Cyclooxygenase-2 (COX-2) has been implicated in a variety of human malignancies and, accordingly, COX-2 selective inhibitors are being investigated as important chemopreventive and therapeutic agents. How COX-2 overexpression results in tumorigenesis and how COX-2 selective agents mediate their chemopreventive effects are issues that remain poorly understood. Here we report that the tumor suppressor p53 upregulates COX-2 expression and that COX-2 can in turn inhibit p53-dependent transcription. Additionally, a COX-2-selective inhibitor potentiates p53-induced apoptosis, which also supports the notion that COX-2 activity appears to interfere with p53 function. Expression of exogenous COX-2 in p53 wildtype cells does not affect the cytoplasmic or nuclear levels of p53, suggesting that COX-2 may not affect p53 turnover or subcellular localization. We further demonstrate that endogenous COX-2 interacts with p53 and that COX-2 and p53 interactions are a physiologically relevant event. Thus, p53 upregulates COX-2 and COX-2 in turn appears to negatively affect p53 activity via mechanisms that could involve physical interactions between COX-2 and p53. Based on our results, we propose that p53-dependent upregulation and activation of COX-2 appear to be yet another novel mechanism by which p53 could abate its own growth-inhibitory and apoptotic effects.

SummaryWe had previously reported that RBEL1A, a novel Ras-like GTPase, was overexpressed in multiple human malignancies and that its depletion suppressed cell growth. However, the underlying molecular mechanism remained to be elucidated. Here we report that depletion of endogenous RBEL1A results in p53 accumulation due to increased p53 half-life whereas increased expression of RBEL1A reduces p53 levels under unstressed and genotoxic stress conditions. RBEL1A directly interacts with p53 and MDM2, and strongly enhances MDM2-dependent p53 ubiquitylation and degradation. We also found that RBEL1A modulation of p53 ubiquitylation by MDM2 does not depend on its GTPase activity. We have also defined the p53 oligomeric domain and RBEL1A GTPase domain to be the crucial regions for p53-RBEL1A interactions. Importantly, we have found that RBEL1A strongly interferes with p53 transactivation function; thus our results indicate that RBEL1A appears to function as a novel p53 negative regulator that facilitates MDM2-dependent p53 ubiquitylation and degradation.

For years, the growth inhibitory effects of the tumor suppressor p53 were thought to be antagonized predominantly by the ubiquitin ligase, MDM2. It has long been established that MDM2 physically associates with p53 and targets this tumor suppressor for proteasomal degradation. In light of recent findings, it now appears that MDM2 may not be the only ubiquitin ligase that negatively controls p53 function. Two recently discovered proteins, Pirh2 and COP1, are also believed to facilitate p53 degradation via the ubiquitin-proteasome pathway. Both proteins are upregulated by p53 as well as genotoxic stress and each has been found to directly promote p53 ubiquitination and degradation. Future studies in this field will now face the challenge of elucidating the physiological significance of three molecules all apparently able to independently facilitate p53 degradation and abrogate its function.

The function of p53 as a tumor suppressor remains undisputed. p53 has a central role in cellular stress responses as well as affecting cancer development and progression. The word "central", however, is becoming increasingly more of an understatement as the list of p53-regulated pathways and processes is ever expanding. Although much focus continues to center on p53-mediated signaling cascades that control cell growth arrest and/or apoptosis, recent work has begun to define a role for p53 in the regulation of metabolic pathways typically thought of as essential for maintaining life. With the first potential link between p53 and glycolysis reported nearly ten years ago, the topic has gained a renewed interest. Recent studies now demonstrate the ability of p53 to regulate the expression of several novel genes including PGM (phosphoglycerate mutase), TIGAR (TP53-induced glycolysis and apoptosis regulator) and, SCO2 (synthesis of cytochrome c oxidase 2), each intimately linked to the processes of glycolysis and oxidative phosphorylation. With this discovery, yet another novel means by which p53 carries out its tumor suppressor function is brought into light.

Thapsigargin (TG), by inducing perturbations in cellular Ca 2+ homeostasis, can induce apoptosis, but the molecular mechanisms remain to be fully elucidated. We have recently reported that TG-induced apoptosis appears to involve the DR5-dependent apoptotic pathway that cross talks with the mitochondrial pathway via TG-induced Bid cleavage. In this study, we have utilized Bax-proficient and -deficient HCT116 human colon cancer cells to investigate the effect of Bax deficiency on TG-induced apoptosis and TG regulation of the DR5 and mitochondrial pathways. Our results indicate that Bax-deficient cells are less sensitive to undergo apoptosis following TG treatment. Our results further demonstrate that TG-induced apoptosis is coupled with DR5 upregulation and caspases 8 and 3 activation, as well as Bid cleavage in both Bax-proficient and -deficient cells, although caspase 3 activation was reduced in Bax-deficient cells. TG also promoted the release of cytochrome c into cytosol and caspase 9 activation in Bax-proficient cells but not in Bax-deficient cells. These findings suggest that although Bax is not absolutely required for death receptor (DR)-dependent signals, it appears to be a key molecule in TGregulated mitochondrial events. Bax-deficient cells were relatively more resistant to Apo2L/TRAIL than the Baxproficient counterparts. However, the combination of Apo2L/TRAIL and TG was more effective in mediating apoptosis in both Bax-proficient and -deficient cells and that was coupled with activation of caspases 8 and 3. Although both agents in combination also induced cytochrome c release into cytosol and caspase 9 activation in Bax-proficient cells, these events were abrogated in Bax-deficient cells. Our results thus suggest that the combination of Apo2L/TRAIL and TG appears to bypass the Bax deficiency-induced defects in the mitochondrial (intrinsic) pathway by engaging the DR5-dependent apoptotic signals (extrinsic pathway).

TRB3 has recently been identified as a potential pro-apoptotic protein that may modulate the Akt/PKB-dependent signaling pathway. Here we report that TRB3 expression is strongly upregulated by endoplasmic reticulum (ER) stress-inducing agents that (1) promote ER Ca 2+ pool depletion or (2) disrupt protein trafficking. Genotoxic stress (DNA damage)-inducing agents, by contrast, downregulate TRB3 expression and appear to do so through both p53-dependent and -independent mechanisms. To the best of our knowledge, TRB3 is the first gene that is upregulated by ER stress and downregulated following genotoxic stress. Collectively, these findings highlight the importance of stress-specific signaling cascades as well as point out the seemingly divergent roles that TRB3 may play in the cellular stress response.

Pirh2 is a newly identified E3 ubiquitin ligase known to inhibit tumor suppressor p53 function via ubiquitination and proteasomal degradation. We have identified two novel Pirh2 splice variants that encode different Pirh2 isoforms and named these Pirh2B and Pirh2C. Accordingly, the full-length protein is now classified as isoform Pirh2A. The central region of Pirh2 harbors a RING finger domain that is critical for its ubiquitin ligase function. The Pirh2B isoform lacks amino acids 171-179, whereas Pirh2C is missing C-terminal amino acids 180 -261, which for each isoform results in a RING domain deletion and the abrogation of ubiquitin ligase activity. Our findings further indicate that the Pirh2B isoform but not the Pirh2C isoform is capable of binding to Pirh2A, suggesting that the C-terminal region absent in Pirh2C is critical for Pirh2-Pirh2 interactions. Similar to Pirh2A, both Pirh2B and Pirh2C interact with p53; however, interactions between p53 and Pirh2B appear stronger than those between p53 and Pirh2C. Interestingly, although both Pirh2B and Pirh2C are not able to promote in vitro p53 ubiquitination, both are capable of negatively regulating p53 protein stability and promoting the intracellular ubiquitination of p53. Furthermore, like Pirh2A, both isoforms are able to inhibit p53 transcriptional activity. We have also for the first time demonstrated that Pirh2A as well as the novel isoforms also interact directly with MDM2 within a region encompassing MDM2 acidic and zinc finger domains. It is therefore possible that Pirh2A and the novel Pirh2 isoforms identified in this study may also modulate p53 function by engaging MDM2.The tumor suppressor p53 has been implicated in a growing number of cellular processes, the most recognized of these being the initiation of signaling cascades leading to growth arrest and apoptosis (1). A large body of evidence supports the notion that p53 is capable of regulating these pathways via distinct mechanisms. It is becoming clear that p53 probably exerts its antiproliferative effects through both its well established transcription factor function as well as a transcription-independent role at the mitochondrion (1-3).Given its potent capacity to control cell fate, p53 in normal cells is held in check at multiple, often interrelated levels, including regulation of p53 protein stability, subcellular localization, and transcriptional activity. It is now well understood that p53 ubiquitination and subsequent proteasomal degradation is one of the principal mechanisms controlling these processes (4 -6). The RING domain containing ubiquitin ligase MDM2 was the first non-viral protein found to be responsible for the ubiquitin-dependent targeting of p53 for proteolysis (7,8). The importance of MDM2 in abrogating p53 function was illustrated in an embryonic lethal MDM2(Ϫ/Ϫ) mouse model in which lethality can be attributed to uncontrolled p53 activity and is rescued by p53 deletion (9, 10).Adding complexity to the once clear role of MDM2 in p53 ubiquitination is the recent discovery...

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