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 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.     

Autophagy and caspase-independent cell death: p19ARF enters the game

ARF, often localized in the nucleolus, controls the p53 pathway and ribosomal biogenesis. In a recent issue of Molecular Cell, Kimchi and colleagues describe a short mitochondrial form of ARF (smARF), produced by internal initiation of translation, that dissipates mitochondrial membrane potential independently of p53 and Bcl-2 family members and triggers caspase-independent cell death. The prodeath function of smARF is dependent on the induction of autophagy.     

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 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.     

Targeting a nucleolar SUMO protease for degradation: A mechanism by which ARF induces SUMO conjugation

The p19^ARF p14^ARF in humans protein acts as a tumour suppressor through p53 dependent and independent mechanisms. A well-established role for ARF is to regulate the post-translational modification of substrate proteins with ubiquitin and ubiquitin-like molecules such as SUMO. It is now evident that induction of ARF causes a dramatic accumulation of SUMO conjugates and this has been related to the p53 independent functions of ARF. The majority of these conjugates appear to accumulate in the nucleolus where most of ARF is also found. An obvious function for ARF, which would result in increase of SUMOylation, is to act as an atypical SUMO E3-ligase. Indeed, initial studies suggested that ARF could directly interact with the SUMO E2-conjugating enzyme Ubc9 and therefore bringing the SUMO conjugation machinery in close proximity to its interacting substrates.¹ However, the highly basic charged nature of ARF makes biochemical analysis difficult and there is no clear demonstration that ARF can fulfill the criteria for an E3-ligase in vitro. Therefore, the mechanism(s) behind this phenomenon are not currently understood. As with ubiquitination, SUMO conjugation is a dynamic process controlled by E3-ligases and proteases that specifically remove SUMO from substrates. In this issue of Cell Cycle studies from the Sherr lab suggest that ARF can increase SUMO conjugation by controlling the stability of the nucleolar SUMO protease SENP3.² Recent studies have shown that SENP3 can deconjugate SUMO-2 and SUMO-3 from substrates including nucleophosmin (NPM). NPM is a nucleolar protein, which among other processes is involved in the processing of rRNA during ribosome biosynthesis. NPM interacts with ARF and this results in increased SUMOylation of NPM. SENP3 can counteract the effect of ARF by deconjugating SUMO from NPM and this appears to be critical for NPM function in rRNA processing.³ The new study now suggests that there is an opposing functional relationship between ARF and SENP3. ARF promotes phosphorylation dependent ubiquitination of SENP3, which results in SENP3 degradation and increase in NPM SUMO conjugation. In this process, NPM seems to act as a "platform" for ARF and SENP3, bringing in close proximity its two regulators. The new study suggests an interesting and complex mechanism by which ARF can control SUMOylation. It is now evident that post-translational modifications cooperate to control protein function. The new data suggest that ARF engages phosphorylation to promote ubiquitination and proteasomal degradation of a SUMO protease. This model would propose the existence of a kinase/phosphatase and an E3-ubiquitin ligase/de-ubiquitinating enzyme set which would cooperate their actions to control the stability of SENP3. Given that ARF has multiple binding partners, it would not be surprising that ARF would interact with components of the above enzymatic steps and control their activity. It would therefore be interesting to identify the role of ARF in this process. It is not clear whether degradation of SENP3 per se is sufficient to induce NPM SUMO conjugation and if this is the case which SUMO E3-ligases drive the forward reaction. Even if in this study an interaction of ARF with Ubc9 could not be demonstrated it may be the case that ARF mediates both the degradation of SENP3 and recruitment of the SUMO conjugation machinery, which will result in fast and efficient accumulation of SUMOylated NPM. Another possibility is the effect of ARF on NPM stability itself. Previous studies have shown that ARF can induce ubiquitin-mediated degradation of NPM.⁴ As NPM is important to prevent destabilisation of SENP3, ARF-mediated degradation of NPM could be part of SENP3 degradation. Another point that arises from this is the site of degradation for SENP3. Nucleoli have been suggested to be deficient for proteasomal activity, suggesting that ARF through the phosphorylation/ubiquitination events may alter the localisation/mobility of SENP3 making it susceptible to nucleoplasmic/cytoplasmic proteasomal degradation. The effect of ARF in controlling protein ubiquitination is now well established. Interaction of ARF with E3-ligases such as Mdm2 and ARF-BP1/Mule inhibits their function resulting in inhibition of p53 proteasomal degradation.^5,6 Therefore, the ability of ARF to induce ubiquitination and proteasomal degradation of SENP3 and NPM shows a complex and diverse role for ARF to control protein stability. Further experiments will show whether the ability of ARF to promote degradation of SENP3 or possibly other SUMO proteases is a general mechanism through which ARF induces SUMO conjugation of its binding partners or that the NPM/SENP3 system is a unique example.

References

1. Rizos H, Woodruff S, Kefford RF. p14ARF interacts with the SUMO-conjugating enzyme Ubc9 and promotes the sumoylation of its binding partners. Cell Cycle 2005; 4:597-603. 2. Kuo ML, den Besten W, Thomas MC, Sherr CJ. Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3. Cell Cycle 2008; 7:In this issue 3. Haindl M, Harasim T, Eick D, Muller S. The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing. EMBO Rep 2008; 9:273-9 4. Itahana K, Bhat KP, Jin A, Itahana Y, Hawke D, Kobayashi R, Zhang Y. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol Cell 2003; 12:1151-64. 5. Xirodimas D, Saville MK, Edling C, Lane DP, LaÃ?Â?Ã?Â?Ã?Â?Ã?­n S. Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo. Oncogene 2001; 20:4972-83. 6. Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 2005; 121:1071-83.     

Signal-Dependent Control of Autophagy and Cell Death in Colorectal Cancer Cell: The Role of the p38 Pathway

Autophagy is a vacuolar process leading to the degradation of long-lived proteins and cytoplasmic organelles in eukaryotes. This process has an important role in normal and cancer cells during adaptation to changing environmental conditions, cellular and tissue remodeling, and cell death.

To date, several signaling cascades have been described to regulate autophagy in a cell type-specific and signal-dependent manner.

We found that pharmacological blockade of the p38 pathway in colorectal cancer cells, either by the inhibitor SB202190 or by genetic ablation of p38α kinase, causes cell cycle arrest and autophagic cell death. In these cells, a complex network of intracellular kinase cascades controls autophagy and survival since the effect of p38α blockade is differentially affected by the pharmacological inhibition of MEK1, PI3K class I and III, and mTOR or by the differentiation status.

Collectively, our results suggest an opportunity for exploiting the pharmacological manipulation of the p38α pathway in the treatment of colorectal cancer. Given the number of drugs, currently available or under development, that target the p38 pathway, it stands to reason that elucidating the molecular mechanisms that link p38 and autophagy might have an impact on the clinical translation of these drugs.

Addendum to:

A Novel Cell Type-Specific Role of p38α in the Control of Autophagy and Cell Death in Colorectal Cancer Cells

F. Comes, A. Matrone, P. Lastella, B. Nico, F.C. Susca, R. Bagnulo, G. Ingravallo, S. Modica, G. Lo Sasso, A. Moschetta, G. Guanti and C. Simone

Cell Death Differ 2007; 14: 693-702     

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.     

Mitoptosis: Different Pathways for Mitochondrial Execution

Mitoptosis was described as a sort of mitochondrial death program. It could be associated with both necrosis and apoptosis, although degenerating mitochondria are also found in autophagic vacuoles. It was demonstrated that several molecules might contribute to the remodeling and rearrangement of mitochondrial membranes, leading to mitochondria rupture and disruption. Here, we hypothesize that, at least in T cells, two main pathways of mitoptosis can occur: an inner membrane mitoptosis (IMM), in which only the internal matrix and cristae are lost while the external mitochondrial envelope remains unaltered, and an outer membrane mitoptosis (OMM) where only swollen internal cristae are detected as remnants. We suggest that the study of these processes could provide useful insights not only to the field of cell death but also to the study of the pathogenic mechanisms of mitochondria-associated human diseases.

Addendum to:

Death Receptor Ligation Triggers Membrane Scrambling Between Golgi and Mitochondria

S. Ouasti, P. Matarrese, R. Paddon, R. Khosravi-Far, M. Sorice, A. Tinari, W. Malorni, M. Degli Esposti

Cell Death Differ 2006; Epub ahead of print     

Roles of ARF tumour suppressor protein in lung cancer: time to hit the nail on the head!

Owing to its poor prognosis, the World Health Organization (WHO) lists lung cancer on top of the list when it comes to growing mortality rates and incidence. Usually, there are two types of lung cancer, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), which also includes adenocarcinoma, squamous cell carcinoma and large cell carcinomas. ARF, also known in humans as p14ARF and in the mouse as p19ARF, is a nucleolar protein and a member of INK4, a family of cyclin-independent kinase inhibitors (CKI). These genes are clustered on chromosome number 9p21 within the locus of CDKN2A. NSCLC has reported the role of p14ARF as a potential target. p14ARF has a basic mechanism to inhibit mouse double minute 2 protein that exhibits inhibitory action on p53, a phosphoprotein tumour suppressor, thus playing a role in various tumour-related activities such as growth inhibition, DNA damage, autophagy, apoptosis, cell cycle arrest and others. Extensive cancer research is ongoing and updated reports regarding the role of ARF in lung cancer are available. This article summarizes the available lung cancer ARF data, its molecular mechanisms and its associated signalling pathways. Attempts have been made to show how p14ARF functions in different types of lung cancer providing a thought to look upon ARF as a new target for treating the debilitating condition of lung cancer.

     

Induction of Autophagy by Concanavalin A and its Application in Anti-Tumor Therapy

Concanavalin A (Con A), a lectin from Jack bean seeds that, once bound to the mannose moiety on the cell membrane glycoprotein, is internalized preferentially to the mitochondria. A BNIP3-mediated mitochondria autophagy is then induced, and causes the tumor cells to undergo autophagic cell death. Con A is also a T cell mitogen that can induce autoimmune hepatitis in mice. Because of the dual properties (autophagic cytotoxicity and immunomodulation) via the specific mannose binding, Con A can exert a potent anti-hepatoma therapeutic effect by inhibiting tumor nodule formation in the liver and prolonging the survival of the tumor-bearing mice. The anti-tumor effect is primarily mediated by activated CD8⁺ T cells, and will also establish a tumor antigen-specific immune memory during the hepatic inflammation. This finding provides a novel mechanism in which Con A can be used as an anti-hepatoma agent, and also gives support for the search for natural lectins as anti-cancer compounds.

Addendum to:

Concanavalin A Induces Autophagy in Hepatoma Cells and has a Therapeutic Effect in a Murine In Situ Hepatoma Model

C.P. Chang, M.C. Yang, H.S. Liu, Y.S. Lin, H.Y. Lei HY.Hepatology 2007; 45:286-296.     

Apoptosis and Autophagy Function Cooperatively for the Efficacious Execution of Programmed Nurse Cell Death During Drosophila virilis Oogenesis

Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle and late oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.

Addendum to:

Mechanisms of Programmed Cell Death During Oogenesis in Drosophila virilis

A.D. Velentzas, I.P. Nezis, D.J. Stravopodis, I.S. Papassideri and L.H. Margaritis

Cell Tissue Res 2006; doi: 10.1007/s00441-006-0298-x     

A Two-Carbon Switch to Sterol-Induced Autophagic Death

Although both cholesterol and plant sterols are abundant in our diets, our intestinal epithelial cells selectively and efficiently rid the body of plant sterols. However, a rare mutation in plant sterol excretion in humans results in the accumulation of plant sterols, particularly sitosterol, in the plasma and tissues. Sitosterol differs from cholesterol only in an extra ethyl group on the sterol side chain. Significantly, sitosterolemia is associated with accelerated atherothrombotic vascular disease, notably myocardial infarction. An important process that promotes atherothrombosis is advanced lesional macrophage death, leading to plaque necrosis. One of the causes of atherosclerotic macrophage death is sterol-induced cytotoxicity. We therefore compared the effects of excess intracellular sitosterol vs. cholesterol on macrophage death. Whereas excess cholesterol kills macrophages by caspase-dependent apoptosis, sitosterol kills macrophages by a caspase-independent pathway involving necroptosis and autophagy. The finding that an ethyl group on the sterol side chain fundamentally alters the way cells respond to excess sterols adds new insight into the mechanisms of sterol-induced cell death and may provide at least one explanation for the excess atherosclerotic heart disease in patients with sitosterolemia.

Addendum to:

Sitosterol-Containing Lipoproteins Trigger Free Sterol-Induced Caspase-Independent Death in ACAT-Competent Macrophages: Implications for Sterol Structure-Dependent Mechanisms of Cell Death and for Atherosclerotic Vascular Disease in Sitosterolemia

L. Bao, Y. Li, S.X. Deng, D. Landry and I. Tabas

J Biol Chem 2006; In press