AREL1 E3 ubiquitin ligase inhibits TNF-induced necroptosis via the ubiquitination of MTX2.

Previously, we reported on a novel anti-apoptotic E3 ubiquitin ligase, apoptosis-resistant E3 ubiquitin protein ligase 1 (AREL1), that ubiquitinates inhibitors of apoptosis proteins antagonists. The present study demonstrated that AREL1 ubiquitinated Metaxin 2 (MTX2), which was involved in TNF-induced necroptosis. MTX2 has been identified as a protein that belongs to the Metaxin family. It interacts with another Metaxin protein, Metaxin 1 (MTX1), which is localized in the outer membrane of mitochondria, and is involved in TNF-induced necroptosis. This study found that AREL1 interacted with MTX2, but not MTX1, while the amino-terminal domain of MTX2 interacted with MTX1, AREL1 interacted with the carboxyl-terminal domain of MTX2. Furthermore, AREL1 expression led to a decrease in the protein expression of MTX2, but not MTX1. However, a mutant form of AREL1, AREL1C790A, which is deficient for E3 activity, did not cause MTX2 degradation. Moreover, the protein levels of MTX2 were increased by AREL1 knockdown. Therefore, these results implied that AREL1 ubiquitinates and promotes the degradation of MTX2. The expression of MTX2, together with MTX1, enhanced TNF-induced necroptosis. However, AREL1 inhibited necroptosis even in cells expressing Metaxin proteins. Therefore, these results suggested that the inhibition of AREL1-dependent ubiquitination of MTX2 could be beneficial to sensitize tumor cells to TNF-induced necroptosis.

Repression of the F-box protein Skp2 is essential for actin damage-induced tetraploid G1 arrest.

We previously reported that p53 plays a role as a key regulator in the tetraploid G1 checkpoint, which is activated by actin damage-induced cytokinesis blockade and then prevents uncoupled DNA replication and nuclear division without cytokinesis. In this study, we investigated a role of Skp2, which targets CDK2 inhibitor p27/Kip1, in actin damage-induced tetraploid G1 arrest. Expression of Skp2 was reduced, but p27/Kip1 was increased, after actin damage-induced cytokinesis blockade. The role of Skp2 repression in tetraploid G1 arrest was investigated by analyzing the effects of ectopic expression of Skp2. After actin damage, ectopic expression of Skp2 resulted in DNA synthesis and accumulation of multinucleated cells, and ultimately, induction of apoptosis. These results suggest that Skp2 repression is important for sustaining tetraploid G1 arrest after cytokinesis blockade and is required to prevent uncoupled DNA replication and nuclear division without cytokinesis. [BMB Reports 2017; 50(7): 379-383].

Inhibition of senescence and promotion of the proliferation of chondrocytes from articular cartilage by CsA and FK506 involves inhibition of p38MAPK.

Cyclosporine A (CsA) and tacrolimus (FK506) are the most important immunosuppressive compounds that block the activation of helper T-cells. In this study, we investigated the effects of CsA and FK506 on growth and senescence of articular chondrocytes. Chondrocytes from young rabbit cartilage entered senescence after 8.6 ± 0.8 population doublings (PDs), while chondrocytes treated with CsA and FK506 entered senescence after 12.3 ± 1.4 and 13.7 ± 0.6 PDs, respectively. Furthermore, chondrocytes from the cartilage of old rabbits were senescent after 2.6 ± 0.9 PDs, whereas those treated with CsA and FK506 were senescent after 8.2 ± 1.8 and 6.9 ± 1.6 PDs, respectively. These compounds also inhibited senescence induction of chondrocytes in a high-cell density pellet culture system. We previously reported that p38MAPK plays a critical role in the onset of senescence in chondrocyte. This study revealed that the phosphorylation of p38MAPK was inhibited by either CsA or FK506. The early onset of senescence in chondrocyte harboring MKK6E, which is a constitutively-active form of MKK6 and increases p38MAPK phosphorylation, was blocked by CsA. These results suggest that CsA and FK506 increase the proliferation and inhibit the senescence of articular chondrocytes through inactivation of p38MAPK.

Identification of a novel anti-apoptotic E3 ubiquitin ligase that ubiquitinates antagonists of inhibitor of apoptosis proteins SMAC, HtrA2, and ARTS.

Identification of new anti-apoptotic genes is important for understanding the molecular mechanisms underlying apoptosis and tumorigenesis. The present study identified a novel anti-apoptotic gene named AREL1, which encodes a HECT (homologous to E6-AP carboxyl terminus) family E3 ubiquitin ligase. AREL1 interacted with and ubiquitinated IAP antagonists such as SMAC, HtrA2, and ARTS. However, AREL1 was cytosolic and did not localize to nuclei or mitochondria. The interactions between AREL1 and the IAP antagonists were specific for apoptosis-stimulated cells, in which the IAP antagonists were released into the cytosol from mitochondria. Furthermore, the ubiquitination and degradation of SMAC, HtrA2, and ARTS were significantly enhanced in AREL1-expressing cells following apoptotic stimulation, indicating that AREL1 binds to and ubiquitinates cytosolic but not mitochondria-associated forms of IAP antagonists. Furthermore, the anti-apoptotic role of AREL1-mediated degradation of SMAC, HtrA2, and ARTS was shown by simultaneous knockdown of three IAP antagonists, which caused the inhibition of caspase-3 cleavage, XIAP degradation, and induction of apoptosis. Therefore, the present study suggests that AREL1-mediated ubiquitination and degradation of cytosolic forms of three IAP antagonists plays an important role in the regulation of apoptosis.

DNA damage induces the IL-6/STAT3 signaling pathway, which has anti-senescence and growth-promoting functions in human tumors.

IL-6 is a multifunctional cytokine that is important for immune responses, cell survival, apoptosis, and proliferation. However, little is known about the correlation between the IL-6 signaling pathway and DNA damage in human tumors. The present study demonstrates the role of the IL-6/STAT3 signaling pathway in human tumor cells exposed to DNA damage. Tumor cells exposed to DNA damage increase the expression and secretion of IL-6 and the phosphorylation of JAK1 and STAT3. The activation of the JAK1-STAT3 signaling pathway is inhibited by knockdown of gp130 or neutralization of soluble IL-6, implying that DNA damage induces the phosphorylation of JAK1 and STAT3 by autocrine IL-6. Interestingly, inhibition of the IL-6/STAT3 signaling pathway impairs the growth of tumor cells exposed to DNA damage and results in the induction of senescence. Therefore, the present study suggests that IL-6 inhibits senescence but promotes the survival and proliferation of tumor cells exposed to DNA damage through the activation of the JAK1-STAT3 signaling pathway.

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest.

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin- damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced- tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA- PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.

NF-Y binds to both G1- and G2-specific cyclin promoters; a possible role in linking CDK2/Cyclin A to CDK1/Cyclin B.

We previously reported that CDK2/Cyclin A can phosphorylate and activate the transcription factor NF-Y. In this study, we investigated a potential regulatory role for NF-Y in the transcription of Cyclin A and other cell cycle regulatory genes. Gel-shift assays demonstrate that NF-Y binds to CCAAT sequences in the Cyclin A promoter, as well as to those in the promoters of cell cycle G2 regulators such as CDC2, Cyclin B and CDC25C. Furthermore, expression of Cyclin A increases NF-Y's affinity for CCAAT sequences in the CDC2 promoter; however, Cyclin A's induction of CDC2 transcription is antagonized by p21, an inhibitor of CDK2/Cyclin A. These results suggest a model wherein NF-Y binds to and activates transcription from the Cyclin A promoter, increasing cellular levels of Cyclin A/CDK2 and potentiating NF-Y's capacity for transcriptional transactivation, and imply a positive feedback loop between NF-Y and Cyclin A/CDK2. Our findings are additionally indicative of a role for Cyclin A in activating Cyclin B/CDK1 through promoting NF-Y dependent transcription of Cyclin B and CDC2; NF-Y mediated crosstalk may therefore help to orchestrate cell-cycle progression.

p41-Arc, a regulatory subunit of Arp2/3 complex, can induce premature senescence in the absence of p53 and Rb.

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the tumor suppressor proteins, such as Rb or p53. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-ß-galactosidase (SA-ß-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. p41-Arc has been known to be a putative regulatory component of the mammalian Arp2/3 complex, which is required for the formation of branched networks of actin filaments at the cell cortex. In this study, we demonstrate that p41-Arc can induce senescent phenotypes when it is overexpressed in human tumor cell line, SaOs-2, which is deficient in p53 and Rb tumor suppressor genes, implying that p41 can induce senescence in a p53-independent way. p41-Arc overexpression causes a change in actin filaments, accumulating actin filaments in nuclei. Therefore, these results imply that a change in actin filament can trigger an intrinsic senescence program in the absence of p53 and Rb tumor suppressor genes.

p53 prevents immature escaping from cell cycle G2 checkpoint arrest through inhibiting cdk2-dependent NF-Y phosphorylation.

PURPOSE: Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and this function is required for the maintenance of genomic integrity. In this study, we addressed a role of p53 in escaping from cell cycle G2 arrest following DNA damage. MATERIALS AND METHODS: Cell cycle checkpoint arrest in the human colon cancer cell line HCT116 and its derivatives carry p53 or p21 deletions, were examined by FACS analysis, immunoprecipitation, Western blot and IP-kinase assay. RESULTS: While the cells with functional p53 were arrested at both the G1 and G2 checkpoints, the p53-deficient cells failed to arrest at G1, but they were arrested at G2. However, the p53-deficient cells failed to sustain G2 checkpoint arrest and they entered mitosis earlier than did the p53-positive cells and so this resulted in extensive cell death. Cdc2 kinase becomes reactivated in p53-deficient cells in association with entry into mitosis, but not in the p53-positive cells. Upon DNA damage, the p21-deficient cells, like the p53-negative cells, not only failed to repress cdk2-dependent NF-Y phosphorylation, but they also failed to repress the expression of such cell cycle G2-regulatory genes as cdc2, cyclin B, RNR-R2 and cdc25C, which have all been previously reported as targets of NF-Y transcription factor. CONCLUSIONS: p53 is essential to prevent immature escaping from cell cycle G2 checkpoint arrest through p21-mediated cdk2 inactivation, and this leads to inhibition of cdk2-dependent NF-Y phosphorylation and NF-Y dependent transcription of the cell cycle G2-regulatory genes, including cdc2 and cyclin B.

Transcription repression of a CCAAT-binding transcription factor CBF/HSP70 by p53.

NF-Y transcription factor binds to CCAAT boxes on promoters of cell cycle regulatory genes such as cdc2, cyclin B, cdc25C, and cyclin A. We previously reported that the DNA binding activity of NF-Y is regulated by p53-p21-cdk2 pathway. CBF/HSP70 was originally identified as a transcription factor binding to the CCAAT box on the hsp70 promoter and mediates transcription repression of hsp70 pro- moter by p53. Recently it was demonstrated that CBF/HSP70 interacts and cooperates with NF-Y. In this study, we found that p53 represses the trans-cription of CBF/HSP70. Since transactivation ability of NF-Y is regulated in a cell cycle-dependent manner, we examined the transcription of CBF/HSP70 during the cell cycle. After synchronization of a human bladder carcinoma cell lacking functional p53 at early S phase, we infect the cells with adenovirus encoding p53. Cells infected with control virus progressed to S and G2 after release from the arrest. In contrast, cells expressing p53 enter S and G2 phases, but arrest at G2/M. The expression of CBF/HSP70 was induced at S/G2 phase in cells infected with a control virus, but kept to be repressed in cells expressing p53. Thus, these results suggest that p53 suppresses the expression of cell cycle regulatory genes though inhibiting both CCAAT binding factors, CBF/HSP70 and NF-Y.

Oocyte-based screening of cytokinesis inhibitors and identification of pectenotoxin-2 that induces Bim/Bax-mediated apoptosis in p53-deficient tumors.

In this study, we demonstrate that a loss of p53 sensitizes tumor cells to actin damage. Using a novel oocyte-based screening system, we identified natural compounds that inhibit cytokinesis. Among these, pectenotoxin-2 (PTX-2), which was first identified as a cytotoxic entity in marine sponges, which depolymerizes actin filaments, was found to be highly effective and more potent to activate an intrinsic pathway of apoptosis in p53-deficient tumor cells compared to those with functional p53 both in vitro and in vivo. Other agents that depolymerize or knot actin filaments were also found to be toxic to p53-deficient tumors. In p53-deficient cells, PTX-2 triggers apoptosis through mitochondrial dysfunction, and this is followed by the release of proapoptotic factors and caspase activation. Furthermore, we observed Bax activation and Bim induction only in p53-deficient cells after PTX-2 treatment. RNA interference of either Bim or Bax resulted in the inhibition of caspases and apoptosis induced by PTX-2. However, the small interfering RNAs (SiRNA) of Bim blocked a conformational change of Bax, but Bax SiRNA did not affect Bim expression. Therefore, these results suggest that Bim triggers apoptosis by activating Bax in p53-deficient tumors upon actin damage, and that actin inhibitors may be potent chemotherapeutic agents against p53-deficient tumors.

Inactivation of p38 kinase delays the onset of senescence in rabbit articular chondrocytes.

Replicative senescence limits cellular proliferation in vivo and in vitro. Recently, other groups and we reported that p38 kinase plays a key role on the onset of senescence. In this study, we demonstrated that replicative senescence can be delayed in rabbit chondrocytes in vitro by that p38 kinase inactivation. We found that the activity of p38 kinase is elevated in senescent chondrocytes as compared to pre-senescent counterparts. To examine the role of p38 kinase on the onset of senescence, we inactivated the kinase pharmacologically or genetically using either a chemical inhibitor, SB203580, or dominant negative mutant forms of MKK6 and p38 (MKK6A and p38dn, respectively). We show that the inactivation of p38 kinase leads to the stimulation of proliferation, the extension of life span, and a delay in the onset of senescence, thus implying that p38 kinase limits the life span of rabbit articular chondrocytes in vitro.

Cdk2-dependent phosphorylation of the NF-Y transcription factor is essential for the expression of the cell cycle-regulatory genes and cell cycle G1/S and G2/M transitions.

We previously reported that cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y transcription factor and this phosphorylation is essential for DNA binding of NF-Y. In this study, we examined the effects of a phosphorylation-deficient mutant form of YA, YA-aa, in which the two serine residues are replaced with alanine, on the cell cycle and expression of the NF-Y target genes. Transient transfection assays show that YA-aa inhibits transcription from the NF-Y target promoters, such as cdc2, cyclin A, and cdc25C. Moreover, this inhibitory function of YA-aa can be suppressed by the expression of wild-type YA, implying that YA-aa inhibits transcription of those NF-Y target genes by inactivating wild-type YA. Since NF-Y target genes include the cell cycle-regulatory genes that ensure orderly progression of the cell cycle, we examined the effects of YA-aa in cell cycle progression. We constructed a recombinant adenovirus encoding YA-aa and found that YA-aa expression leads to repression of cell cycle-regulatory genes, such as cyclin A, RNR R2, DNA polymerase alpha, cdc2, cyclin B, and cdc25C. Consistently, YA-aa expression results in the inactivation of both cdc2 and cdk2. Furthermore, cell cycle analysis reveals that YA-aa induces cell cycle arrest at both G1 and G2/M. These results suggest that cdk2-dependent phosphorylation of NF-Y is essential for the expression of the cell cycle-regulatory genes and therefore for cell cycle progression at both G1/S and G2/M.

Bcl-xL and E1B-19K proteins inhibit p53-induced irreversible growth arrest and senescence by preventing reactive oxygen species-dependent p38 activation.

In this study, we describe novel functions of the anti-apoptotic Bcl-2 family proteins. Bcl-x(L) and E1B-19K were found to inhibit p53-induced irreversible growth arrest and senescence, but not to inhibit transient growth arrest, implying that Bcl-x(L) and E1B-19K are specifically involved in senescence without participating in growth arrest. We provide several lines of evidences showing that the functions of Bcl-x(L) and E1B-19K to prevent generation of reactive oxygen species (ROS) are important to inhibit senescence induction. First, we found that that ROS are increased during p53-induced senescence. Moreover, Bcl-x(L) and E1B-19K inhibit this p53-induced ROS generation. Second, antioxidants prevent the induction of senescence and ROS by p53, but not the persistence of the senescence phenotype. Third, the anti-senescence functions of Bcl-x(L) and E1B-19K were suppressed by adding exogenous ROS. These results suggest that Bcl-x(L) and E1B-19K inhibit senescence induction by preventing ROS generation. Furthermore, p38 kinase was found to be activated during p53-induced senescence, but not in cells expressing Bcl-x(L) or E1B-19K, or in cells treated with anti-oxidants. Consistently, a chemical inhibitor of p38 kinase, SB203580, was found to inhibit p53-induced senescence, but only when treated before the cellular commitment to senescence, implying that p38 kinase is necessary for senescence induction. Therefore, Bcl-x(L) and E1B-19K inhibit p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 kinase. These results also suggest that the oncogenic potential of Bcl-2 is due to its ability to inhibit senescence as well as apoptosis.

Roscovitine sensitizes glioma cells to TRAIL-mediated apoptosis by downregulation of survivin and XIAP.

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in many glioma cell lines. However, treatment with TRAIL in combination with subtoxic doses of roscovitine, a specific inhibitor of Cdc2 and Cdk2, induced rapid apoptosis in TRAIL-resistant glioma cells. Roscovitine could sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells, but not human astrocytes, to TRAIL-induced apoptosis, offering an attractive strategy for safely treating resistant gliomas. Treatment with roscovitine significantly inhibited Cdc2 activity, and expression of a dominant-negative Cdc2 mutant sensitized glioma cells to TRAIL-induced apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in U87MG and T98 glioma cells, treatment with roscovitine recovered TRAIL-induced activation of caspases very efficiently in these cells. We found that treatment with roscovitine or expression of a dominant-negative Cdc2 mutant downregulated the protein levels of survivin and XIAP, two major caspase inhibitors. Overexpression of survivin or XIAP attenuated the apoptosis induced by roscovitine and TRAIL. Taken together, these results suggest that downregulation of survivin and XIAP by subtoxic doses of roscovitine contributes to the amplification of caspase cascades, thereby overcoming glioma cell resistance to TRAIL-mediated apoptosis.

Cdk2-dependent phosphorylation of the NF-Y transcription factor and its involvement in the p53-p21 signaling pathway.

Recent studies have suggested that the NF-Y transcription factor is involved in transcription repression of the cell cycle regulatory genes in a response to p53 induction or DNA damage. Here we demonstrate the cdk2-dependent phosphorylation of NF-Y and its involvement in transcription repression by the p53-p21 signaling pathway. Cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y. Cyclin A-cdk2 appears to associate with NF-Y both in vitro and in vivo. Furthermore, YA protein is phosphorylated in parallel with a cell cycle-dependent activation of cdk2 kinase and cyclin A expression. YA phosphorylation is unnecessary for heterotrimer formation with the YB-YC dimer. However, NF-Y containing a phosphorylation-deficient mutant form of YA, YA-aa, has its DNA binding activity impaired. Consistently, YA-aa inhibits transcription activation of a NF-Y target promoter, cdc2, by cdk2. These results facilitate the elucidation of the regulatory mechanisms of cell cycle progression involving the p21-cdk2-NF-Y signaling pathway.

p38 kinase regulates nitric oxide-induced apoptosis of articular chondrocytes by accumulating p53 via NFkappa B-dependent transcription and stabilization by serine 15 phosphorylation.

Nitric oxide (NO) during primary culture of articular chondrocytes causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation, and differentiation status. In this study, we characterized the molecular mechanism by which p38 kinase induces apoptosis through activation of p53. We report here that NO-induced activation of p38 kinase leads to activation of NFkappaB, which in turn induces transcription of the p53 gene. Activated p38 kinase also physically associates and phosphorylates the serine 15 residue of p53, which results in accumulation of p53 protein during NO-induced apoptosis. Ectopic expression of wild-type p53 enhanced NO-induced apoptosis, whereas expression of a dominant negative p53 blocked it, indicating that p53 plays an essential role in NO-induced apoptosis of chondrocytes. The increased accumulation of p53 caused expression of Bax, a pro-apoptotic member of the Bcl-2 family that is known to cause apoptosis via release of cytochrome c and caspase activation. These results suggest that NO-activated p38 kinase activates p53 function in two different ways, transcriptional activation by NFkappaB and direct phosphorylation of p53 protein, leading to apoptosis of articular chondrocytes.