p73 induces apoptosis by different mechanisms

p73, like its homologue, the tumor suppressor p53, is able to induce apoptosis in several cell types. This property is important for the involvement of p73 in cancer development and therapy. However, in contrast with p53, the TAp73 gene has two distinct promoters coding for two protein isoforms with opposite effects: while the transactivation proficient TAp73 shows pro-apoptotic effects, the amino-terminal-deleted DeltaNp73 has an anti-apoptotic function. Indeed, the relative expression of these two proteins is related to the prognosis of several cancers. Here we discuss recent developments in the control of p73-induced apoptosis. First, TAp73 induces ER stress via the direct transactivation of Scotin. Second, TAp73 induces the mitochondrial pathway by directly transactivating both Bax and the BH3 only protein PUMA promoters. While the first transactivation is weak, and not sufficient to trigger apoptosis (at least in the in vitro cellular models so far evaluated), the induction of PUMA is strong and lethal. Third, the promoter of the death receptor CD95 contains a p53 responsive element and preliminary experiments suggest that TAp73 also activates the death receptor pathway. In addition, TAp73 is able to transactivate its own second promoter, thus inducing the expression of the anti-apoptotic DeltaNp73 isoform. Therefore, the balance between TAp73 and DeltaNp73 finely regulates cellular sensitivity to death.     

p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.     

Inhibition of apoptosis via CHIP-mediated proteasomal degradation of TAp73α

TAp73, a homologous of tumor suppressor p53, regulates apoptosis in a p53-independent manner and its suppressive as well as stimulatory role in promoting angiogenesis has been reported. It exists in multiple isoforms which varies structurally in their N-terminus and C-terminus region and crucial interplay among them guides the decision of cell survival and death. As molecular chaperones control both stability and degradation of TAp73, selective regulation of p73 isoforms has implication upon developing new therapeutic for hypoxic tumor. We have discovered that under DNA damage carboxy terminus Hsp70 interacting protein (CHIP's) antiapoptotic function is displayed via its E3 ligase activity that inhibits exclusively TAp73α-mediated apoptosis in cancer cell. The decrease in TAp73α level by CHIP as it is supported by increased ubiquitination pattern is reverted back by sh-CHIP. Further, the transactivation of p53-downstream apoptotic genes BAX, PUMA and PIG3 by TAp73α is also shown to be subsequently inhibited by CHIP. The tetratricopeptide TPR-domain of CHIP in its amino-terminus interacts with the carboxy-terminus of TAp73α and ΔNp73α and as a result, U-BOX domain of CHIP in the carboxy-terminus is able to ubiquitinate TAp73α for proteasomal degradation. Due to lack of C-terminus in TAp73β, CHIP fails to interact with and degrade it. In conclusion, we have thus uncovered for the first time a novel mechanism of chaperone-assisted regulation of p73 stability as well as its apoptotic functions by CHIP that might be utilized to develop new anticancer strategies.     

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.     

p63 overexpression induces the expression of Sonic Hedgehog

p63 and p73 are members of the p53 protein family and have been shown to play an important role in cell death, development, and tumorigenesis. In particular, p63 has been shown to be involved in the maintenance of epidermal stem cells and in the stratification of the epidermis. Sonic Hedgehog (Shh) is a morphogen that has also been implicated to play a role in epithelial stem cell proliferation and in the development of organs. Recently, Shh has also been shown to play an important role in the progression of a variety of cancers. In this report, we show that p63 and p73 but not p53 overexpression induces Shh expression. In particular, p63gamma and p63beta (both TA and DeltaN isoforms) and TAp73beta isoform induce Shh. Expression of Shh was found to be significantly reduced in mouse embryo fibroblasts obtained from p63-/- mice. The naturally occurring p63 mutant TAp63gamma(R279H) and the tumor suppressor protein p14(ARF) inhibited the TAp63gamma-mediated transactivation of Shh. The region -228 to -102 bp of Shh promoter was found to be responsive to TAp63gamma-induced transactivation and TAp63gamma binds to regions within the Shh promoter in vivo. The results presented in this study implicate p63 in the regulation of the Shh signaling pathway.