Expression profiles and functional implications of p53-like transcription factors in thymic epithelial cell subtypes
Tomoki Kikuchi,
Shingo Ichimiya,
Takashi Kojima,
Laura Crisa1,
Shigeru Koshiba,
Akiko Tonooka,
Nobuhiko Kondo,
Paul T. van der Saag2,
Shigeaki Yokoyama and
Noriyuki Sato
Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 0608556, Japan 1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA 2 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands
Correspondence to: S. Ichimiya; E-mail: ichimiya{at}sapmed.ac.jp
Transmitting editor: C. Terhorst
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Abstract
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In this study, we investigated the localization and functional significance of p53 tumor suppressor-like molecules, p63 and p73, in human thymic epithelial cells (TECs). Immunohistochemical studies showed particular distribution profiles of p63 and p73 in thymic epithelium, in which cortical TECs preferentially expressed p63 in their nuclei whereas subcapsular and medullary TECs expressed both p63 and p73 in their nuclei. The wide distribution of p63 in TECs was further suggested by studies using TECs of primary culture. In vitro studies using two human TEC lines demonstrated that p63 was capable of up-regulating intercellular adhesion molecule-1 (ICAM-1) and enhancing the production of IL-6 and IL-8. Moreover, in vitro studies also indicated that p73, but not p63, had the capacity to induce granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in the TEC lines. These findings suggest that p63 would regulate the cell adhesive property through ICAM-1/LFA-1 interaction and the production of IL-6 and IL-8, probably in all TEC subtypes. p73 in subcapslar and medullary TECs was suggested to play a role in the regulation of the production of GM-CSF and G-CSF, which might stimulate other stromal cells such as dendritic cells, macrophages and endothelial cells around these regions.
Keywords: colony stimulating factors, human thymic epithelial cells, ICAM-1, interleukins, p63, p73
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Introduction
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The thymus is the primary lymphoid organ that provides an optimal milieu for the development of a functional T cell repertoire via positive and negative selection (1). This inductive environment is constituted by various stromal cells, including thymic epithelial cells (TECs), which compose largely stationary stromal cells to make the three-dimensional scaffolding framework within which all other thymic cell types reside. TECs have roles in the homing, migration and differentiation of T cell precursors through the establishment of adhesive interactions and the release of various cytokines (2).
According to anatomically characterized localization, TECs are classified into three broad groups (35). From the exterior to the interior, subcapsular TECs (sTECs) lie along the capsule or the septa with the outermost layer on the basement membrane. TECs in the cortex (cTECs) make a complex framework with long cytoplasmic processes for intercellular interaction with the vast majority of immature CD4+CD8+ (double positive, DP) T cells. TECs in the medulla (mTECs) are conspicuously scattered and constitute a loose network surrounding CD4+CD8 and CD4CD8+ (single positive, SP) T cells. As suggested by murine experiments, the earliest lymphoid progenitors migrate toward sTECs. After the gene rearrangement of the TCRß chain, the lymphoid progenitors develop into DP-T cells which cTECs tightly surround. Followed by MHC-based selection, SP-T cells migrate through the network of mTECs to the periphery (6). To determine the functional significance of these TECs, many studies have been attempted to identify different TEC populations using anti-cytokeratin-subtype antibodies or anti-neuroendocrine- related molecule antibodies (7,8). However, our knowledge about the regulatory mechanisms of these TEC species is still limited.
We have recently reported that a member of the p53 family of transcriptional factors, p73, is expressed in the nuclei of human TECs by immunohistochemical analyses with p73 specific antibodies (9). Another p53-related transcription factor is known as p63, which has an important role to specify stem cell property in epithelium (10,11). It is also recognized that mutations of the p63 gene lead to developmental defects of ectodermal structures of ectrodactylyectodermal dysplasiacleft lip/palate (EEC) and its related syndromes (12,13). Together with our reported evidence, these findings indicate epitheliotropism of p63 and p73, suggesting their possible roles in the regulation of epithelium even in the thymus.
Herein, we demonstrate for the first time the particular expression profiles of p63 and p73 in human TECs. Immunohistochemical studies of thymic tissues and TECs of primary culture show the broad distribution of p63 in the nuclei of TEC subtypes. In contrast to p63, p73 is mainly localized in the nuclei of TECs in the subcapsular and medullary regions, indicating that the p53 family members could be utilized as a new marker discriminating TEC subtypes. By using human TEC lines, we also define a novel role of p63 as a potent regulator of the ICAM-1 gene in TECs, which encodes a complimentary ligand of LFA-1 abundantly expressed on the thymocyte (14). More interestingly, it is suggested that the production of immunoregulatory cytokines, including IL-6, IL-8 and colony stimulating factors, is regulated by p63 or p73 in TEC lines.
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Methods
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Thymic tissues
Normal human thymuses were obtained from patients <3 years of age undergoing cardiovascular surgery for congenital heart disease through the Hokkaido Childrens Hospital and Medical Center in Japan. Before cryostat sections were prepared, these were stored at 80°C. Six-micrometer sections of the tissues were cut and placed on glass slides. Then they were fixed in ice-cold acetone for 30 s and dried. Formalin-fixed paraffin-embedded tissue sections of the thymuses were also used in this study of 6 µm sections. All tissue was obtained with informed consent and approval by institutional review boards.
Cell cultures and transfections
Two human TEC lines, P1.4D6 and F2.5B6, were cultured in D-valine containing MEM (Invitrogen, Carlsbad, CA) supplemented with 2 mmol/l of L-glutamine, 10 mmol/l HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin and 10% fetal bovine serum in a humidified atmosphere at 37°C in 5% CO2 as previously described (15). These cells were transfected by using Lipofectaine 2000 reagent (Invitrogen) with TAp63
cDNA recombined in expression vector pIRES-hyg2 (Clontech, Palo Alto, CA), named pIRES-hyg2-TAp63
or TAp73
cDNA in pIRES-puro (Clontech), named pIRES-puro-TAp73
. Transfected cells with pIRES-hyg2-TAp63
or pIRES-puro-TAp73
were selected in medium containing 500 µg/ml hygromycin B or 10 µg/ml puromycin (Sigma, St Louis, MO), respectively. During these procedures, mock-transfectants were simultaneously established as a control. HaCaT cells were maintained in DMEM (Sigma) supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin and 10% fetal bovine serum. Jurkat, Molt-4 and Ball-1 cells were cultured in RPMI 1640 (Sigma) with 100 U/ml penicillin G, 100 µg/ml streptomycin and 10% fetal bovine serum.
Primary culture of thymic epithelial cells
Procedures of primary thymic epithelial cell culture were described as reported previously with several modifications (16,17). In brief, human thymus was minced into pieces 2 3 mm3 in volume and washed with phosphate-buffered saline (PBS) five times. These fragments were suspended in 20 ml of dispersing solution with 0.4 mg/ml DNase I (Sigma) and 0.14 mg/ml Liberase Blenzyme 3 (Roche, Basel, Switzerland) in PBS and then incubated at 37°C for 30 min. The dissociated fragments were subsequently filtrated with 300 µm mesh followed by filtration with 40 µm mesh. After washing with PBS three times, cells were cultured in calcium-free WAJC404 medium (pH 7.5; Kyokuto Pharmaceutical Industrial, Tokyo, Japan) supplemented with 28 mmol/l HEPES, 14 mmol/l sodium bicarbonate, 10 µg/ml of cholera toxin, 10 µg/ml of insulin, 10 ng/ml epidermal growth factor, 10 µg/ml transferrin, 10 nM dexamethasone and 25 µg protein/ml bovine pituitary extract and 2% horse serum (Sigma) in collagen-coated dishes at 37°C. After incubation for 15 h, adherent cells were generously washed with PBS three times and maintained in the same medium without serum for several days.
Antibodies
A mouse monoclonal antibody (mAb) and a rabbit polyclonal antibody (pAb) against all p63 variants were 4A4 (LabVision, Fremont, CA) and H-137 (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. ST-2G was a rabbit pAb specific for p73
as previously described (9). An anti-cytokeratin mAb (KL1) obtained from Immunotech (Marseille, France), and mouse TE3 and TE4 mAbs, specific for human thymic cortical and medullary epithelium, respectively (18), were from American Type Culture Collection (ATCC; Rockville, MD). The following mAbs were purchased from BD Biosciences: a mouse mAb specific for CD3 and mouse mAbs conjugated with PE for CD11a, CD54, CD58 and CD152 for flow cytometry. For detecting CD54 molecules by western blot analysis, a mouse mAb was employed (clone 28; BD Biosciences).
Immunohistochemistry
Phenotypic analyses of thymic stromal cells were performed using indirect immunohistochemical procedures (9). Briefly, frozen sections of the tissue were incubated with the optimal diluted antibody overnight at 4°C. After washing with PBS three times, slides were then incubated with the secondary antibody conjugated with peroxidase at room temperature for 1 h. Peroxidase activities were revealed with diaminobenzidine tetrahydrochloride (DAB) as the chromogen in the presence of hydrogen peroxide (Nichirei, Tokyo, Japan). For double-staining analysis, tissue sections of normal thymuses were subjected to staining with a mouse mAb and a rabbit pAb. These were visualized with Alexa 488 (green)-conjugated goat anti-mouse IgG and Alexa 594 (red)-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and examined with a fluorescence microscope (IX71; Olympus, Tokyo, Japan) or a laser scanning confocal microscope (MRC 1024; Bio-Rad, Hercules, CA).
Northern blot analysis
Northern blot membranes with 2 µg of poly A+ RNA derived from various lymphoid tissues were used (Human Immune System MTN Blot II; Clontech). As a probe, an EcoRI-fragment 614 bp in length from pcDNA3-TAp63
, which included the N-terminal sequence of TAp63
, was labeled with [
-32P]dCTP (3000 Ci/mmol; Amersham, Piscataway, NJ). Hybridization was performed as specified in the manufacturers protocol using ExpressHyb solution (Clontech). Briefly, the membrane filter was hybridized with radiolabeled probes at 68°C for 15 h. Then the filter was washed in SSC containing 0.1% SDS solution with stepwise decreased salt concentrations followed by 2x SSC, 1x SSC and 0.5x SSC at 50°C for 30 min. The filter was subjected to autoradiography for consecutive days for up to one month at most. For investigating transcripts of transformed cells, 10 µg of total RNA was subjected to electorophoresis in 1% formalin gel and transferred onto Hybond N+ membranes (Amersham). A probe for detecting ICAM-1 transcripts was generated by the RTPCR method on human thymic cDNA with specific primers for ICAM-1 cDNA (R&D Systems, Minneapolis, MN). The amplified products 750 bp in length were ligated into pGEM-T easy vector (Promega, Madison, WI) and the nucleotide sequence of the insert DNA was confirmed using an ABI-PRISM 310 sequencer (PE Applied Biosystems, Foster City, CA). The labeling of the insert DNA digested with EcoRI and hybridization were performed as described above.
Western blot 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 TrisHCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and protease inhibitors (Roche) for 30 min at 4°C as previously described (9). Aliquots of the supernatants were applied to SDS10% polyacrylamide gels under reducing conditions and transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). After blocking non- specific protein binding using Tris-buffered salineTween 20 (TBST) containing 5% fat-free dry milk, the membrane was incubated with antibodies for 1 h at room temperature, followed by reaction with a secondary antibody conjugated with peroxidase. After washing with TBST three times, signals were detected by chemiluminescence with an ECL kit (Amersham).
Flow cytometry
For detecting surface adhesion molecules, cultured cells were detached from the tissue culture bottles by using PBSEDTA solution to prepare 5 x 106 cells. Cells were stained with a series of mAbs conjugated with PE and subjected to analysis with a FACScan flow cytometer (10 000 events/sample; BD Biosciences). All data shown were obtained with gates set on living cells and analyzed with CellQuest software (BD Biosciences). Prior to the staining with TE3 and TE4 mAbs which bind with intracytoplasmic antigens, cells were permeabilized with lysolecithin as described previously (19). Followed by staining with a secondary antibody conjugated with FITC, cells were analyzed with a FACScan flow cytometer.
Luciferace activity assay
Four human ICAM-1 promoter-reporter constructs in a luciferase pGL3-basic vector (Promega), including pIC1352 (containing full-length promoter, 1352/+1), pIC339 (339/+1), pIC135 (135/+1) and pIC135-
AP2 (a deleted form of pIC135 without GAS, Sp-1 and AP-2 sites), were used as described previously (20,21). Cells were plated in 12-well plates at a density of 2 x 105 cells/well. After 24 h, 250 ng of a reporter gene plasmid in combination with 25 ng of pSV-ß-galactosidase (Promega) was transiently transfected by using Lipofectamine 2000 reagent (Invitrogen). Transfections were performed in triplicate, and cells were harvested after 48 h. Luciferase activity was measured according to the manufacturers protocol (Promega) and normalized for transformation efficiency with the ß-galactosidase assay (Stratagene, La Jolla, CA).
Cytokine assays
Cytokine concentrations in culture supernatants were analyzed with enzyme-linked immunosorbent assay (ELISA) kits as described in the manufacturers protocol (Genzyme, Cambridge, MA). Samples were assayed in triplicate and cytokine concentrations were calculated with calibration curves made with serial dilutions of the respective cytokines in the kit. All data were subjected to Students t-test to determine whether differences between two groups were statistically significant. Significance was assigned to P < 0.05.
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Results
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Expression of p63 in the thymus
To investigate the gene expression of p63 in the thymus at the transcriptional level, we initially conducted northern blot analysis. The results demonstrated that p63 transcripts were present in the thymus as a single band (Fig. 1A). In other immunological tissues or cells, including spleen, lymph node, peripheral blood leukocytes and bone marrow, no transcriptional species of p63 were detected after exposure for over one month, as was also the case in fetal liver. As a positive control of the hybridization, we performed northern blot analysis using several human cell lines, including HaCaT cells, which were previously reported to express p63 (22). The results showed that the HaCaT epidermal cells demonstrated transcripts of p63 under the same hybridization conditions (Fig. 1B). In contrast, the transcripts of p63 were not detected in Jurkat and Molt-4 T cells, or in Ball-1 B cells. These findings indicated that p63 was transcriptionally expressed in certain types of stromal cells in the human thymus.
We next examined the tissue distribution of p63 in the thymus by immunohistochemical analyses. While structural similarities between p63 and p53 have been noted, p63 proteins are constituted from transcriptional variants caused by alternative splicing at both termini of the p63 gene (23). Results using an antibody reacting to all of the variant isoforms of p63 (pan-p63 mAb, 4A4) showed that p63 was expressed in the nuclei of cells localizing in all regions including the subcapsule, cortex and medulla (Fig. 2). The histological distribution of p63 in the thymus demonstrated that p63 was likely to be expressed in stromal cells, not lymphoid cells. These observations were supported by double staining analysis of the thymus with antibodies specific for p63 and cytokeratin or CD3, indicating that cells expressing p63 in the thymus were epithelial cells, not immature T lymphocytes (Fig. 3A and B). To further confirm the expression of p63 in TECs, we conducted immunohistochemical studies of TECs in primary cultures. In accordance with the immunohistochemical studies as shown in Figs 2 and 3, all epithelial cells of the primary culture of the thymus expressed p63 in their nuclei (Fig. 4). This further provided evidence that p63 was broadly expressed in TECs in vivo.

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Fig. 2. Immunohistochemical analysis of human thymus with pan-p63 mAb (4A4). Stromal cells with nuclear expression of p63 are widely observed in the cortical and medullary regions (depicted as C and M, respectively) as well as in the subcapsular region (arrowheads indicate stained cells arrayed in a line). Note that p63 is not expressed in thymocytes of the cortex and medulla. Hassalls corpuscles are shown by arrows in the medullary region. Original magnification: x200.
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Fig. 3. Confocal laser microscopy after double staining of the thymic tissue sections with pan-p63 pAb (H-137) and anti-cytokeratin or anti-CD3 mAb. (A) Cytokeratin-positive cells (green) showing nuclear expression of p63 (red) in the cortical region. (B) CD3-positive cells (green) not showing nuclear expression of p63 (red) in the cortical and subcapsular regions. Original magnification: x500.
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Fig. 4. Immunofluorescence microscopy after double staining of thymic epithelial cells of primary culture with pan-p63 pAb (H-137) and anti-cytokeratin mAb. All of the epithelial cells are positive for cytokeratin (green) express p63 (red). The nuclear localization of p63 in these cells is recognized by staining with 4',6-diamidino-2-phenylindole (DAPI; in blue). Original magnification: x450.
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Tissue distribution of p63 and p73 in the thymus
As we previously reported epitheliotropism of p73 in the human thymus, we examined the distribution of p63 and p73 of the p53 family in thymic epithelium of subcapsular, cortical and medullary regions. When the immunohistochamical pattern of p63 was compared to that of p73, sTECs and mTECs seemed to express both p63 and p73 in their nuclei (Fig. 5). Most cTECs, however, appeared to show only nuclear expression of p63, not both p63 and p73. Next, double-staining analysis was performed to exactly identify the endogenous colocalization of p63 and p73 in the subcapsular and medullary regions. The results demonstrated that p63 was clearly merged with p73 in cell nuclei of the subcapsular region, indicating the expression of both p63 and p73 in sTECs (Fig. 6). As was observed in sTECs, most mTECs expressed both p63 and p73. It was also of interest that, different from sTECs, their intensities in some mTECs were varied, implying heterogeneity of epithelial cells in the medulla in terms of expression intensities of p53 transcription factors.

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Fig. 5. Distribution of p63 and p73 in the thymic tissue sections investigated with pan-p63 mAb (4A4) and anti-p73 pAb (ST-2G). p63 is widely distributed throughout the subcapsule (arrowheads), cortex (C) and medulla (M). On the other hand, more p73 is expressed in subcapsular and medullary regions than in the cortical region. Left panel, a hematoxylineosin stained section as a control; center panel, p63; right panel, p73, counterstained with hematoxylin after development with DAB. Hassalls corpuscles are indicated by arrows in the medullary region. These were examined by light microscopy. Original magnification: x220.
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Fig. 6. Confocal laser microscopy after double staining of the thymic tissue sections with pan-p63 mAb (4A4) and anti-p73 pAb (ST-2G). In the subcapsule, TECs express p63 and p73 in the nuclei with the same intensities. The expression pattern of p63 and p73 in the medullary region is similar to that of subcapsular TEC. Note that some mTECs show varied expression of p73. Upper panel, subcapsular region; lower panel, medullary region. Original magnification: x350.
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Induction of ICAM-1 by p63 in thymic epithelial cells
To assess the functional role of p63 in TECs, we established and analyzed two stably transfected human thymic epithelial cell lines, P1.4D6 and F2.5B6 (15). Since their origins were described as the thymic cortex, we further confirmed that they were cTECs by using a cTEC-specific mAb, TE3 (Fig. 7A). The expression of TAp63
(p63
isoform with transactivation domain) in P1.4D6 and F2.5B6 cells transfected with pIRES-hyg2-TAp63
(named P1.4-p63 and F2.5-p63, respectively) was verified by immunohistochemical and western blot analyses (Fig. 7B and C). It was noted that endogenous p63 proteins were not detected in mock-transfected cells of P1.4D6 and F2.5B6 (named P1.4-mock and F2.5-mock, respectively).
First, we investigated P1.4-p63 and F2.5-p63 cell lines in terms of the surface expression of adhesion molecules with which TECs were capable of anchoring immature T lymphocytes and stromal cells (24,25). Flow cytometric analyses suggested that the expression of ICAM-1 (CD54) was up-regulated in P1.4-p63 and F2.5-p63 cells in comparison to P1.4-mock and F2.5-mock cells (Fig. 8). The intensity of the expression of other adhesion molecules, including LFA-1 (CD11a), LFA-3 (CD58) and CTLA-4 (CD152), was not significantly changed by the introduction of TAp63
in P1.4D6 and F2.5B6 cells. In accordance with these results from flow cytometric analyses, the enhancement of ICAM-1 expression was observed in P1.4-p63 and F2.5-p63 cells at protein as well as transcriptional levels (Fig. 9A and B).
To test the promoter activity of the ICAM-1 gene in TEC lines, a series of luciferase reporter vectors with the 5'-regulatory region of the ICAM-1 gene were transiently transfected into P1.4-mock or P1.4-p63 cells and investigated (20, 21). When we used pIC1352 (1352/+1) containing the full-length human ICAM-1 promoter, a 2.0-fold increase of the luciferase activity in P1.4-p63 cells was observed as compared to P1.4-mock cells (Fig. 10). The luciferase activity of P1.4-mock cells was gradually decreased from the activity of pIC1352 when we used shorter forms of pIC339 (339/+1) and pIC135 (135/+1). In contrast, the luciferase activity of P1.4-p63 cells was still preserved even when we employed pIC135. These findings indicated that cis-acting elements within pIC135, including IFN-
activating sequences (GAS), Sp-1 and AP-2, were responsible for mediating the induction of ICAM-1 expression in P1.4-p63 cells.
Induction of cytokines by p63 and p73 in thymic epithelial cells
TECs are known as an important source of various cytokines required for the maturation of immature T cells and maintenance of other stromal cells. By using ELISA analyses of the culture supernatants of transfected TEC lines, we assessed cytokines affected by p63 or p73 in TECs. As shown in Table 1, P1.4-p63 cells as well as F2.5-p63 cells exhibited increased secretion of IL-6 and IL-8 when compared to mock transfectants (15,26). The levels of IL-4, IL-7, stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF) in the supernatants were not influenced by the introduction of p63 in these two types of cells. Nor were the levels of IL-1ß and IL-5 influenced in TEC lines by p63.
To examine the functional significance of p73, which was suggested to be localized in sTECs and mTECs, we established and analyzed TEC lines transfected with pIRES-puro-TAp73
(p73
isoform with transactivation domain). In F2.5 cells, the levels of GM-CSF, G-CSF and macrophage colony stimulating factor (M-CSF) were markedly increased by the introduction of p73 (Table 2). These cytokines are known to stimulate dendritic cells, macrophages and endothelial cells (2729). Transforming growth factor ß1 (TGFß1) is a pleiotropic cytokine involved in T cell development and hematopoiesis, levels of which in F2.5 cells were not significantly changed by p73 (30).
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Discussion
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In this study, we have provided new evidence on the expression patterns of the p53-like transcription factors and their possible roles in thymic epithelium. To the best of our knowledge, no other family of transcription factors with expression profiles similar to the p53 family in human postnatal thymus has been described. The fact that p63 is widely distributed in all TEC subtypes suggests its fundamental role in the thymus, further supported by the evidence that thymic hypoplasia and depletion of cells in the T zones of lymph nodes were observed in an autopsy case with EEC-related syndrome (31).
We inferred possible involvement of p63 in the production of IL-6 and IL-8 as well as the induction of ICAM-1 in TECs. IL-6 is a multifunctional cytokine that regulates T and B cell functions, acute phase immune reactions and hematopoiesis. In thymus, IL-6 is known to cause immature T cells to proliferate and differentiate, and is especially suggested to be involved in the differentiation of CD4+ T cells from DP-T cells (32). IL-8 is postulated to be an inflammatory cytokine acting as a neutrophil chemoattractant and activating factor. Interest ingly, it has been proposed that a granulocyte-based mechanism for the mobilization of hematopoietic precursor cells is induced by IL-8 (33). Because the P1.4D6 and F2.5B6 cells used in this study possibly originate from cortical TECs and do not express endogenous p63, phenotypic analyses of these cells may clarify the proper roles of p63 in cTEC. In the cortex, the level of ICAM-1 expression influences the effectiveness of MHC-based selection of DP-T cells forming a multifocal immunological synapse with cTECs (34,35). In addition to immature T cells, macrophages express LFA-1 on the cell surface as well. Therefore, macrophageepithelial interaction in the thymus might also be regulated by p63. At present, how p63 affects the locus to initiate ICAM-1 transcription has not been elucidated, although p63 was found to be associated with the regulation of the proximal locus 135 bp upstream of the transcription initiation site among a number of cis-acting elements in the ICAM-1 promoter regions. In the 135 bp region are Sp-1, AP-2 and GAS, which is thought to be important for the induction of ICAM-1 transcription by IFN-
(36).
Different from p63, p73 was preferentially expressed in TECs of the subcapsular or medullary regions. We should consider the origin of TEC lines, suggested to be cTECs, as the results from the p73-transformed TEC lines would help account for the possible function of p73 in mTECs and sTECs. In this context, a certain amount of GM-CSF, G-CSF and M-CSF induced by p73 might be present around mTECs and sTECs. These cytokines may have roles in maintaining stromal cells such as dendritic cells, macrophages and endothelial cells around the subcapsule and medulla (2729). Dendritic cells localized in the medulla are important stromal cell compartments for negative selection of immature T cells. In the cortico-medullary border, there are vascular-rich sites through which prothymocytes enter and SP-T cells exit. Perivascular TECs (pTECs) are reported to have the same phenotype as sTECs, suggesting that p73 might be also present in the nuclei of pTECs (4). It was also of interest that some mTECs showed variable expression of p63 and p73. This implies that the expression levels of p63 and p73 are probably regulated by different mechanisms (37). Such variation of the p53 family in mTECs would be in accord with reported evidence obtained by immunohistochemical and ultrastructural analyses indicating that there are heterogeneous TEC populations in the medullary region (3).
The overlapping functions of p53 transcription factors have been documented. p73 in subcapsular and medullary TECs modifies the discharge of p63 and vice versa, whereas only p63 has a role in cTECs. It is also known that p63 and p73 are capable of inducing p53-responsive genes and can elicit cell cycle arrest and apoptosis (23,34,38). Accordingly, we failed to establish TEC lines of P1.4D6 or F2.5B6 expressing both p63 and p73 for the purpose of studying the roles of these two transcription factors. These results imply that mTECs and cTECs might contain a mechanism for preventing cell death induced by the expression of both p63 and p73 in their nuclei.
Our results in the current study are not sufficient to fully explain the reason why thymic hypoplasia occurs in EEC-related syndrome, because mice deficient in ICAM-1 or cytokines induced by p63 or p73 show insignificant abnormalities of T cell development (39). Accumulating evidence suggests that p63 and p73 play essential roles in epidermal development and cellular proliferation in contrast to the archetypal tumor suppressor p53 (11,23,40). In particular, it is interesting that p63 is necessary for the maintenance of epithelial stem cell precursors as indicated by many studies. Although the molecular mechanism regulated by p63 for preserving the stem cell identity remains to be elucidated, such mechanisms might be associated with the regulation of epithelium residing all over the human thymus.
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Abbreviations
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DPdouble positive
G-CSFgranulocyte colony stimulating factor
GM-CSFgranulocyte macrophage colony stimulating factor
ICAM-1intercellular adhesion molecule-1
IRESinternal ribosome entry site
LFA-1leukocyte function-associated antigen-1
M-CSFmacrophage colony stimulating factor
SPsingle positive
TECthymic epithelial cell
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Acknowledgements
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We are grateful to Gerry Melino (University of Rome Tor Vergata, Italy) for providing the p63 and p73 plasmid cDNA, María L. Toribio (Universidad Autónoma de Madrid, Spain) for the P1.4D6 and F2.5B6 cells and Tetsu Akiyama (University of Tokyo, Japan) for the HaCaT cells. We also thank Jenny P.-Y. Ting (University of North Carolina, NC) and Akira Nakagawara (Chiba Cancer Center Research Institute, Japan) for helpful advice. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to S. I. (No. 13670182).
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