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Transcription factor Trp63, a homolog of the tumor suppressor p53, is pivotal in the development of stratified epithelial tissues, including the epidermis, breast, prostate, and thymus.1 The p63 gene encodes multiple products (isoforms). Specifically, its transcription, initiated from two different promoters, produces isoforms containing (TAp63) or lacking (ΔNp63) an N-terminal transactivation domain. Both transcripts undergo alternative splicing at the C-terminus leading to α, β, and γ isoforms of TAp63 and ΔNp63.2 Thus, p63 executes complex molecular functions to regulate various and sometimes paradoxical phenotypes. Although the exact roles of each p63 isoform are still not clear, two fundamental functions have emerged: (i) tumor suppression through the induction of tumor cell senescence and apoptosis,3, 4, 5 associated mainly with the TAp63 isoform and (ii) epithelial stem cell maintenance1, 6, 7, 8 through the regulation of self-renewal and proliferation, associated mainly with the ΔNp63 isoform.

The role of p63 in thymic development is considered to be essential for the proliferation potential of thymic epithelial stem/progenitor cells, but it could be dispensable for lineage commitment and differentiation.9, 10 Generally, thymic development appears to be regulated by the ΔNp63 isoform rather than by the TAp63 isoform through the maintenance of epithelial progenitor ‘stemness’. This was demonstrated in vivo by introducing the ΔNp63 or the TAp63 transgene into p63-knockout mice. The results show that ΔNp63, but not TAp63, could rescue defective thymus development in the p63-knockout mice.9 However, the role of TAp63 in the thymus in vivo is largely unknown.

TAp63, has been shown to possess opposing functions—prevention of aging11 and promotion of cellular senescence,4 but studies of pan-p63 ’s roles in epithelial cell and organ aging show that reduction in p63 expression caused cellular senescence and led to accelerated aging.11, 12 Similar paradoxical effects were observed in tumor studies as well. For example, p63 was initially considered to be a tumor suppressor as it overlapped with p53 in targeting genes.2 Later, p63 was found to function as a putative oncogene, as its expression was increased in early neoplasia.13 This may be due to the molecular complexity of p63, which has both transactivating and transcriptional repressor activities that bind over 5800 target sites14 to regulate a wide spectrum of target genes.1 Thus, to determine whether TAp63 is associated with regulation of thymic epithelial cell (TEC) senescence3, 4, 5 during thymic aging, both changes in expression and gain-of-function of TAp63 in the thymus under physiological aging conditions should be investigated.

Cellular replicative senescence was originally referred to as a proliferative end stage in cultured somatic cells (mostly fibroblasts).15, 16 It can be induced by telomere erosion, DNA damage, oxidative stress, and oncogene activation.17, 18 The concept of senescence in vitro may be applied to in vivo tissue homeostasis as it is related to natural aging, and could also have a role in organismal aging and age-related pathology.19 For example, aged organs are considered to be sites of accumulated cellular senescence.20, 21 In the aged thymus, it is possible that there is an accumulation of senescent TECs as implied by senescence-associated β-galactosidase (SA-β-gal) staining activity22 in a previously characterized premature aging mouse model.28 and are available from Jackson Laboratories (Bar Harbor, ME, USA) (no. 012941). Mouse age groups are young (±2-month), late young (>3 months), early middle-aged (6∼9-month), middle-aged (±12-month), and aged (≥18-month), based on WT mouse thymic size in our previous experiments.24, 45 All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center at Fort Worth, in accordance with guidelines from the National Institutes on Aging, Bethesda, MD, USA.

Intrathymic transformation of TAp63-cDNA or FoxN1-cDNA

TAp63γ and TAp63β cDNAs (kindly provided by Dr. Mills4) were sub-cloned into the CMV promoter-driven pADTrack vector (Supplementary Figure S2). FoxN1-cDNA placed in the CMV promoter-driven pADTrack vector was kindly provided by Dr. Brissette.46 The control vector was an empty pADTrack plasmid. The cDNA plasmid was delivered in vivo by a nonviral PEI-mediated method, as described previously.29 A mixture of plasmid and PEI (VWR, no. 201-20G) at ionic balance N/P ratio=8, in ∼25 μl volume was intrathymically injected into young (TAp63γ TAp63β or TAp63βγ mixed cDNAs, as the TAp63βγ isoforms were shown to be the most robust senescence inducers4) and aged (FoxN1-cDNA) mice under anesthesia using suprasternal notch surgery.29 Each mouse was injected twice over a 2-week interval, and 4 weeks after the first injection the thymi were isolated for analyses.

Senescence-associated β-galactosidase assay

Cryosections of differently aged mouse thymus tissues (16 μm thick) were analyzed for SA-β-gal activity using a Senescence β-Galactosidase Staining Kit according to the manufacturer’s protocol (Cell Signaling Technology, Inc., Danvers, MA, USA, no. 9860), and counterstained with nuclear fast red (RICCA Chemical no. R5463200) solution.

IF staining

Cryosections (6 μm thick) were fixed in cold acetone, blocked with 10% donkey serum in Tris-buffered saline (TBS), and stained with optimized dilutions of dual primary antibodies, followed by optimized dilutions of fluorochrome-conjugated dual secondary antibodies. The primary antibodies used were Pan-p63 (4A4) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-8431), ΔNp63 (BioLegend, San Diego, CA, USA, no. 619001),11 TAp63 (D-20) (Santa Cruz, sc-8608), rabbit anti-mouse claudin-3,4 (Invitrogen, Grand Island, NY, USA, no. 34–1700 and no. 36–4800), p21 (Santa Cruz, F-5, sc-6346), and Keratin-8 (Troma-1 supernatant). The secondary antibodies used were Cy3-conjugated donkey anti-mouse, -goat, or -rabbit IgG (Jackson ImmunoResearch Lab), or Alexa-Fluor-488-conjugated donkey anti-rat IgG (Invitrogen). IF labeled samples were mounted using anti-fade aqueous mounting medium, which usually contains 4',6-diamidino-2-phenylindole (DAPI). The positively stained areas were quantified by NIH Image-J software. The magnification in the figures indicates the objective lens of a Nikon Eclipse Ti-U fluorescence microscope.

Real-time RT-PCR

Total RNA from fluorescence-activated cell sorting (FACS)-sorted TECs (gate CD45−MHC-II+ population) was prepared and reverse transcribed with the SuperScriptIII cDNA kit (Invitrogen). Real-time RT-PCR was performed in a Step-One-Plus thermal cycler system (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) with SYBR-green reagents. The sequences of ΔNp63 and TAp63 primers were as previously published.4 The relative expression levels of TAp63 and ΔNp63 mRNAs from aged animals were compared with those from young animals. The average ΔΔCT value from multiple young animals was always arbitrarily set as 1.0 in each real-time PCR reaction. Samples were also internally normalized to GAPDH controls.

Western blot analysis and immunoprecipitation (IP)

The whole thymus was subjected to homogenization and protein extraction in RIPA lysis buffer (Sigma, St Louis, MO, USA, #R0278). Protein, ∼25 μg/lane, was loaded under reducing condition for direct western blot assay with TAp63 antibody (D-20, Santa Cruz, sc-8608), and GAPDH was used as an internal loading control. Alternatively, the protein was precipitated with Pan-p63 antibody (4A4) using protein A/G PLUS-agarose (Santa Cruz, sc-2003) at 4 °C overnight and subjected to western blot analysis with TAp63 antibody.

Flow cytometry assays

FoxN1flox mice were injected intraperitoneally with TM (2 mg/mouse/day) for 4 successive days.28 On the fourth day after the last TM injection, the mice were killed for flow cytometry assay of p-p53 in TAp63+ population. The thymi were torn apart in PBS to release thymocytes, and dissociated by incubation through three enzyme cycles (Collagenase-V/DNase-I) to enrich TECs.25, 47 The single cell suspension of thymic cells was stained with combinations of fluorochrome-conjugated antibodies against cell surface markers: anti-mouse-PE/Cy5-CD45 and PE-MHC-II (M5/114) (BioLegend). Cells were then fixed with 2% PFA/PBS, permeabilized with 0.1% TritonX-100, and intracellularly stained for TAp63 with D-20 antibody (goat), followed by incubation with APC-anti-goat IgG, and then further intracellularly stained with p-p53 antibody (Ser-15, rabbit, Cell Signaling Technology Inc., Cat no. 12571). FoxN1 cDNA vector-injected aged thymi were also subjected to flow cytometry assay to analyze proliferation using intracellular staining of ΔNp63 (BioLegend, no. 619001) and Ki67 (BioLegend, clone 16A8). Data were acquired using a BD LSRII Flow Cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (FlowJo Home: Tree Star, Inc., Ashland, OR, USA).

Statistics

Statistical significance was analyzed by unpaired Student’s t-test. Differences were considered statistically significant at values of P<0.05.