1Cell Biology Section, Division of Intramural Research, and 2Laboratory of Pathology, National Institute of Environmental Health Sciences, Research Triangle Park 27709; 3Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina, Chapel Hill, North Carolina 27599; 4Experimental Pathology Section, Cell and Cancer Biology Branch, National Cancer Institute, Rockville, Maryland 20850; and 5Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel 76100
Submitted 20 June 2003 ; accepted in final form 22 February 2004
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ABSTRACT |
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retinoic acid; stem cell; carcinoma; basal cell; differentiation
Recent studies of p63-null mice revealed that during embryonic development, p63 plays a critical role in the morphogenesis of several tissues (38, 70). Mice deficient in p63 die soon after birth and display a number of striking developmental defects, including the absence of epidermis, hair follicles, teeth, prostate, and mammary glands, as well as several abnormalities in limb development (38, 58, 70). p63 is particularly highly expressed in progenitor or stem cell populations of a variety of epithelial tissues (38, 43, 58, 65, 70). It is highly expressed in epidermal stem cells, and the lack of a stratified epidermis in p63/ mice suggests a role in the regulation of differentiation and/or maintenance of epidermal progenitor cells (2, 33, 38, 43, 47, 70). Recent studies (29) have indicated distinct functions for TAp63 and Np63 in the initiation of epithelial stratification and the maintenance of the proliferative potential of basal keratinocytes. Evidence indicating a regulatory role for p63 in development was derived from patients with various autosomal dominant syndromes (7, 9, 37, 62). These disorders have phenotypes reminiscent of p63 knockout mice and have been linked to the presence of heterozygous mutations in the p63 gene (7, 63). Although the role of p53 in cancer is well established, the involvement of p63 in cancer remains relatively unclear (67). The p63 gene maps to human chromosome 3q27-ter, a region known to be amplified frequently in human cancer, particularly in patients with squamous cell carcinomas (18, 25, 35, 40, 61, 64).
In this study, we investigated the expression of p63 during development of the esophagus and trachea and examined how the lack of p63 expression affects the morphogenesis of these epithelia (67). The trachea and the esophagus have a common origin and develop from the foregut endoderm at embryonic day 9.5 (E9.5) in the mouse (66). At the time of tracheoesophageal separation (E10.5E11), the epithelia consist of two to four layers of cells (45) and are likely derived from a common stem cell. Stem cells, defined by the International Society of Stem Cell Research as cells that have the capacity to self-renew as well as to differentiate in more mature cells (http://www.isscr.org/glossary/index.htm), are also responsible for regenerating injured tissue and maintaining tissue homeostasis. Complicating this issue are recent changes in stem cell concepts: stem cells in adult tissues have been reported to have much greater plasticity and differentiation potential than previously thought, and cells well along a differentiation pathway can revert to stem cells (6). In both the tracheobronchial and esophageal epithelia, stem cells differentiate first into ciliated cells, followed by basal cells (50, 66). After birth, the esophageal epithelium becomes a stratified squamous epithelium that in Barrett's metaplasia transforms into a simple columnar, mucosecretory epithelium as a result of gastroesophageal reflux disease (20). The tracheobronchial epithelium differentiates after birth into a pseudostratified mucociliary epithelium that, during injury and vitamin A deficiency, transforms into a stratified, squamous epithelium (22). However, the stem cell-progeny relationships in these epithelia have not yet been determined with certainty.
In this study, we demonstrate that p63 is abundant in early progenitor cells of both the esophageal and tracheobronchial epithelia in E15.5 embryos and sequentially becomes confined to the K14+/K5+/BS-I-B4+ basal cells and in increasingly reduced levels in transient amplifying cells. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar, ciliated epithelium that is deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny.
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MATERIALS AND METHODS |
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Immunohistochemistry.
Formalin-fixed, paraffin-embedded specimens of human tumors and normal tissues were obtained from the Cooperative Human Tissue Network and the archives of the Laboratory of Pathology of the National Cancer Institute (Bethesda, MD). Sections of human tracheal explants cultured in the presence or absence of 0.1 µM retinoic acid (60) were obtained from Dr. Jonathan Kurie (Program in Cancer Biology, M.D. Anderson Cancer Center, Houston, TX). All human tissues used were collected after approval was obtained from the appropriate institutional review boards at the individual institutions. Mouse tissues were fixed in 4% paraformaldehyde in PBS for 4 h at 4°C, dehydrated, and embedded in paraffin. Sections were examined by immunohistochemistry for lectin binding with the use of peroxidase-labeled lectin Bandeiraea simplicifolia (BS-I-B4; Sigma, St. Louis, MO) or with mouse monoclonal antibodies specific for p63 (4A4; 1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), the basal cell marker keratin 14 (K14; 1:300; Novocastra Laboratories, Newcastle-upon-Tyne, UK), keratin 5 (K5; 1:200; Research Diagnostics, Flanders, NJ), and a marker for ciliated cells, -tubulin IV (AM-2510-01, 1:1,500; InnoGenex, San Ramon, CA). Staining was performed using a Vectastain Elite ABC system (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAKO, Carpinteria, CA). Sections were either counterstained with a 1:5 dilution of eosin phloxine stain (Poly Scientific, Bay Shore, NY) or methyl green or stained with hematoxylin and eosin.
For dual staining of p63 and K5, sections were incubated simultaneously with both primary antibodies, and, after washing, Cy3- or Cy2-conjugated donkey anti-mouse and anti-guinea pig secondary antibodies, respectively, were added (no species cross-reaction grade; Jackson ImmunoResearch, West Grove, PA). Slides were viewed on a Zeiss 510 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY).
Western blot analysis. Cells were washed in PBS and then collected in sample buffer (60 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 10 mM DTT, 1 mM phenylmethylsulfonyl fluoride, aprotinin, and leupeptin) and phosphatase inhibitor mixture I and II (Sigma). Proteins were examined by Western blot analysis with the use of the 4A4 mouse monoclonal anti-p63 or rabbit anti-cornifin (34) antibody. Peroxidase-conjugated anti-mouse or anti-rabbit IgG purchased from Chemicon (Temecula, CA) was used as secondary antibody. Antibodies were diluted in PBS containing 1% milk powder and 0.05% Tween 20. Detection was performed with SuperSignal chemiluminescent substrate (Pierce, Rockford, IL), and luminol and peroxide were purchased from Pierce.
Tissue culture. Human tracheobronchial epithelial cells were obtained form Clonetics (Walkersville, MD) and cultured in complete human tracheobronchial epithelial (TBE) medium as described previously (39). Cells were induced to undergo squamous cell differentiation by the addition of TPA (30 ng/ml).
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RESULTS |
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In humans, the basal layer of the esophageal epithelium consists of two zones: one overlying the papillae [i.e., the papillary basal layer (PBL)] and the other between the papillae [i.e., the interpapillary basal layer (IBL)] (Fig. 3, E and F). Previous studies have provided evidence that stem cells are located mainly in the IBL (55, 56). In both the IBL and the PBL, the basal cells closest to the basement membrane stained most intensely for p63 (Fig. 3F) and K14 (not shown). Some heterogeneity was observed within this layer, however, with some cells staining more intensely than others. Staining for p63 generally declined in the epibasal layers, which consist mainly of transient amplifying cells that transit further into the more suprabasal layers, where they undergo squamous differentiation. Therefore, the highest level of p63 expression appears to be associated with cells that are the least committed to terminal differentiation. This is in agreement with findings in the epidermis, where epidermal stem cells express the highest level of p63, and expression is diminished in transient amplifying cells (38, 43, 70).
In the setting of Barrett's metaplasia, a disorder in which the stratified epithelium is replaced by a simple columnar epithelium consisting of mucosecretory cells, none of the cells were p63+ (Fig. 3G). Basal cells in submucosal glands and cells in the basal layer of certain multilayered glandular epithelia were p63+ (Fig. 3H). These p63+ cells differed from the p63+ basal cells in stratified squamous esophageal epithelium in that they were negative for K14 and BS-I-B4 (not shown). Barrett's metaplasia is mainly a result of duodenogastroesophageal reflux disease (20). The origin of the columnar cells in Barrett's metaplasia is still controversial. It has been suggested that Barrett's metaplasia arises from esophageal stem or basal cells through reprogramming of their differentiation process, from the glandular ducts lining the esophagus, or from cells at the gastroesophageal junction (20, 55). Barrett's metaplasia has a high probability of developing into esophageal adenocarcinoma. Adenocarcinomas (n = 3) of esophageal origin did not stain positively or contained few weakly positive cells (not shown); in contrast, all squamous cell carcinomas (n = 9) stained strongly for p63 (Fig. 3H), although with different intensities. Some disagreement exists about the expression of p63 in adenocarcinomas. The absence of p63 staining in Barrett's metaplasia and adenocarcinoma reported in this study is consistent with recent observations by Glickman et al. (16) but appears to contrast with those of Hall et al. (17), who demonstrated weak staining in Barrett's metaplasia and strong staining in adenocarcinomas.
As in the esophagus, the tracheobronchial epithelium in E15.5 mice consists of a layer of two to three cells (Fig. 1E). Only the putative stem cells adherent to the basement membrane were p63+ and did not stain for K14, -tubulin IV (Fig. 1, FH), K5, or BS-I-B4 (not shown). During development, stem cells first differentiate into ciliated (p63/
-tubulin IV+) and secretory cells and later, at birth, begin to differentiate into basal cells (p63+/K14+/K5+/BS-I-B4+), thereby generating a pseudostratified epithelium (Fig. 4, A and C) (49). Similarly to basal cells in the esophagus, the basal cells in the tracheal epithelium of newborn mice stained strongly for p63 (Fig. 4B) and sporadically and weakly for K14, K5, and BS-I-B4 (Fig. 4D and data not shown) but began to stain more intensely for K14, K5, and BS-I-B4 after birth, in agreement with findings described in previous reports (49). In adult mice as well as in humans, the tracheobronchial lining consists of a pseudostratified epithelium containing ciliated, basal, and mucosecretory cells. K14+ cells stained positively for p63, whereas ciliated and mucous cells were negative (Fig. 4 and Fig. 5, A and B), in agreement with recent studies (8). Figure 6, BD, shows that in the normal adult mouse trachea, the expression of p63 also correlates closely with the expression of the basal cell marker K5, in agreement with observations in squamous cell carcinomas (24). Basal cells in mucous cell hyperplasia stained positively for p63 (Fig. 5D), as did basal cells in the submucosal glands of human and mouse (Figs. 5C and 6A). In human basal cell hyperplasia, many cells in basal and suprabasal layers stain positively for p63 (Fig. 5E). Examination of sections from human lung tumors with different histologies showed that expression of p63 is highly correlated with squamous cell carcinomas (6 of 6 squamous cell, 0 of 3 large cell, 0 of 5 adenocarcinoma, and 0 of 4 small cell lung carcinoma samples were p63 positive; not shown), in agreement with previous observations (35, 44, 64). In human lung carcinomas containing mixed tumor types, p63 immunoreactivity was confined predominantly to the region with squamous histology. In squamous cell carcinomas, p63 was localized to the nuclei of less differentiated cells, whereas more differentiated regions did not stain for p63.
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To obtain more insight into the role of p63 in the morphogenesis of these epithelia, we examined the effect of the lack of p63 expression on the development of these epithelia in p63/ mice. Because p63/ mice die shortly after birth (38, 70), we compared the structure of these epithelia in newborn wild type and p63/ mice. Mice deficient in p63 developed a trachea and an esophagus, suggesting that the lack of p63 does not appear to affect the early stages of development of these tissues, including budding from the foregut endoderm or the tracheoesophageal separation. However, in contrast to the esophagus of newborn wild-type mice, which consists of several layers of cells with few ciliated cells and many K14+ basal cells (Fig. 2, AD), the esophageal epithelium of p63/ mice consisted of a columnar epithelium of largely ciliated cells that lacked K14+ basal cells (Fig. 2, EH), in agreement with previous observations (70). In contrast to the cells in wild-type mice, these ciliated cells made contact with the basement membrane. Similar changes were observed in the tracheobronchial epithelium of newborn p63/ mice: the number of ciliated cells was greatly enhanced, and the epithelial lining consisted of a well-organized, columnar, ciliated epithelium lacking basal cells, as indicated by the abundant staining for -tubulin IV and the total absence of K14 staining (Fig. 4, DH).
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DISCUSSION |
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During injury and under vitamin A deficiency, the normal pseudostratified tracheobronchial epithelium is transformed into a stratified squamous epithelium (22). This involves enhanced proliferation of p63+/K14+/K5+/BS-I-B4+ basal cells and their differentiation into transient amplifying cells and subsequently into terminally differentiated squamous cells (Fig. 8). The latter program of differentiation is similar to that of squamous differentiation of basal cells in the esophagus. Squamous differentiation in vivo and in cultured cells is accompanied by downregulation of p63 expression. The precise functions of p63 in basal keratinocytes and the downregulation of p63 during differentiation are not precisely understood (2, 4, 12, 33, 47). It has been suggested that p63 might be required for maintaining the undifferentiated phenotype of basal keratinocytes and that its downregulation might be necessary for basal cells to differentiate into squamous cells and/or in the execution of the differentiation program after cells become committed. The reduced expression of p63 in transient amplifying cells would be in agreement with such a hypothesis but would not prove a role for p63 in the control of this differentiation process. Activation of phosphoinositide 3-kinase, which inhibits epidermal differentiation, recently was reported to positively regulate the expression of Np63
(4), in agreement with a role for
Np63
in the survival and proliferative capacity of keratinocytes. Recent studies (12, 26, 29) revealed distinct roles for TAp63 or
Np63
in epidermal keratinocytes and demonstrated a role for p63 in the maintenance of the proliferative potential of basal cells and in the initiation of the epithelial stratification program. The association between strong expression of p63 and hyperplasia and/or squamous metaplasia and squamous cell carcinomas may suggest a role for p63 in these pathological processes and is consistent with these proposed functions (10, 16, 19, 24, 25, 35, 40, 42, 44, 53, 64). Defects in the control mechanisms regulating the expression of p63 may promote the undifferentiated phenotype, proliferation, and/or inhibition of apoptosis and therefore may play a role in tumorigenesis (4, 10, 41, 69).
As a result of gastroesophageal reflux disease, the esophagus transforms into Barrett's metaplasia, consisting of a simple, columnar mucosecretory epithelium (20) and lacking p63 expression. The stem cell-progeny relationships in the esophagus are still poorly understood. Barrett's metaplasia may arise via reprogramming of the differentiation program of basal cells, from the glandular ducts, or from cells at the gastroesophageal junction (20, 55). It is interesting to note that the basal layer of the glands lining the human esophagus stain positively for p63 but do not stain for the basal cell markers K14 or BS-I-B4+ (not shown) (16). This phenotype is similar to that of the early stem cells in E15.5 mouse esophagus. One might speculate that these p63+/K14/BS-I-B4 cells in the glands might be closely related to the early stem cells and might have the ability to differentiate into mucosecretory cells. Through reprogramming of their differentiation program and loss of p63 expression, these p63+/K14/BS-I-B4 glandular cells could function as the progenitors in Barrett's metaplasia. However, such a mechanism would not explain the development of Barrett's-like metaplasia in rodents that do not have glands. Alternatively, reprogramming (i.e., dedifferentiation) of p63+/K14+/BS-I-B4+ basal cells into p63/K14/BS-I-B4 stem cells might function as the progenitors in Barrett's metaplasia (20, 55).
In summary, our study demonstrates that p63 plays a critical role in the normal morphogenesis of the tracheobronchial and esophageal epithelia. The lack of basal cells in p63/ mice suggests that p63 is required for the differentiation of early stem cells into basal cell progeny and/or the maintenance and/or survival of the basal cell population. Although expression of p63 has been reported to be critical to the survival of stem cells in a number of epithelia (38, 58, 70), the esophagus and trachea of p63/ mice still contain p63/K14/BS-I-B4 stem cells that are able to differentiate into ciliated and mucosecretory cells (70). This raises the question of whether p63 is needed only to generate the basal cell progeny. Based on this idea, an alternative stem cell progeny model (Fig. 8C) may be considered in which the adult trachea retains few p63/K14/BS-I-B4 stem cells that are able to generate basal, mucous, and ciliated cells. In this model, the p63+/K14/BS-I-B4 cells constitute prebasal cells, an intermediate step in the differentiation program to mature p63+/K14+/BS-I-B4+ basal cells. A recent study provides support for this hypothesis (29). To date, however, no evidence exists indicating that p63/K14/BS-I-B4 stem cells remain in the adult tracheal epithelium. Future studies using additional stem cell markers are needed to distinguish these two possibilities.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bamberger C, Pollet D, and Schmale H. Retinoic acid inhibits downregulation of Np63
expression during terminal differentiation of human primary keratinocytes. J Invest Dermatol 118: 133138, 2002.
3. Barbareschi M, Pecciarini L, Cangi MG, Macri E, Rizzo A, Viale G, and Doglioni C. p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol 25: 10541060, 2001.[CrossRef][ISI][Medline]
4. Barbieri CE, Barton CE, and Pietenpol JA. Np63
expression is regulated by the phosphoinositide 3-kinase pathway. J Biol Chem 278: 5140851414, 2003.
5. Barrandon Y and Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84: 23022306, 1987.[Abstract]
6. Blau HM, Brazelton TR, and Weimann JM. The evolving concept of a stem cell: entity or function? Cell 105: 829841, 2001.[CrossRef][ISI][Medline]
7. Brunner HG, Hamel BCJ, and van Bokhoven H. p63 gene mutations and human developmental syndromes. Am J Med Genet 112: 284290, 2002.[CrossRef][ISI][Medline]
8. Chilosi M and Doglioni C. Constitutive p63 expression in airway basal cells. A molecular target in diffuse lung diseases. Sarcoidosis Vasc Diffuse Lung Dis 18: 2326, 2001.[ISI][Medline]
9. Chilosi M, Poletti V, Murer B, Lestani M, Cancellieri A, Montagna L, Piccoli P, Cangi G, Semenzato G, and Doglioni C. Abnormal re-epithelialization and lung remodeling in idiopathic pulmonary fibrosis: the role of N-p63. Lab Invest 82: 13351345, 2002.[ISI][Medline]
10. Choi HR, Batsakis JG, Zhan F, Sturgis E, Luna MA, and El-Naggar AK. Differential expression of p53 gene family members p63 and p73 in head and neck squamous tumorigenesis. Hum Pathol 33: 158164, 2002.[CrossRef][ISI][Medline]
11. Dohn M, Zhang S, and Chen X. p63 and
Np63
can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes. Oncogene 20: 31933205, 2001.[CrossRef][ISI][Medline]
12. Ellisen LW, Ramsayer KD, Johannessen CM, Yang A, Beppu H, Minda K, Oliner JD, McKeon F, and Haber DA. REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol Cell 10: 9951005, 2002.[ISI][Medline]
13. Floyd EE and Jetten AM. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids. Mol Cell Biol 9: 48464851, 1989.[ISI][Medline]
14. Fuchs E. Epidermal differentiation and keratin gene expression. J Cell Sci Suppl 17: 197208, 1993.[Medline]
15. Ghioni P, Bolognese F, Duijf PH, van Bokhoven H, Mantovani R, and Guerrini L. Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains. Mol Cell Biol 22: 86598668, 2002.
16. Glickman JN, Yang A, Shahsafaei A, McKeon F, and Odze RD. Expression of p53-related protein p63 in the gastrointestinal tract and in esophageal metaplastic and neoplastic disorders. Hum Pathol 32: 11571165, 2001.[CrossRef][ISI][Medline]
17. Hall PA, Woodman AC, Campbell SJ, and Shepherd NA. Expression of the p53 homologue p63 and
Np63
in the neoplastic sequence of Barrett's oesophagus: correlation with morphology and p53 protein. Gut 49: 618623, 2001.
18. Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, Ratovitski EA, Jen J, and Sidransky D. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 97: 54625467, 2000.
19. Hu H, Xia SH, Li AD, Xu X, Cai Y, Han YL, Wei F, Chen BS, Huang XP, Han YS, Zhang JW, Zhang X, Wu M, and Wang MR. Elevated expression of p63 protein in human esophageal squamous cell carcinomas. Int J Cancer 102: 580583, 2002.[CrossRef][ISI][Medline]
20. Jankowski JA, Harrison RF, Perry I, Balkwill F, and Tselepis C. Barrett's metaplasia. Lancet 356: 20792085, 2000.[CrossRef][ISI][Medline]
21. Jetten AM, George MA, Smits HL, and Vollberg TM. Keratin 13 expression is linked to squamous differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid. Exp Cell Res 182: 622634, 1989.[ISI][Medline]
22. Jetten AM, Nervi C, and Vollberg TM. Control of squamous differentiation in tracheobronchial and epidermal epithelial cells: role of retinoids. J Natl Cancer Inst Monogr 13: 93100, 1992.[Medline]
23. Jones PH. Epithelial stem cells. Bioessays 19: 683690, 1997.[ISI][Medline]
24. Kaufmann O, Fietze E, Mengs J, and Dietel M. Value of p63 and cytokeratin 5/6 as immunohistochemical markers for the differential diagnosis of poorly differentiated and undifferentiated carcinomas. Am J Clin Pathol 116: 823830, 2001.[CrossRef][ISI][Medline]
25. Kettunen E, el-Rifai W, Bjorkqvist AM, Wolff H, Karjalainen A, Anttila S, Mattson K, Husgafvel-Pursiainen K, and Knuutila S. A broad amplification pattern at 3q in squamous cell lung cancer: a fluorescence in situ hybridization study. Cancer Genet Cytogenet 117: 6670, 2000.[CrossRef][ISI][Medline]
26. King KE, Ponnamperuma RM, Yamashita T, Tokino T, Lee LA, Young MF, and Weinberg WC. Np63
functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes. Oncogene 22: 36353644, 2003.[CrossRef][ISI][Medline]
27. Koo JS, Yoon JH, Gray T, Norford D, Jetten AM, and Nettesheim P. Restoration of the mucous phenotype by retinoic acid in retinoid-deficient human bronchial cell cultures: changes in mucin gene expression. Am J Respir Cell Mol Biol 20: 4352, 1999.
28. Koster MI, Huntzinger KA, and Roop DR. Epidermal differentiation: transgenic/knockout mouse models reveal genes involved in stem cell fate decisions and commitment to differentiation. J Investig Dermatol Symp Proc 7: 4145, 2002.[CrossRef][ISI][Medline]
29. Koster MI, Kim S, Mills AA, DeMayo FJ, and Roop DR. p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 18: 126131, 2004.
30. Kurita T and Cunha GR. Roles of p63 in differentiation of Mullerian duct epithelial cells. Ann NY Acad Sci 948: 912, 2001.
31. Lancillotti F, Darwiche N, Celli G, and De Luca LM. Retinoid status and the control of keratin expression and adhesion during the histogenesis of squamous metaplasia of tracheal epithelium. Cancer Res 52: 61446152, 1992.[Abstract]
32. Levrero M, De Laurenzi V, Costanzo A, Gong J, Wang JY, and Melino G. The p53/p63/p73 family of transcription factors: overlapping and distinct functions. J Cell Sci 113: 16611670, 2000.
33. Liefer KM, Koster MI, Wang XJ, Yang A, McKeon F, and Roop DR. Down-regulation of p63 is required for epidermal UV-B-induced apoptosis. Cancer Res 60: 40164020, 2000.
34. Marvin KW, George MD, Fujimoto W, Saunders NA, Bernacki SH, and Jetten AM. Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids. Proc Natl Acad Sci USA 89: 1102611030, 1992.[Abstract]
35. Massion PP, Taflan PM, Jamshedur Rahman SM, Yildiz P, Shyr Y, Edgerton ME, Westfall MD, Roberts JR, Pietenpol JA, Carbone DP, and Gonzalez AL. Significance of p63 amplification and overexpression in lung cancer development and prognosis. Cancer Res 63: 71137121, 2003.
36. Matsuura H, Adachi H, Smart RC, Xu X, Arata J, and Jetten AM. Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells. Mol Cell Endocrinol 147: 8592, 1999.[CrossRef][ISI][Medline]
37. McGrath JA, Duijf PH, Doetsch V, Irvine AD, de Waal R, Vanmolkot KR, Wessagowit V, Kelly A, Atherton DJ, Griffiths WA, Orlow SJ, van Haeringen A, Ausems MG, Yang A, McKeon F, Bamshad MA, Brunner HG, Hamel BC, and van Bokhoven H. Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63. Hum Mol Genet 10: 221229, 2001.
38. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, and Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398: 708713, 1999.[CrossRef][ISI][Medline]
39. Nervi C, Vollberg TM, George MD, Zelent A, Chambon P, and Jetten AM. Expression of nuclear retinoic acid receptors in normal tracheobronchial cells and in lung carcinoma cells. Exp Cell Res 195: 163170, 1991.[ISI][Medline]
40. Nylander K, Coates PJ, and Hall PA. Characterization of the expression pattern of p63 and
Np63
in benign and malignant oral epithelial lesions. Int J Cancer 87: 368372, 2000.[CrossRef][ISI][Medline]
41. Park JJ, Sun D, Quade BJ, Flynn C, Sheets EE, Yang A, McKeon F, and Crum CP. Stratified mucin-producing intraepithelial lesions of the cervix: adenosquamous or columnar cell neoplasia? Am J Surg Pathol 24: 14141419, 2000.[CrossRef][ISI][Medline]
42. Parsa R, Yang A, McKeon F, and Green H. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113: 10991105, 1999.
43. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, and De Luca M. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98: 31563161, 2001.
44. Pelosi G, Pasini F, Olsen Stenholm C, Pastorino U, Maisonneuve P, Sonzogni A, Maffini F, Pruneri G, Fraggetta F, Cavallon A, Roz E, Iannucci A, Bresaola E, and Viale G. p63 immunoreactivity in lung cancer: yet another player in the development of squamous cell carcinomas? J Pathol 198: 100109, 2002.[CrossRef][ISI][Medline]
45. Qi BQ and Beasley SW. Stages of normal tracheo-bronchial development in rat embryos: resolution of a controversy. Dev Growth Differ 42: 145153, 2000.[CrossRef][ISI][Medline]
46. Quade BJ, Yang A, Wang Y, Sun D, Park J, Sheets EE, Cviko A, Federschneider JM, Peters R, McKeon FD, and Crum CP. Expression of the p53 homologue p63 in early cervical neoplasia. Gynecol Oncol 80: 2429, 2001.[CrossRef][ISI][Medline]
47. Radu E, Simionescu O, Regalia T, Dumitrescu D, and Popescu LM. Stem cells p63+ in keratinocyte cultures from human adult skin. J Cell Mol Med 6: 593598, 2002.[ISI][Medline]
48. Randell SH, Comment CE, Ramaekers FC, and Nettesheim P. Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl end groups. Am J Respir Cell Mol Biol 4: 544554, 1991.[ISI][Medline]
49. Randell SH, Shimizu T, Bakewell W, Ramaekers FC, and Nettesheim P. Phenotypic marker expression during fetal and neonatal differentiation of rat tracheal epithelial cells. Am J Respir Cell Mol Biol 8: 546555, 1993.[ISI][Medline]
50. Raymond C, Anne V, and Millane G. Development of esophageal epithelium in the fetal and neonatal mouse. Anat Rec 230: 225234, 1991.[ISI][Medline]
51. Rearick JI and Jetten AM. Effect of substratum and retinoids upon the mucosecretory differentiation of airway epithelial cells in vitro. Environ Health Perspect 80: 229237, 1989.[ISI][Medline]
52. Reddy SP, Chuu YJ, Lao PN, Donn J, Ann DK, and Wu R. Expression of human squamous cell differentiation marker, SPR1, in tracheobronchial epithelium depends on JUN and TRE motifs. J Biol Chem 270: 2645126459, 1995.
53. Reis-Filho JS, Torio B, Albergaria A, and Schmitt FC. p63 expression in normal skin and usual cutaneous carcinomas. J Cutan Pathol 29: 517523, 2002.[CrossRef][ISI][Medline]
54. Schoch KG, Lori A, Burns KA, Eldred T, Olsen JC, and Randell SH. A subset of mouse tracheal epithelial basal cells generates large colonies in vitro. Am J Physiol Lung Cell Mol Physiol 286: L631L642, 2004.
55. Seery JP. Stem cells of the oesophageal epithelium. J Cell Sci 115: 17831789, 2002.
56. Seery JP and Watt FM. Asymmetric stem-cell divisions define the architecture of human oesophageal epithelium. Curr Biol 10: 14471450, 2000.[CrossRef][ISI][Medline]
57. Senoo M, Matsumura Y, and Habu S. TAp63 (p51A) and
Np63
(p73L), two major isoforms of the p63 gene, exert opposite effects on the vascular endothelial growth factor (VEGF) gene expression. Oncogene 21: 24552465, 2002.[CrossRef][ISI][Medline]
58. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A, Montironi R, McKeon F, and Loda M. p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 157: 17691775, 2000.
59. Stapleton D, Balan I, Pawson T, and Sicheri F. The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nat Struct Biol 6: 4449, 1999.[CrossRef][ISI][Medline]
60. Sueoka N, Lee HY, Walsh GL, Fang B, Ji L, Roth JA, LaPushin R, Hong WK, Cohen P, and Kurie JM. Insulin-like growth factor binding protein-6 inhibits the growth of human bronchial epithelial cells and increases in abundance with all-trans-retinoic acid treatment. Am J Respir Cell Mol Biol 23: 297303, 2000.
61. Urist MJ, Di Como CJ, Lu ML, Charytonowicz E, Verbel D, Crum CP, Ince TA, McKeon FD, and Cordon-Cardo C. Loss of p63 expression is associated with tumor progression in bladder cancer. Am J Pathol 161: 11991206, 2002.
62. Van Bokhoven H, Hamel BC, Bamshad M, Sangiorgi E, Gurrieri F, Duijf PH, Vanmolkot KR, van Beusekom E, van Beersum SE, Celli J, Merkx GF, Tenconi R, Fryns JP, Verloes A, Newbury-Ecob RA, Raas-Rotschild A, Majewski F, Beemer FA, Janecke A, Chitayat D, Crisponi G, Kayserili H, Yates JR, Neri G, and Brunner HG. p63 gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation. Am J Hum Genet 69: 481492, 2001.[CrossRef][Medline]
63. Van Bokhoven H and McKeon F. Mutations in the p53 homolog p63: allele-specific developmental syndromes in humans. Trends Mol Med 8: 133139, 2002.[CrossRef][ISI][Medline]
64. Wang BY, Gil J, Kaufman D, Gan L, Kohtz DS, and Burstein DE. p63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors. Hum Pathol 33: 921926, 2002.[CrossRef][ISI][Medline]
65. Wang X, Mori I, Tang W, Nakamura M, Nakamura Y, Sato M, Sakurai T, and Kakudo K. p63 expression in normal, hyperplastic and malignant breast tissues. Breast Cancer 9: 216219, 2002.[Medline]
66. Wells JM and Melton DA. Vertebrate endoderm development. Annu Rev Cell Dev Biol 15: 393410, 1999.[CrossRef][ISI][Medline]
67. Yang A, Kaghad M, Caput D, and McKeon F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet 18: 9095, 2002.[CrossRef][ISI][Medline]
68. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, and McKeon F. p63, a p53 homolog at 3q2729, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 2: 305316, 1998.[ISI][Medline]
69. Yang A and McKeon F. P63 and P73: P53 mimics, menaces and more. Nat Rev Mol Cell Biol 1: 199207, 2000.[CrossRef][ISI][Medline]
70. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, and McKeon F. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398: 714718, 1999.[CrossRef][ISI][Medline]