Putative function of TAP63α during endochondral bone formation

Abstract

P63, a member of the P53 tumor suppressor family, is known to play important functions in cancer and development. Interestingly, previous studies have shown that p63 null mice are absent or have truncated limbs, while mutations in human P63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in long bone development. Indeed, we detected increased level of p63 transcript in hypertrophic MCT cells (an established cell model of chondrocyte maturation) than in proliferative MCT cells. To investigate the in vivo role of P63 upon endochondral bone formation, we have established transgenic mouse lines in which HA- and Flag-tagged TAP63α (the longest P63 isoform) is driven by the hypertrophic chondrocyte-specific Col10a1 regulatory elements. Skeletal staining of Col10a1-TAP63α transgenic mice at either embryonic day 17.5 (E17.5) or postnatal day 1 (P1) observed accelerated ossification in long bone, digit and tail bones compared to their wild-type littermates, suggesting a putative function of P63 during skeletal development. We also detected decreased level of Sox9 and Bcl-2 transcripts, while Alp and Ank are slightly upregulated in Col10a1-TAP63α transgenic mouse limbs. Further immunohistochemical analysis confirmed the decreased Sox9 expression in the proliferative and hypertrophic zone of these mice. Von Kossa staining suggests increased mineralization in hypertrophic zone of transgenic mice compared to littermate controls. Together, our results suggest a role of TAP63α upon skeletal development. TAP63a may promote endochondral ossification through interaction with genes relevant to matrix mineralization and chondrocyte maturation or apoptosis

Distinct function of P63 isoforms during embryonic skeletal development

“…1A). This fragment was blunted and cloned into the HindIII site of the pcDNA3.1(−) downstream of the hypertrophic chondrocyte-specific Col10a1 promoter element, which contains four copies of the 300-bp Col10a1 cis-enhancer and a short basal promoter (265 bp) as previously described (Zheng et al, 2009; Li et al, 2012). The junction sequence and the insert orientation of Col10a1-ΔNP63 α were confirmed by sequencing using Δ NP63 α-specific primers.…”

“…After genotyping, transgenic ( TG ) mice or their wild-type ( WT ) littermates at embryonic day 15.5 (E15.5), 17.5 (E17.5), and postnatal day 1 (P1) stages were subject to skeletal phenotypic analysis. Mouse skeletons were prepared and stained with Alcian blue (8GX, Sigma, St. Louis, MO) and Alizarin red (Sigma, St. Louis, MO) according to published protocol and as previously described (Ovchinnikov D, 2009, Li et al, 2012). The signal intensity of the Alizarin red staining indicating ossification status were evaluated for mouse limb, digit, and tail bones (ossified caudal vertebrae numbers).…”

“…At least 30 longitudinal (sagittal) sections (5-µm-thick) of the limb growth plate from both TG and WT littermates were collected and analyzed with the microscope (Nikon Eclipse 80i, Nikon Instruments Inc., Melville, NY USA) and the Qcapture Suite software (version, 2.95.0, Quantitative Imaging Corp., USA). For immunohistochemical (IHC) analysis, detailed protocol using paraffin-embedded sections and Sox9 antibody (H-90, sc-20095, Santa Cruz) has been described previously (Li et al, 2012). The concentration used for the primary anti-Sox9 antibody was at 1:3000 dilution.…”

“…Tissue sections were further incubated with biotinylated secondary antibody (anti-rabbit IgG, Santa Cruz). Detection using the ABC kit (Elite PK-6200 Universal, VECTOR laboratories) and microscopic analysis was as previously described (Li et al, 2012). …”

“…We have recently performed transgenic studies by targeting TAP63 α expression using the hypertrophic chondrocyte-specific Col10a1 promoter elements. Accelerate ossification was observed in long bone, digit and tail bones of the Col10a1-TAP63 α transgenic mice at both E17.5 and P1 stages, suggesting its positive role in late embryonic skeletal development (Li et al, 2012). In this manuscript, we report generation of multiple transgenic mouse lines in which P63 transcript variants Δ NP63 α and TAP63 α were driven by either the hypertrophic chondrocyte-specific Col10a1 -, or the chondrocyte-specific Col2a1 -promoter elements.…”