithelia.25 p63 plays a critical role in squamous epithelia by initiating epithelial hyperstratification26 and contributes to squamous metaplasia-induced pulmonary fibrosis.27 In our previous reports7,9 and the current study, p63 expression dramatically increased in airlift cultures of both human limbal and conjunctival tissues, indicating that it contributes to the hyperproliferation of ocular surface epithelia. Even though the transcriptome changes underlying squamous metaplasia development are still not clear, this disease is likely related to gene abnormalities.26 
Interestingly, the hyperproliferation of conjunctival epithelial cells was largely blocked with APR-246 treatment. p63 expression was also significantly reduced in conjunctival epithelium. We presume the downregulation of p63 expression may be related to restoration of normal p63 function. APR-246 targets both mutant p53,10,28,29 and mutant p63.30 In the case of mutant p53, APR-246 restores wild-type conformation of mutant p53 and inhibits growth of a wide range of tumor cell lines,10 but it has no effect on normal p53 cells.31 In cells expressing mutant p63, APR-246 restores p63-dependent transdifferentiation in keratinocytes derived from EEC syndrome patients.16 Furthermore, APR-246 restores a normal p63-related signaling pathway in iPSCs obtained from EEC patients.16 Future studies are needed to further elucidate the mechanism through which APR-246 affects p63 expression. 
Another distinguishing characteristic of squamous metaplasia is abnormal differentiation. In airlifted conjunctival tissue cultures, conjunctival epithelial cells lost their normal phenotype and acquired an epidermal epithelial phenotype, along with gradual declines in goblet cell differentiation. However, APR-246 treatment inhibited this abnormal transdifferentiation process as well as declines in gel-forming mucin MUC5AC expression that were evident in the untreated group at day 12. This protective effect indicates that APR-246 treatment partially preserved goblet cell differentiation. 
The Wnt signaling pathway is an important modulator of cell proliferation and differentiation in various cell types.32 Whole-genome analysis has shown that p63 directly binds to genes including those controlling cell death and stem cell signaling and molecular expression involved in the Wnt signaling pathway.33 Wnt pathway activation is involved in squamous metaplasia development.32,34 In the current study, Wnt pathway activation occurred as evidenced by nuclear translocation of β-catenin and a high level of TCF-4 gene expression at day 12. In contrast, β-catenin remained at a relatively low level in conjunctival explants cultured with APR-246 as compared to those cultured without APR-246. These results suggest that APR-246–induced restoration of normal p63 function may reduce squamous metaplasia development through Wnt signaling inhibition. 
Furthermore, our study demonstrated that APR-246 reversed squamous metaplasia in pterygial tissue. These epithelial cells express the keratinocyte marker K10 and are hyperproliferative as evidenced by K14 and p63 levels that were higher than those in the normal conjunctival epithelium. APR-246 inhibited hyperproliferation as evidenced by p63 downregulation relative to its levels in pterygial explants cultured without APR-246. Similarly, β-catenin and TCF-4 gene expression decreased dramatically to relatively low levels in tissues cultured with APR-246. These effects suggest that APR-246 can reduce cell hyperproliferation along with squamous metaplasia in pterygium through inhibition of Wnt signaling by restoring normal p63 function. 
Taken together, the preventive effects of APR-246 on squamous metaplasia development in airlifted conjunctival explants and reversal of this pathologic condition in pterygial tissues agree with earlier studies on nonocular tissues, indicating that these changes may be a consequence of restoring normal p63 function. It is noteworthy that APR-246 had this restorative effect by reversing abnormal p63 expression irrespective of whether it was induced by airlifting or by an unknown mediator of pterygium pathology. The mechanism underlying how APR-246 restores normal p63 function requires future clarification. Our results point to the possibility that APR-246 may be of therapeutic benefit in a clinical setting for a host of ocular surface diseases in which squamous metaplasia contributes to the underlying pathology compromising normal vision. 
Acknowledgments
The authors are grateful to Peter Sol Reinach for his critical reading and valuable suggestions on the manuscript. 
Supported in part by grants from the National Natural Science Foundation of China (NSFC No. 81470601 [CL], U1205025 [ZL], 81270978 [ZL], 30931160432 [ZL], 81470600 [ND], and 81470602 [WL]), and Chinese National Key Scientific Research Project (No. 2013CB967003 [WL]). 
Disclosure: J. Li, None; C. Li, None; G. Wang, None; Z. Liu, None; P. Chen, None; Q. Yang, None; N. Dong, None; H. Wu, None; Z. Liu, None; W. Li, None 
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Figure 1
 
Histology of conjunctival tissue. (A) Schematic drawing of airlift culture. Conjunctival tissue was cut into pieces and placed on the inserts with epithelial side up and cultured in SHEM with or without APR-246 for different durations. (B) Histology of conjunctival tissues cultured under the airlifting condition without APR-246 exhibited hyperproliferation of epithelial cells, undulation, and digital invasion of epithelium. In contrast, incubation with either 3 or 15 μM APR-246 prevented hyperproliferation. In the presence of 15 μM APR-246, the epithelial surface maintained near smooth surface after 12 days in culture. Scale bar: 100 μm. (C) Conjunctival epithelial cell layer counting showed dramatic increase of cell layers in control group at day 12 in airlift culture, while it significantly decreased in APR-246–treated groups (***P < 0.001).
Figure 1
 
Histology of conjunctival tissue. (A) Schematic drawing of airlift culture. Conjunctival tissue was cut into pieces and placed on the inserts with epithelial side up and cultured in SHEM with or without APR-246 for different durations. (B) Histology of conjunctival tissues cultured under the airlifting condition without APR-246 exhibited hyperproliferation of epithelial cells, undulation, and digital invasion of epithelium. In contrast, incubation with either 3 or 15 μM APR-246 prevented hyperproliferation. In the presence of 15 μM APR-246, the epithelial surface maintained near smooth surface after 12 days in culture. Scale bar: 100 μm. (C) Conjunctival epithelial cell layer counting showed dramatic increase of cell layers in control group at day 12 in airlift culture, while it significantly decreased in APR-246–treated groups (***P < 0.001).
Figure 2
 
Cytokeratin 19 expression in conjunctival tissue. (A) Cytokeratin 19 was expressed in the full thickness of normal conjunctival epithelium, while its expression gradually decreased from days 4 to 12 in airlift culture. With 3 μM or 15 μM APR-246, K19 expression was well maintained in the conjunctival epithelium at day 12. Scale bar: 100 μm. Both relative IOD analysis (B) and real-time PCR (C) showed that there was significant decrease of K19 expression in the conjunctival tissue cultured in the airlift condition without APR-246 for 12 days as compared with the normal conjunctiva (*P < 0.05, ***P < 0.001).
Figure 2
 
Cytokeratin 19 expression in conjunctival tissue. (A) Cytokeratin 19 was expressed in the full thickness of normal conjunctival epithelium, while its expression gradually decreased from days 4 to 12 in airlift culture. With 3 μM or 15 μM APR-246, K19 expression was well maintained in the conjunctival epithelium at day 12. Scale bar: 100 μm. Both relative IOD analysis (B) and real-time PCR (C) showed that there was significant decrease of K19 expression in the conjunctival tissue cultured in the airlift condition without APR-246 for 12 days as compared with the normal conjunctiva (*P < 0.05, ***P < 0.001).
Figure 3
 
Cytokeratin 10 expression in conjunctival tissue. (A) Cytokeratin 10 expression was negative in normal conjunctival epithelium. In airlift cultures, K10-positive cells gradually increased from days 8 to 12 in the superficial cell layers. There were fewer K10-positive epithelial cells in cultures with 3 μM APR-246 at days 8 and 12. Furthermore, with 15 μM APR-246, there were no K10-positive cells throughout the culture duration. Scale bar: 100 μm. The IOD analysis (B) and quantitative real-time PCR (C) confirmed a significant increase of K10 expression level in airlift culture, while it was downregulated dramatically at day 12 in the APR-246 treated groups (***P < 0.001).
Figure 3
 
Cytokeratin 10 expression in conjunctival tissue. (A) Cytokeratin 10 expression was negative in normal conjunctival epithelium. In airlift cultures, K10-positive cells gradually increased from days 8 to 12 in the superficial cell layers. There were fewer K10-positive epithelial cells in cultures with 3 μM APR-246 at days 8 and 12. Furthermore, with 15 μM APR-246, there were no K10-positive cells throughout the culture duration. Scale bar: 100 μm. The IOD analysis (B) and quantitative real-time PCR (C) confirmed a significant increase of K10 expression level in airlift culture, while it was downregulated dramatically at day 12 in the APR-246 treated groups (***P < 0.001).
Figure 4
 
MUC5AC expression in conjunctival tissue. (A) MUC5AC became negative after 4 days of airlift culture without APR-246. In the 3 μM drug-treated group, MUC5AC was undetectable at day 4, but reappeared at day 8 and had increased by day 12. MUC5AC expression also decreased in conjunctival epithelium at day 4 with 15 μM APR-246, but it was rescued from days 8 to 12. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that MUC5AC expression was significantly reduced in the control group, whereas it was higher in APR-246 treated groups on day 12 in airlift cultures (***P < 0.001).
Figure 4
 
MUC5AC expression in conjunctival tissue. (A) MUC5AC became negative after 4 days of airlift culture without APR-246. In the 3 μM drug-treated group, MUC5AC was undetectable at day 4, but reappeared at day 8 and had increased by day 12. MUC5AC expression also decreased in conjunctival epithelium at day 4 with 15 μM APR-246, but it was rescued from days 8 to 12. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that MUC5AC expression was significantly reduced in the control group, whereas it was higher in APR-246 treated groups on day 12 in airlift cultures (***P < 0.001).
Figure 5
 
Pax6 expression in conjunctival tissue. (A) Normal conjunctival epithelial cells exhibit widespread positive Pax6 cells throughout the whole epithelial thickness. Under the airlifting condition, Pax6 expression underwent dramatic decrease in the superficial and basal layers from days 4 to 12, and become almost undetectable at day 12 in the airlift culture without APR-246. However, Pax6 expression in culture with 3 or 15 μM APR-246 remained at a level similar to that in the normal freshly isolated conjunctival epithelial cells. Scale bar: 100 μm. This protective effect of 3 or 15 μM APR-246 against declines in Pax6 expression was also confirmed by relative IOD analysis (B) and real-time PCR (C) (***P < 0.001).
Figure 5
 
Pax6 expression in conjunctival tissue. (A) Normal conjunctival epithelial cells exhibit widespread positive Pax6 cells throughout the whole epithelial thickness. Under the airlifting condition, Pax6 expression underwent dramatic decrease in the superficial and basal layers from days 4 to 12, and become almost undetectable at day 12 in the airlift culture without APR-246. However, Pax6 expression in culture with 3 or 15 μM APR-246 remained at a level similar to that in the normal freshly isolated conjunctival epithelial cells. Scale bar: 100 μm. This protective effect of 3 or 15 μM APR-246 against declines in Pax6 expression was also confirmed by relative IOD analysis (B) and real-time PCR (C) (***P < 0.001).
Figure 6
 
p63 expression in conjunctival tissue. (A) p63 was expressed in basal and some suprabasal epithelial cell nuclei. p63 expression increased from days 4 to 12 in the airlifting condition without APR-246, while its expression was relatively stable during exposure to either 3 or 15 μM APR-246. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that APR exposure suppressed increases and spreading of p63 expression at day 12 (***P < 0.001).
Figure 6
 
p63 expression in conjunctival tissue. (A) p63 was expressed in basal and some suprabasal epithelial cell nuclei. p63 expression increased from days 4 to 12 in the airlifting condition without APR-246, while its expression was relatively stable during exposure to either 3 or 15 μM APR-246. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that APR exposure suppressed increases and spreading of p63 expression at day 12 (***P < 0.001).
Figure 7
 
Cytokeratin 14 expression in conjunctival tissue. (A) There was a slight increase in K14 expression from days 4 to 12 under airlifting conditions, while K14 expression in 3 and 15 μM APR-246–treated groups was stable and similar to the expression level in normal freshly isolated conjunctival epithelium. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that K14 expression level was significantly higher in conjunctiva cultured without APR-246 than in normal conjunctiva and explants cultured with either 3 or 15 μM APR-246 (*P < 0.05, ***P < 0.001).
Figure 7
 
Cytokeratin 14 expression in conjunctival tissue. (A) There was a slight increase in K14 expression from days 4 to 12 under airlifting conditions, while K14 expression in 3 and 15 μM APR-246–treated groups was stable and similar to the expression level in normal freshly isolated conjunctival epithelium. Scale bar: 100 μm. Relative IOD analysis (B) and real-time PCR (C) showed that K14 expression level was significantly higher in conjunctiva cultured without APR-246 than in normal conjunctiva and explants cultured with either 3 or 15 μM APR-246 (*P < 0.05, ***P < 0.001).
Figure 8
 
Wnt signaling pathway activation in conjunctival tissue. (A) In normal freshly isolated conjunctival epithelium, β-catenin expression was essentially cell membrane delimited throughout all tissue layers. During culturing from days 4 to 12 under airlifting conditions, there is some β-catenin translocation from the cell membrane to cytoplasmic and nuclear domains. The number of cells that show β-catenin nuclear translocation gradually increased. Meanwhile, there was also sporadic nuclear translocation of β-catenin from days 4 to 12 when cultured with 3 μM APR-246, which was slightly less with 15 μM APR-246. Scale bar: 100 μm. Inset: High-magnification picture represents nuclear translocation of β-catenin. (B) Percentage of cells with nuclear-positive β-catenin expression in the control group was greater than that in the APR-246 group, which was nearly the same as in normal conjunctival epithelium (*P < 0.05, ***P < 0.001). (C) Real-time PCR analysis of the TCF-4 gene expression changes also showed that APR-246 protects the tissue against Wnt signaling activation induced by tissue airlifting (***P < 0.001).
Figure 8
 
Wnt signaling pathway activation in conjunctival tissue. (A) In normal freshly isolated conjunctival epithelium, β-catenin expression was essentially cell membrane delimited throughout all tissue layers. During culturing from days 4 to 12 under airlifting conditions, there is some β-catenin translocation from the cell membrane to cytoplasmic and nuclear domains. The number of cells that show β-catenin nuclear translocation gradually increased. Meanwhile, there was also sporadic nuclear translocation of β-catenin from days 4 to 12 when cultured with 3 μM APR-246, which was slightly less with 15 μM APR-246. Scale bar: 100 μm. Inset: High-magnification picture represents nuclear translocation of β-catenin. (B) Percentage of cells with nuclear-positive β-catenin expression in the control group was greater than that in the APR-246 group, which was nearly the same as in normal conjunctival epithelium (*P < 0.05, ***P < 0.001). (C) Real-time PCR analysis of the TCF-4 gene expression changes also showed that APR-246 protects the tissue against Wnt signaling activation induced by tissue airlifting (***P < 0.001).
Figure 9
 
Pterygium tissues treated with APR-246. (A) The H&E staining showed a mild increase of epithelial thickness after 4 days airlift culture of pterygium without APR-246, while there was an obvious decrease of epithelial thickness in pterygial tissues cultured with APR-246. p63 was strongly expressed in pterygium epithelium before culture, while it dramatically decreased in pterygial tissues cultured with APR-246. K10 was expressed in superficial epithelial cell layers in pterygial samples. However, in pterygial tissues cultured with APR-246, K10-positive cells were instead sparse. β-catenin membrane expression and nuclear translocation in pterygial epithelium were detected after 4 days. However, in explants cultured with APR-246, there was little detectable β-catenin nuclear translocation on day 4. Scale bar: 100 μm. (B) The number of epithelial cell layers in pterygium cultured with APR-246 was significantly less than that in the control group (*P < 0.05). (C) Real-time PCR analysis detected a significant difference in p63, K10, and TCF-4 gene expression between APR-246–cultured pterygial tissue and that without APR-246 on day 4 (***P < 0.001).
Figure 9
 
Pterygium tissues treated with APR-246. (A) The H&E staining showed a mild increase of epithelial thickness after 4 days airlift culture of pterygium without APR-246, while there was an obvious decrease of epithelial thickness in pterygial tissues cultured with APR-246. p63 was strongly expressed in pterygium epithelium before culture, while it dramatically decreased in pterygial tissues cultured with APR-246. K10 was expressed in superficial epithelial cell layers in pterygial samples. However, in pterygial tissues cultured with APR-246, K10-positive cells were instead sparse. β-catenin membrane expression and nuclear translocation in pterygial epithelium were detected after 4 days. However, in explants cultured with APR-246, there was little detectable β-catenin nuclear translocation on day 4. Scale bar: 100 μm. (B) The number of epithelial cell layers in pterygium cultured with APR-246 was significantly less than that in the control group (*P < 0.05). (C) Real-time PCR analysis detected a significant difference in p63, K10, and TCF-4 gene expression between APR-246–cultured pterygial tissue and that without APR-246 on day 4 (***P < 0.001).
Table
 
Primer Sequence Pairs Used for Quantitative Real-Time PCR
Table
 
Primer Sequence Pairs Used for Quantitative Real-Time PCR
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