Nucleolar p19ARF unlike mitochondrial smARF is incapable of inducing p53-independent autophagy

ARF mRNA encodes two distinct proteins, the nucleolar p19ARF and ashorter mitochondrial isoform, named smARF. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19ARF and smARF proteins. The two ARF isoforms differ not only in their localization but also in their functions. Nucleolar p19ARF inhibits cell growth mainly by activating p53 or by inhibiting ribosomal biogenesis. In contrast, mitochondrial smARF caninduce dissipation of the mitochondrial membrane potential and autophagy in a p53 independent manner. Recently, it was proposed by Abida et. al., that similar to smARF, the nucleolar p19ARF can also induce p53 independent autophagy, but in contrast to smARF it does so from within the nucleolus. Our current work shown here indicates however, that if the ectopic expression of p19ARF is restricted to thenucleolus it cannot induce autophagic vesicle formation. Only upon extreme overexpression, when p19ARF is localized to extra nuclear compartments, it can trigger p53-independent autophagic vesicle formation. Thus, our experiments indicate that the nucleolar p19ARF is incapable to induce autophagy from within the nucleolus.     

Nucleolar p19ARF, unlike mitochondrial smARF, is incapable of inducing p53-independent autophagy

ARF mRNA encodes two distinct proteins, the nucleolar p19(ARF), and a shorter mitochondrial isoform, named smARF. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19(ARF) and smARF proteins. The two ARF isoforms differ not only in their localization but also in their functions. Nucleolar p19(ARF) inhibits cell growth mainly by activating p53 or by inhibiting ribosomal biogenesis. In contrast, mitochondrial smARF can induce dissipation of the mitochondrial membrane potential and autophagy in a p53 independent manner. Recently, it was proposed by Abida et al., that similar to smARF, the nucleolar p19(ARF) can also induce p53 independent autophagy, but in contrast to smARF it does so from within the nucleolus. Our current work shown here indicates, however, that if the ectopic expression of p19(ARF) is restricted to the nucleolus it cannot induce autophagic vesicle formation. Only upon extreme overexpression, when p19(ARF) is localized to extra nuclear compartments, can it trigger p53-independent autophagic vesicle formation. Thus, our experiments indicate that the nucleolar p19(ARF) is incapable of inducing autophagy from within the nucleolus.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.     

A smARF way to die: a novel short isoform of p19ARF is linked to autophagic cell death

We recently revealed a novel mechanism by which p19ARF can induce cell death. We found that the p19ARF mRNA encodes an additional shorter isoform from the same open reading frame, named smARF. smARF is a short lived protein, which is rapidly degraded by the proteasome, but accumulates after inappropriate proliferative signals generated by oncogenes. Surprisingly, smARF translocates to the mitochondria, impairs the structure of the mitochondria, and dissipates the mitochondrial membrane potential in a p53 and Bcl-2 family independent manner, ultimately inducing type II caspase-independent autophagic cell death.     

A smARF Way to Die: A Novel Short Isoform of p19ARF is Linked to Autophagic Cell Death

We recently revealed a novel mechanism by which p19ARF can induce cell death. We found that the p19ARF mRNA encodes an additional shorter isoform from the same open reading frame, named smARF. smARF is a short lived protein, which is rapidly degraded by the proteasome, but accumulates after inappropriate proliferative signals generated by oncogenes. Surprisingly, smARF translocates to the mitochondria, impairs the structure of the mitochondria and dissipates the mitochondrial membrane potential in a p53 and Bcl-2 family independent manner, ultimately inducing type II caspase-independent autophagic cell death.

Addendum to:

A Short Mitochondrial form of p19(ARF) Induces Autophagy and Caspase-Independent Cell Death

S. Reef, E. Zalckvar, O. Shifman, S. Bialik, H. Sabanay, M. Oren and A. Kimchi

Mol Cell 2006; 22:463-75     

A conserved domain in exon 2 coding for the human and murine ARF tumor suppressor protein is required for autophagy induction

The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1β of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF’s autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5′ end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF’s tumor suppressor function.     

Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy

Parkinson disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Multiple genes have been associated with PD, including Parkin and PINK1. Recent studies have established that the Parkin and PINK1 proteins function in a common mitochondrial quality control pathway, whereby disruption of the mitochondrial membrane potential leads to PINK1 stabilization at the mitochondrial outer surface. PINK1 accumulation leads to Parkin recruitment from the cytosol, which in turn promotes the degradation of the damaged mitochondria by autophagy (mitophagy). Most studies characterizing PINK1/Parkin mitophagy have relied on high concentrations of chemical uncouplers to trigger mitochondrial depolarization, a stimulus that has been difficult to adapt to neuronal systems and one unlikely to faithfully model the mitochondrial damage that occurs in PD. Here, we report that the short mitochondrial isoform of ARF (smARF), previously identified as an alternate translation product of the tumor suppressor p19ARF, depolarizes mitochondria and promotes mitophagy in a Parkin/PINK1-dependent manner, both in cell lines and in neurons. The work positions smARF upstream of PINK1 and Parkin and demonstrates that mitophagy can be triggered by intrinsic signaling cascades.     

Amino terminal hydrophobic import signals target the p14ARF tumor suppressor to the mitochondria

The p14ARF tumour suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. This allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.     

ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.     

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

ARF in the mitochondria: The last frontier?

The tumor suppressor ARF carries out different functions in different cellular compartments. In the nucleus, ARF interacts physically and functionally with Mdm2 to inhibit cell cycle progression through activation of p53. In the nucleolus, ARF interacts with B23/NPM to inhibit ribosomal biogenesis through control of rRNA processing. Recent studies have expanded ARF's territory into the mitochondria. New data have shown that ARF interacts with the mitochondrial protein p32/C1QBP and that the interaction is critical in order for ARF to localize to the mitochondria and induce apoptosis. Remarkably, the ARF-p32 interaction, and hence ARF's pro-apoptotic function, can be interrupted by human cancer-derived mutations in exon2 of the p14ARF-p16INK4a gene locus. Here, we discuss the implications of these studies and their potential relevance to human cancer.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell,recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression.Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results revealed that, compared with control cells, the WI-38 cells in which p19ARFgene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10-12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells.These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

p19ARF-induced p53-independent apoptosis largely occurs through BAX

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).