ARTICLE |
Correspondence to: Noriyuki Sato, Dept. of Pathology, Sapporo Medical U. School of Medicine, South-1,West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail: nsatou@sapmed.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis. (J Histochem Cytochem 50:455462, 2002)
Key Words: p73, p53, human thymic epithelial cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
T-LYMPHOCYTES, which are the major regulatory cells of the immune system, derive from precursors in hematopoietic tissues. Before seeding to the peripheral lymphoid tissues, immature T-cells (thymocytes) undergo differentiation and precise selection to acquire specific immunological responses (, IL-6, and IL-7 for their subsequent maturation (
TECs have characteristics similar to those observed in keratinocytes of the epidermis, e.g., expression of cytokeratin molecules in addition to a morphological resemblance (
p73 has been identified as a homologous molecule of the archetypal tumor suppressor p53 (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissues and Cell Lines
Normal human thymuses were obtained through the Hokkaido Children's Hospital and Medical Center from patients <3 years of age undergoing cardiovascular surgery for congenital heart disease. These were stored at -80C before cryostat sections were prepared. Six-µm sections of the tissues were cut and placed on glass slides. Then they were fixed in ice-cold acetone for 30 sec and dried. All tissue was obtained with approval by institutional review boards.
Human embryo kidney 293 cells were maintained with DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere at 37C and 5% CO2. These cells were transiently transformed by using the Lipofectamine 2000 reagent (Invitrogen; Carlsbad, CA) with p73 cDNA recombined in expression vector pcDNA3 (courtesy of Dr. G. Melino; University of Tor Vergata, Rome, Italy). TEC lines were cultured in D-valine-containing MEM (Sigma; St Louis, MO) with supplements as previously described (
Antibodies
A rabbit polyclonal antibody against p73 was obtained from New Zealand White rabbits, which were immunized with a synthetic peptide of 18 amino acid residues, CKARKQPIKEEFTEAEIH, at the C-terminal portion of human p73. The IgG fraction purified from the antiserum for the following analyses was named ST-2G. H-79 is a rabbit anti-p73 polyclonal antibody against the N-terminal portion of p73 (Santa Cruz Biotechnology; Santa Cruz, CA). Anti-human cytokeratin-19 and anti-human CD3 monoclonal antibodies were purchased from DAKO (Carpinteria, CA), and the anti-human CD34 monoclonal antibody was from Nichirei (Tokyo, Japan).
Northern Blotting Analysis
A Northern blot membrane on which 2 µg of poly A+ RNA derived from various lymphoid tissues was used (Human Immune System MTN Blot II; Clontech, Palo Alto, CA). An EcoRI fragment of 1380 bp from pcDNA3-p73 was used as a probe. It included exons 714 and the 3' untranslated region. Hybridization was performed as specified in the manufacturer's protocol. Briefly, the membrane was hybridized with radiolabeled probes in ExpressHyb solution (Clontech) at 68C for 20 hr. Then the membrane was washed in SSC containing 0.1% SDS solution with stepwise decreased salt concentrations followed by 2 x SSC, 1 x SSC, and 0.5 x SSC at 50C for 30 min. The membrane was subjected to autoradiography for consecutive days for up to 1 month at most.
Western Blotting Analysis
Cell lysis was performed using subconfluent cultures by incubating 10-cm2 dishes with 1 ml of lysis buffer containing 0.5% NP-40, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and protease inhibitors (Roche; Basel, Switzerland) for 30 min at 4C. Aliquots of the supernatants were applied to 10% SDS-PAGE gels under reducing conditions and transferred onto a polyvinylidene fluoride membrane. After blocking nonspecific protein binding using Tris-buffered salineTween-20 (TBST) containing 5% fat-free dry milk, the membrane was incubated with antibodies overnight at 4C, followed by reaction with a secondary antibody conjugated with peroxidase (Kirkegaard & Perry; Gaithersburg, MD). After washing with TBST, signals were detected by chemiluminescence with an ECL kit (Amersham; Piscataway, NJ).
Immunohistochemical Analysis
In the immunohistochemical analysis with two different types of antibodies (double-staining method), frozen sections of normal thymic tissues were fixed with ice-cold acetone and stained with ST-2G and a certain monoclonal antibody as previously described (
For immunofluorescent microscopic examination of cultured cells, cells were fixed with ice-cold acetone and stained with an anti-p73 polyclonal antibody (ST-2G), followed by reaction with FITC-conjugated anti-rabbit IgG (Kirkegaard & Perry). These specimens were analyzed with a fluorescence microscope (Carl Zeiss; Göttingen, Germany).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
p73 Transcripts Expressed in Normal Thymus
To examine p73 expression in the thymus, we performed Northern blotting analysis of multiple lymphoid tissue blots. As previously noted, the p73 gene is constituted of 14 exons at chromosome 1p36.33, of which the last four exons are mainly responsible for its alternative splicing, and the dominant spliced form is p73, having all exons (
cDNA as a probe demonstrated transcripts in the thymus of 2.5 kb and 5.6 kb (Fig 1). According to the cDNA length of p73 transcripts in the database, the 2.5-kb band was expected to be a p73
transcript, as shown by RT-PCR analyses in somatic tissues, including thymus (
isoform. Although we could not fully explain the nature of the 5.6-kb band, it might have been due to cross reaction with transcripts from the p63 gene, as previously reported (
|
A Polyclonal Antibody Against p73
Human thymic tissues consist of various types of cells, such as developing T-cells, epithelial cells, macrophages, endothelial cells, and other stromal cells ( cDNA (293-p73) (Fig 2). Seemingly, polyclonal antibody H-79 (purchased from Santa Cruz Biotechnology), which was generated with the N-terminal region of p73
, exhibited the same band as that found in ST-2G. These results indicated that the two antibodies recognized the p73 molecule. Although the molecular weight of the core protein of p73
was expected to be 69.5 kD, this subtle discrepancy in the molecular size can probably be explained by post-translational modification of p73
, such as phosphorylation in cells (
in the nucleus (Fig 3). Therefore, we concluded that ST-2G was available for the following immunohistochemical studies of p73.
|
|
p73 Expression in the Nuclei of Human TECs
Microscopic examination of human thymus immunostained with ST-2G revealed that p73 appeared in the nuclei of cells that were widely localized in the subcapsular region, cortex, and medulla (Fig 4). This distribution of p73 throughout the thymus was like that of TECs. We also found that Hassall's corpuscles, a particular structure formed by TECs in the medulla, expressed p73. To further identify cells presenting the p73 molecule in the thymus, we employed confocal laser microscopy for double immunostaining analysis with ST-2G and several monoclonal antibodies specific for certain cell types. The results demonstrated that p73 was localized in the nuclei of cells expressing cytokeratin-19 (CK-19), which is a ubiquitously expressed cytokeratin in human TEC (Fig 5) (
|
|
|
p73 Expression of TEC Lines
To further study p73 expression in TECs, two human TEC lines were subjected to an immunohistochemical analysis.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we demonstrated the nuclear expression of p73 in epithelial cells of the human thymus as well as in TEC lines. The antibody used in this study, ST-2G, was against an 18-mer peptide of the C-terminal region of p73 observed in both p73 (a full-length form with 636 aa) and a somewhat shorter form, p73
(a spliced-out form of exon 11 and 13 with 555 aa) among spliced-variant species of the p73 gene (
is probably dominant because Northern blotting analysis showed a band presumably corresponding to a p73
transcript (Fig 1), in addition to reported evidence that p73
is the main isoform in the thymus (
may be regulated by its SAM domain in the C-terminal, which is a regulatory element in cells (
to other p73 isoforms may also affect its transcriptional activities (
It has been reported that a substantial number of genes are activated by p73, like p53. They include mdm2, p21, gadd45, cyclin G, bax, and insulin-like growth factor binding protein (IGFBP3), which regulate the cell cycle and apoptosis (
Most CK-19-positive cells whose subtype is distributed over thymic tissues showed nuclear localization of p73, though some CK-19-positive cells exhibited faint or no expression of p73 in the nucleus. This may imply that there is some population of TECs not under the control of p73. Ontogenically, the thymic epithelial rudiment in humans is widely recognized to be formed from the endoderm of the third pharyngeal pouch and ectoderm of the corresponding branchial clefts, indicating heterogenous epithelial components in the thymus (
In summary, p73 is expressed in human TECs and may have a role in regulating the cell cycle of these cells. p73 null mice have neurological and immunological defects but no obvious abnormalities of lymphoid populations (
![]() |
Acknowledgments |
---|
Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology of Japan and by the Akiyama Foundation of Japan.
We are grateful to Gerry Melino (University Tor Vergata; Rome, Italy) for p73 cDNA. We also thank Avinash Bhandoola (University of Pennsylvania) for helpful discussions, and Laura Crisá (Scripps Research Institute) and Shigeo Yokoyama (Hokkaido Children's Hospital and Medical Center, Japan) for generous assistance.
Received for publication October 8, 2001; accepted November 21, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agami R, Blandino G, Oren M, Shaul Y (1999) Interaction of c-Abl and p73alpha and their collaboration to induce apoptosis. Nature 399:809-813[Medline]
Annunziato F, Romagnani P, Cosmi L, Lazzeri E, Romagnani S (2001) Chemokines and lymphopoiesis in human thymus. Trends Immunol 5:277-281
Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, Smits AP, NewburyEcob R, Hennekam RC, Van Buggenhout G, van Haeringen A, Woods CG, van Essen AJ, de Waal R, Vriend G, Haber DA, Yang A, McKeon F, Brunner HG, van Bokhoven H (1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99:143-153[Medline]
Chi SW, Ayed A, Arrowsmith CH (1999) Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. EMBO J 18:4438-4445
De Laurenzi V, Rossi A, Terrinoni A, Barcaroli D, Levrero M, Costanzo A, Knight RA, Guerrieri P, Melino G (2000) p63 and p73 transactivate differentiation gene promoters in human keratinocytes. Biochem Biophys Res Commun 273:342-346[Medline]
Fernandez E, Vicente A, Zapata A, Brera B, Lozano JJ, Martinez C, Toribio ML (1994) Establishment and characterization of cloned human thymic epithelial cell lines. Analysis of adhesion molecule expression and cytokine production. Blood 83:3245-3254
Frank J, Pignata C, Panteleyev AA, Prowse DM, Baden H, Weiner L, Gaetaniello L, Ahmad W, Pozzi N, CserhalmiFriedman PB, Aita VM, Uyttendaele H, Gordon D, Ott J, Brissette JL, Christiano AM (1999) Exposing the human nude phenotype. Nature 398:473-474[Medline]
Gobin SJ, van den Elsen PJ (2000) Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G. Hum Immunol 61:1102-1107[Medline]
Haynes BF, Denning SM, Le PT, Singer KH (1990) Human intrathymic T cell differentiation. Semin Immunol 1:67-77
Ichimiya S, Nakagawara A, Sakuma Y, Kimura S, Ikeda T, Satoh M, Takahashi N, Sato N, Mori M (2000) p73: structure and function. Pathol Int 50:589-593[Medline]
Ichimiya S, Nimura Y, Kageyama H, Takada N, Sunahara M, Shishikura T, Nakamura Y, Sakiyama S, Seki N, Ohira M, Kaneko Y, McKeon F, Caput D, Nakagawara A (1999) p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent. Oncogene 18:1061-1066[Medline]
Irwin MS, Kaelin WG (2001) p53 family update: p73 and p63 develop their own identities. Cell Growth Differ 7:337-349
Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F, Caput D (1997) Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90:809-819[Medline]
Klug DB, Carter C, Crouch E, Roop D, Conti CJ, Richie ER (1998) Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment. Proc Natl Acad Sci USA 95:11822-11827
Kojima T, Kokai Y, Chiba H, Yamamoto M, Mochizuki Y, Sawada N (2001) Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes. Exp Cell Res 263:193-201[Medline]
Lee LA, Walsh P, Prater CA, Su LJ, Marchbank A, Egbert TB, Dellavalle RP, Targoff IN, Kaufman KM, Chorzelski TP, Jablonska S (1999) Characterization of an autoantigen associated with chronic ulcerative stomatitis: the CUSP autoantigen is a member of the p53 family. J Invest Dermatol 113:146-151
Manley NR (2000) Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation. Semin Immunol 5:421-428
Marx A, MüllerHermelink HK (1997) Epithelial Tumors of the Thymus. New York, Plenum Press
Nishio H, Matsui K, Tsuji H, Tamura A, Suzuki K (2000) Immunolocalization of calcineurin and FKBP12, the FK506-binding protein, in Hassall's corpuscles of human thymus and epidermis. Histochem Cell Biol 114:9-14[Medline]
Osada M, Ohba M, Kawahara C, Ishioka C, Kanamaru R, Katoh I, Ikawa Y, Nimura Y, Nakagawara A, Obinata M, Ikawa S (1998) Cloning and functional analysis of human p51, which structurally and functionally resembles p53. Nature Med 4:839-843[Medline]
Ozaki T, Naka M, Takada N, Tada M, Sakiyama S, Nakagawara A (1999) Deletion of the COOH-terminal region of p73alpha enhances both its transactivation function and DNA-binding activity but inhibits induction of apoptosis in mammalian cells. Cancer Res 59:5902-5907
Pantelouris EM (1973) Athymic development in the mouse. Differentiation 6:437-450
Parsa R, Yang A, McKeon F, Green H (1999) Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J Invest Dermatol 113:1099-1105
Plum J, De Smedt M, Verhasselt B, Kerre T, Vanhecke D, Vandekerckhove B, Leclercq G (2000) Human T lymphopoiesis. In vitro and in vivo study models. Ann NY Acad Sci 917:724-731
Ritter MA, Boyd RL (1993) Development in the thymus: it takes two to tango. Immunol Today 9:62-69
Robles AI, Larcher F, Whalin RB, Murillas R, Richie E, GimenezConti IB, Jorcano JL, Conti CJ (1996) Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia. Proc Natl Acad Sci USA 93:7634-7638
Sait SN, Brooks JJ, Ashraf M, Zhang PJ (2001) A novel t(1;8)(p13;p11) in a thymic carcinoma with unusual giant cell features and renal metastasis. Cancer Genet Cytogenet 124:140-143[Medline]
Senoo M, Seki N, Ohira M, Sugano S, Watanabe M, Inuzuka S, Okamoto T, Tachibana M, Tanaka T, Shinkai Y, Kato H (1998) A second p53-related protein, p73L, with high homology to p73. Biochem Biophys Res Commun 248:603-607[Medline]
Shimosato Y, Mukai K (1997) Tumor of the Mediastinum. 3rd ser. Washington, DC, Armed Forces Institute of Pathology
Su D, Ellis S, Napier A, Lee K, Manley NR (2001) Hoxa3 and pax1 regulate epithelial cell death and proliferation during thymus and parathyroid organogenesis. Dev Biol 236:316-329[Medline]
Takada N, Ozaki T, Ichimiya S, Todo S, Nakagawara A (1999) Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas. Cancer Res 59:2810-2814
Ueda Y, Hijikata M, Takagi S, Chiba T, Shimotohno K (1999) New p73 variants with altered C-terminal structures have varied transcriptional activities. Oncogene 18:4993-4998[Medline]
Ueda Y, Hijikata M, Takagi S, Chiba T, Shimotohno K (2001) Transcriptional activities of p73 splicing variants are regulated by inter-variant association. Biochem J 356:859-866[Medline]
van den Elsen PJ, Gobin SJ, van Eggermond MC, Peijnenburg A (1998) Regulation of MHC class I and II gene transcription: differences and similarities. Immunogenetics 48:208-221[Medline]
Viret C, Janeway CA (1999) MHC and T cell development. Rev Immunogenet 1:91-104[Medline]
Yang A, McKeon F (2000) p63 and p73: p53 mimics, menaces and more. Nat Rev Mol Cell Biol 3:199-207
Yang A, Walker N, Bronson R, Kaghad M, Oosterwegel M, Bonnin J, Vagner C, Bonnet H, Dikkes P, Sharpe A, McKeon F, Caput D (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404:99-103[Medline]
Yun J, Chae HD, Choy HE, Chung J, Yoo HS, Han MH, Shin DY (1999) p53 negatively regulates cdc2 transcription via the CCAAT-binding NF-Y transcription factor. J Biol Chem 274:29677-29682
Zettl A, Strobel P, Wagner K, Katzenberger T, Ott G, Rosenwald A, Peters K, Krein A, Semik M, MullerHermelink HK, Marx A (2000) Recurrent genetic aberrations in thymoma and thymic carcinoma. Am J Pathol 157:257-266