P63 staining on human ocular surface

              ARCH. SOC. CANAR. OFTAL., 2003; 14: 15-19
P63 staining on human ocular
surface
CHENZHUO L, MD, PhD1, MURUBE J, MD, PhD2
1 Department of Mollecular and Cell Biology. Baylor College of Medicine. Houston, USA.
2 Department of Ophthalmology. Hospital Ramón y Cajal. University of Alcalá. Madrid. Spain.
Correspondencia:
Juan Murube del Castillo
Fundación Rizal de Investigación Oftalmológica
Universidad de Alcalá de Henares
C/. Moralzarzal, 43
28034 Madrid
España
RESUMEN
Propósito: Recientemente se ha hipotetizado que la proteína nuclear p63 es el primer marca-dor
de las células madres del epitelio corneal durante el desarrollo. La presente publicación
determina la localización de las células queratolimbales madres del epitelio corneal por el
patrón de expresión p63 en la superficie ocular.
Métodos: Se usó tejido queratolimbal humano para detectar por inmunohistoquimia el patrón
de expresión de la proteína p63. Se desarrolló la hibridización in situ bidireccional para
confirmar la expresión p63 mRNA en el anillo queratolimbal.
Resultados: La proteína p63 y ARNm se expresaron sólo en los núcleos de la capa basal de
las células epiteliales limbales.
Conclusión: De acuerdo con nuestros hallazgos, la proteína p63 localizada en la células basa-les
del epitelio queratolimbal es un buen marcador para detectar las células madres del epi-telio
corneal. La p63 podría utilizarse en práctica clínica para determinar la actividad de las
células madres del epitelio corneal.
Palabras clave: P63, limbo corneal, células madre, epitelio corneal.
SUMMARY
Purpose: It has been recently hypothesized that transcription factor p63 may be the earlier
marker of epithelial stem cells during development. This paper evaluates the p63 location
of the corneal and limbal area to find out the p63 expression pattern in the ocular surface.
Methods: Human cornea and limbal tissue were used to detect p63 protein expression by
immunohistochemistry. In situ hybridization with sense and anti-sense p63 probes were
performed on the corneal and limbal tissue to confirm the p63 mRNA express pattern at the
cornea and limbus.
Results: P63 protein and mRNA were expressed only in the nuclei of the limbal basal epithe-lial
cells layer. There was no staining on the corneal epithelial cells and limbal superficial
epithelial cells.
INVESTIGACIÓN
The corneal epithelial stem cells are belie-ved
to be located at the limbus (1-3). The
supporting data shows that: 1) The limbal
basal cells lack the corneal epithelial diffe-rentiation-
associated keratin pair K3 (4) and
K12 (5); 2) The limbal basal epithelium con-tains
proliferate characteristics such as slow-cycling
cells identified as the «label-retai-ning
cells» following pulse-chase labeling of
all cells with DNA precursor, such as [3H]-
thymidine or bromodeoxyuridine (BrdU) (6)
and also the limbal basal epithelium exhibits
high proliferative potential in culture (6-9);
3) Experimental studies and clinical observa-tions
show abnormal corneal epithelial
wound healing as conjunctivalization, vascu-larization
and chronic inflammation when
the limbal epithelium is partially (10,11) or
completely defected (12,13); 4) The limbal
location of corneal epithelial stem cells can
account for the relative preponderance of
limbal neoplasms and the scarcity of corneal
epithelial tumors, assuming that neoplasms
arise mainly from relatively «undifferentia-ted
» cells (14); 5) Limbal cells are essential
for the long-term maintenance of the central
corneal epithelium and they can be used to
reconstitute the entire corneal epithelium in
patients with limbal stem cell deficiencies
(3,15). Collectively, these data leave little
doubt that corneal epithelial stem cells are
located at the limbal area.
A major challenge in stem cell biology is
the ability to identify stem cells in situ. A
variety of «limbal stem cells markers» have
been suggested. The ability of a small popu-lation
of cells located at the corneal limbus to
retain tritiated thymidine label for long
periods of time has been accepted as indica-tive
of a stem cell population (16), but could
not be accepted as a stem cell marker.
Very recently, the p63 nuclear protein, a
member of the p53 family including p73,
was suggested to be a marker of epithelial
stem cells. Demonstrations show that p63
identifies basal epithelial cells (including
stem cells) in the prostate (17), breast (18),
bronchi (19), epidermis and corneal limbus
(20). Previous work showed that p63 is
highly expressed in the basal cells of many
human epithelial cells, especially in progeni-tor
or stem cell populations of epithelium tis-sues
(21,22).
In this work we use an immunohistoche-mical
method and in situ hybridization to
evaluate the locations of p63 protein and
mRNA express positive cells on the ocular
surface in humans.
MATHERIAL AND METHODS
Human corneal and limbal tissue
preparation
Normal human corneal and limbal tissues
were collected from Texas Lion Eye Bank,
Houston. Paraffin sections were made by the
following procedure: human limbal tissues
were fixed in 10% phosphate buffered for-malin
for one or two days, then transferred to
70% ethanol, dehydrated, paraffin embedded
and 5 μm thick sections were cut. Haema-toxylin-
eosin staining, immunohistoche-mistry
staining and in situ hybridization were
done on the sections.
Immunohistochemistry staining
An antibody against human p63 (NeoMar-kers)
was used for immunohistochemistry.
Sections were first boiled in 10 mM citrate
buffer (pH 6.0) for 20 min, and then blocked
with 10% horse serum in PBS for 1 h to
decrease nonspecific antibody interactions.
16 ARCH. SOC. CANAR. OFTAL., 2003; 14: 15-19
CHENZHUO L, et al.
Conclusion: According to our findings, p63 which is located in limbal basal epithelial cells is
a good marker for corneal stem cells, P63 could be used in the clinic practice to evaluate
corneal stem cell activity.
Key words: P63. Corneal limbus. Stem cell. Epithelium.
This same solution was used to dilute the
antibody at 1:1000. After rinsing in PBS,
biotinylated anti-mouse IgG antibody was
biotinylated with horseradish peroxidase rea-gent
(Vectastain ABC kit, Vector Laborato-ries)
and DAB to give a brown stain.
In situ hybridization
Transcript containing p63 sequence were
detected using [35S]UTP labeled riboprobes.
The 2.0 kb p63a probe was generated from
plasmid p63-Bluescript KS and synthesized
using EcoRI digestion and T3 RNA polyme-rase
(Promega) for the anti-sense probe and
HindIII digestion plus T7 RNA polymerase
(Promega) for the sense probe. Hybridiza-tions
were done on 5 μm thick ocular sec-tions
and were exposed to photographic
emulsion for 6-10 days before developing
and haematoxylin counterstaining.
RESULTS
P63 proteins were only immunodetected
in the nuclei of limbal basal epithelial cells.
There was no staining on the corneal epithe-lial
cells and limbal superficial epithelial
cells (fig. 1). To confirm the p63 expression
pattern in the limbal and corneal area, in situ
hybridization of p63a was performed on cor-neal
and limbal tissue. As a control, there is
no staining of p63 in the corneal and limbal
areas with p63 sense probe (fig. 2a). While
using anti-sense riboprobe showed that the
p63 mRNA is clearly located only on the
basal limbal epithelial cells nuclei; there was
no signal in the superficial limbal epithelium
and cornea (fig. 2b). These results indicated
that TAp63-a mRNA was strongly expressed
only in the limbal basal epithelium.
DISCUSSION
Anatomically, the limbus acts as a junc-tion
or the transition zone between the cor-neal
and conjunctival epithelia. Phenotypic
expression is intermediate between the cor-neal
and conjunctival epithelium.
The specific location of corneal epithelial
stem cells in the limbus provides several
functional advantages (ChenZhuo L, 1998):
1) Limbal basal cells are heavily pigmented
and are thus well protected; 2) Limbal epit-helium
is extremely resistant to shearing for-ces,
and in many species displays a highly
undulating epithelial-stromal junction
(Hogan MJ, 1971) with pegs of stroma
extending upward that are interconnected
with anchoring fibrils linking to the base-ment
membrane (23); 3) Limbal epithelium
is adjacent to a rich vascular network that
provides ample nutrients and other supporti-ve
factors. The blood vessels form part of the
ARCH. SOC. CANAR. OFTAL., 2003; 14: 15-19 17
P63 staining on human ocular surface
Fig. 1: P63
detected at the
limbal basal
epithelial cells.
Fig. 2: In situ
hybridization with
p63a riboprobe on
limbal area. With
sense riboprobe
(a, c): no signal
on the limbal
area. With
antisense
riboprobe (b, d):
strong p63 mRNA
express on the
limbal basal
epithelium.
palisades of Vogt which allow close approxi-mation
between blood vessels and the epit-helium,
potentially providing increased
levels of nutrition and blood-borne cytokines
to the cells at the limbus (23); 4) Limbal epit-helium
serves as the junctional epithelium to
prevent conjunctival epithelial ingrowth onto
the corneal surface during the healing of a
large corneal epithelial defect (12).
The p63 transcription factor belongs to a
family that includes two structurally related
proteins, p53 and p73. Whereas p53 plays a
well-stablished role in tumor suppression, p63
and p73 play unique roles in morphogenesis
and key regulators in the development of neu-ronal
and pheromonal (24) and p63 in limbus,
epithelial, and craniofacial development path-ways
(22,25,26). To date, p63 is the first gene
product definitely distinguishing stem cells
from their transient amplifying progeny in
stratified squamous epithelia. Identification of
p63 as a stem cell marker is consistent with
the phenotype of p63-/- mice (22,25). It was
known that p63-/- mice lack stratified epithelia
and contain clusters of terminally differentia-ted
keratinocytes on the exposed dermis (25),
and that p63 is expressed in the nuclei of kera-tinocytes
with proliferative potential (27). In
addition, it has been demonstrated that p63
expression is gradually reduced from the basal
cells to the terminally differentiated kerati-nocytes
(20,28). These findings correspond to
those observed in our study: strong p63 is spe-cifically
expressed in limbal basal cell, and
lack of p63 staining in corneal basal cells and
limbal superficial epithelial cell. It strongly
suggests that the phenotype of p63-/- mice
could contribute to a failure to maintain stem
cells (25) rather than to the inability of the
p63-/- ectoderm to form epithelial lineages
during development (22). We show here that
p63 is not expressed in the basal TACs of cor-neal
epithelium, but only in limbal basal epit-helial
cells. These observations show that pos-session
of p63 is not simply a property of mul-tiplying
cells (27), but a property of stem
cells.
Generally, in this work, the findings
strongly suggest that p63 may be the best
currently identified marker for corneal/lim-bal
stem cells.
REFERENCES
1. Watt FM, Hogan BL. Out of Eden: stem cells
and their niches. Science 2000; 287(5457):
1427-1430.
2. Dua HS, Azuara-Blanco A. Limbal stem cells
of the corneal epithelium. Surv Ophthalmol
2000; 44(5): 415-425.
3. Tseng SC. Concept and application of limbal
stem cells. Eye 1989; 3 (Pt 2): 141-157.
4. Schermer A, Galvin S, Sun T-T. Differentia-tion-
related expression of a major 64K corneal
keratin in vivo and in culture suggests limbal
location of corneal epithelial stem cells. J Cell
Biol 1986; 103: 49-62.
5. Kurpakus MA, Maniaci MT, Esco M. Expres-sion
of keratins K12, K4, and K14 during deve-lopment
of ocular surface epithelium. Curr Eye
Res 1994; 13: 805-14.
6. Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lav-ker
RM. Existence of slow-cycling limbal epit-helial
basal cells that can be preferentially sti-mulated
to proliferate: implications on epithe-lial
stem cells. Cell 1989; 57(2): 201-209.
7. Ebato B, Friend J, Thoft RA. Comparison of
limbal and peripheral human corneal epithelium
in tissue culture. Invest Ophthalmol Vis Sci
1988; 29(10): 1533-1537.
8. Kruse FE, Tseng SC. A tumor promoter-resis-tant
subpopulation of progenitor cells is larger
in limbal epithelium than in corneal epithelium.
Invest Ophthalmol Vis Sci 1993; 34(8): 2501-
2511.
9. Pellegrini G, Golisano O, Paterna P, Lambiase
A, Bonini S, Rama P et al. Location and clonal
analysis of stem cells and their differentiated
progeny in the human ocular surface. J Cell
Biol 1999; 145(4): 769-782.
10. Chen JJ, Tseng SC. Corneal epithelial wound
healing in partial limbal deficiency. Invest
Ophthalmol Vis Sci 1990; 31(7): 1301-1314.
11. Chen JJ, Tseng SC. Abnormal corneal epithelial
wound healing in partial-thickness removal of
limbal epithelium. Invest Ophthalmol Vis Sci
1991; 32(8): 2219-2233.
12. Huang AJ, Tseng SC. Corneal epithelial wound
healing in the absence of limbal epithelium.
Invest Ophthalmol Vis Sci 1991; 32(1): 96-105.
13. Jenkins C, Tuft S, Liu C, Buckley R. Limbal
transplantation in the management of chronic
contact-lens-associated epitheliopathy. Eye
1993; 7: 629-633.
14. Pizzarello LD, Jakobiec FA. Bowen's disease of
conjunctiva: a misnomer. In: Jakobiec FA, edi-tor.
Ocular and adnexal tumors. Birmingham:
Acculapius, 1978: 553-571.
15. Tseng SCG, Sun T-T. In Stem Cells: Ocular
Surface Maintenance, ed. Brightbill, F.S.
Mosby, St. Louis: 1999; 9-18.
18 ARCH. SOC. CANAR. OFTAL., 2003; 14: 15-19
CHENZHUO L, et al.
16. 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 19): 2867-2875.
17. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin
D, Garraway L et al. p63 is a prostate basal cell
marker and is required for prostate develop-ment.
Am J Pathol 2000; 157(6): 1769-1775.
18. Barbareschi M, Pecciarini L, Cangi MG, Macri
E, Rizzo A, Viale G et al. p63, a p53 homolo-gue,
is a selective nuclear marker of myoepithe-lial
cells of the human breast. Am J Surg Pathol
2001; 25(8): 1054-1060.
19. Chilosi M, Doglioni C. Constitutive p63
expression in airway basal cells. A molecular
target in diffuse lung diseases. Sarcoidosis Vasc
Diffuse Lung Dis 2001; 18(1): 23-26.
20. Pellegrini G, Dellambra E, Golisano O, Marti-nelli
E, Fantozzi I, Bondanza S et al. p63 iden-tifies
keratinocyte stem cells. Proc Natl Acad
Sci U S A 2001; 98(6): 3156-3161.
21. Yang A, Kaghad M, Wang Y, Gillett E, Fle-ming
MD, Dotsch V et al. p63, a p53 homolog
at 3q27-29, encodes multiple products with
transactivating, death-inducing, and dominant-negative
activities. Mol Cell 1998; 2(3): 305-
316.
22. Mills AA, Zheng B, Wang XJ, Vogel H, Roop
DR, Bradley A. p63 is a p53 homologue requi-red
for limb and epidermal morphogenesis.
Nature 1999; 398(6729): 708-713.
23. Gipson IK. The epithelial basement membrane
zone of the limbus. Eye 1989; 3 ( Pt 2): 132-140.
24. Yang A, McKeon F. P63 and P73: P53 mimics,
menaces and more. Nat Rev Mol Cell Biol
2000; 1(3): 199-207.
25. Yang A, Schweitzer R, Sun D, Kaghad M, Wal-ker
N, Bronson RT et al. p63 is essential for
regenerative proliferation in limb, craniofacial
and epithelial development. Nature 1999;
398(6729): 714-718.
26. Celli J, Duijf P, Hamel BC, Bamshad M, Kra-mer
B, Smits AP et al. Heterozygous germline
mutations in the p53 homolog p63 are the cause
of EEC syndrome. Cell 1999; 99(2): 143-153.
27. Parsa R, Yang A, McKeon F, Green H. Asso-ciation
of p63 with proliferative potential in
normal and neoplastic human keratinocytes. J
Invest Dermatol 1999; 113(6): 1099-1105.
28. De LV, Rossi A, Terrinoni A, Barcaroli D, Lev-rero
M, Costanzo A et al. P63 and p73 transac-tivate
differentiation gene promoters in human
keratinocytes. Biochem Biophys Res Commun
2000; 273(1): 342-346.
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