Pin1 and JNK1 cooperatively modulate TAp63γ.

The p63 gene encodes at least 10 isoforms, which can be classified into TA and ∆N isotypes (TAp63 and ∆Np63 proteins) according to their differences at the N termini. TAp63γ is an important transcription factor. We previously reported that peptidyl-prolyl isomerase (PPI) Pin1 directly binds to TAp63γ protein and identified that serine 12 (S12 ) in the transactivation domain (TAD) of TAp63γ is required for regulation of its transcriptional activity. In the present study, we report that Pin1 stimulates transcriptional and pro-apoptotic activities of TAp63γ; this Pin1-mediated stimulation may depend on phosphorylation of S12 mediated by JNK1 and results in striking activation of TAp63γ. JNK1 represses transactivity of TAp63γ in cells without abundant Pin1 proteins and enhances it in the presence of sufficient levels of Pin1. Collectively, our data suggest a novel mechanism for regulation of TAp63γ transactivity: TAp63γ with unphosphorylated S12 is moderately active, phosphorylation at this residue (pS12 ) makes it hypoactive, and Pin1 binds to the pS12 -P13 motif and makes TAp63γ hyperactive. Our findings will aid in the elucidation of the mechanism underlying modulation of TAp63γ.

TAp63γ influences mouse cartilage development.

Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63γ. To investigate the role of TAp63γ in chondrocyte differentiation and maturation, we developed stable TAp63γ expressing ATDC5 cells. Compared to control cells, TAp63γ cells showed significant upregulation of Col10a1 after 4 and 7 days in culture. Moreover, alkaline phosphatase, Alizarin red, and Alcian blue staining were stronger in TAp63γ cells, suggesting that TAp63γ promotes chondrocyte proliferation, hypertrophic differentiation, and possibly matrix mineralization. To investigate the in vivo function of TAp63γ during endochondral bone formation, we established transgenic mice that express flag-tagged TAp63γ driven by Col10a1 regulatory elements. Skeletal staining of transgenic mice at postnatal day 1 showed accelerated ossification in long bone, tail, and digit bones compared to wild-type littermates. Furthermore, Sox9 expression was reduced and Runx2 expression was increased in the proliferative and/or hypertrophic zones of these mice. Altogether, these results suggest that TAp63γ promotes endochondral ossification and skeletal development, at least partially via controlling chondrocyte differentiation and maturation.

JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

TAp63γ is a homologue of tumor suppressor p53 and functions as a transcriptional factor playing key roles in cell cycle and cell apoptosis. In the present work, we find that JNK1 can physically interact with N-terminal transactivation domain (TAD) of TAp63. Overexpression of JNK1 inhibits TAp63γ-mediated transcription, while knockdown or inhibition of endogenous JNK1 increases transactivity of TAp63γ. Further study reveals that Ser12 site in TAD is critical for JNK1-mediated inhibition of TAp63γ. This JNK1-mediated inhibition can impair pro-apoptotic activity of TAp63γ. Together, we report a novel regulation of TAp63γ transactivity and pro-apoptotic activity mediated by JNK1.

Mouse p63 variants and chondrogenesis.

As a critical member of the p53 family of transcription factors, p63 has been implicated a role in development than in tumor formation, because p63 is seldom mutated in human cancers, while p63 null mice exhibit severe developmental abnormalities without increasing cancer susceptibility. Notably, besides the major epithelial and cardiac defect, p63 deficient mice show severe limb and craniofacial abnormalities. In addition, humans with p63 mutations also show severe limb and digit defects, suggesting a putative role of p63 in skeletal development. There are eight p63 variants which encode for the TAp63 and ΔNp63 isoforms by alternative promoters. How these isoforms function during skeletal development is currently largely unknown. Our recent transgenic studies suggest a role of TAP63α, but not ΔNP63α, during embryonic long bone development. However, the moderate skeletal phenotypes in the TAP63α transgenic mice suggest requirement of additional p63 isoform(s) for the limb defects in p63 null mice. Here, we report analysis of mouse p63 variants in MCT and ATDC5 cells, two cell models undergo hypertrophic differentiation and mimic the process of endochondral bone formation upon growth arrest or induction. We detected increased level of p63 variants in hypertrophic MCT cells by regular RT-PCR analysis. Further analysis by qRT-PCR, we detected significantly upregulated level of γ variant (p<0.05), but not α or ß variant (p>0.05), in hypertrophic MCT cells than in proliferative MCT cells. Moreover, we detected upregulated TAP63γ in ATDC5 cells undergoing hypertrophic differentiation. Our results suggest that TAp63γ plays a positive role during endochondral bone formation.