p53 and MDM2 are involved in the regulation of osteocalcin gene expression

Osteocalcin (OC) is a major noncollagenous bone matrix protein and an osteoblast marker whose expression is limited to mature osteoblasts during the late differentiation stage. In previous studies we have shown osteosarcomas to lose p53 function with a corresponding loss of osteocalcin gene expression. Introduction of wild type p53 resulted in re expression of the osteocalcin gene. Using gel shift and chromatin immunoprecipitation assays, we have identified a putative p53 binding site within the rat OC promoter region and observed an increase in OC promoter activity when p53 accumulates using a CAT assay. The p53 inducible gene Mdm2 is a well-known downstream regulator of p53 levels. Our results showed a synergistic increase in the OC promoter activity when both p53 and MDM2 were transiently overexpressed. We further demonstrate that p53 is not degraded during overexpression of MDM2 protein. Increased OC expression was observed with concomitantly increased p53, VDR, and MDM2 levels in ROS17/2.8 cells during treatment with differentiation promoting (DP) media, but was significantly decreased when co-treated with DP media and the small molecule inhibitor of MDM2-p53 interaction, Nutlin-3. We have also observed a dramatic increase of the OC promoter activity in the presence of p53 and Mdm2 with inclusion of Cbfa-1 and p300 factors. Our results suggest that under some physiological conditions the oncoprotein MDM2 may cooperate with p53 to regulate the osteocalcin gene during osteoblastic differentiation.     

c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis

Activation of Bax following diverse cytotoxic stress has been shown to be an essential gateway to mitochondrial dysfunction and activation of the intrinsic apoptotic pathway characterized by cytochrome c release with caspase-9/-3 activation. Interestingly, c-Myc has been reported to promote apoptosis by destabilizing mitochondrial integrity in a Bax-dependent manner. Stress-induced activation of caspase-2 may also induce permeabilization of mitochondria with activation of the intrinsic death pathway. To test whether c-Myc and caspase-2 cooperate to activate Bax and thereby mediate intrinsic apoptosis, small interfering RNA was used to efficiently knock down the expression of c-Myc, caspase-2, and Apaf-1, an activating component in the apoptosome, in two human cancer cell lines, lung adenocarcinoma A-549 and osteosarcoma U2-OS cells. Under conditions when the expression of endogenous c-Myc, caspase-2, or Apaf-1 is reduced 80-90%, cisplatin (or etoposide)-induced apoptosis is significantly decreased. Biochemical studies reveal that the expression of c-Myc and caspase-2 is crucial for cytochrome c release from mitochondria during cytotoxic stress and that Apaf-1 is only required following cytochrome c release to activate caspases-9/-3. Although knockdown of c-Myc or caspase-2 does not affect Bax expression, caspase-2 is important for cytosolic Bax to integrate into the outer mitochondrial membrane, and c-Myc is critical for oligomerization of Bax once integrated into the membrane.     

Translocation and oligomerization of Bax is regulated independently by activation of p38 MAPK and caspase-2 during MN9D dopaminergic neurodegeneration

Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.     

p53, Apaf-1, caspase-3, and -9 are dispensable for Cdk5 activation during cell death

Cyclin-dependent kinase 5 (Cdk5) is a member of the cyclin-dependent kinase family that is mostly seen in neurons, does not vary with cell cycle, and is activated in many neurodegenerative disorders and other non-neuronal pathologies, but its relationship to non-neuronal apoptosis is not understood, nor is the control of the activation of Cdk5 by its activators. The most widely studied activator of Cdk5, p35, is cleaved to p25 by calpain, an event that has been linked with activation of Cdk5 and neuronal death. Here we report that calpain-mediated Cdk5/p25 activation accompanies non-neuronal as well as neuronal cell death, suggesting that the p35/calpain/p25/Cdk5 activation sequence is a general feature of cell death. We further demonstrate that Cdk5 can be activated in the absence of p53, Apaf-1, caspase-9, and -3 during cell death, indicating that its activation relates more to cell death than to a specific pathway of apoptosis.     

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.     

Thioredoxin-dependent redox regulation of p53-mediated p21 activation

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-kappaB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1. cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.     

Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.     

Mathematical modeling of the regulation of caspase-3 activation and degradation

Caspases are thought to be important players in the execution process of apoptosis. Inhibitors of apoptosis (IAPs) are able to block caspases and therefore apoptosis. The fact that a subgroup of the IAP family inhibits active caspases implies that not each caspase activation necessarily leads to apoptosis. In such a scenario, however, processed and enzymically active caspases should somehow be removed. Indeed, IAP-caspase complexes covalently bind ubiquitin, resulting in degradation by the 26S proteasome. Following release from mitochondria, IAP antagonists (e.g. second mitochondrial activator of caspases (Smac)) inactivate IAPs. Moreover, although pro-apoptotic factors such as irradiation or anti-cancer drugs may release Smac from mitochondria in tumor cells, high cytoplasmic survivin and ML-IAP levels might be able to neutralize it and, consequently, IAPs would further be able to bind activated caspases. Here, we propose a simple mathematical model, describing the molecular interactions between Smac deactivators, Smac, IAPs, and caspase-3, including the requirements for both induction and prevention of apoptosis, respectively. In addition, we predict a novel mechanism of caspase-3 degradation that might be particularly relevant in long-living cells.     

p53 gene expression and activation of p53-dependent transcription in melanoma cell lines

Malignant melanoma has poor prognosis because of its high metastatic potential and resistance to chemotherapy. A possible approach to more effective therapy is induction of p53-dependent apoptosis. This approach is promising, since the wild-type p53 is expressed in most melanomas. An attempt was made to estimate the functional activity of p53 in several malignant melanoma cell lines. Most lines were characterized by a high protein level and nuclear localization of p53. All cell lines expressing the wild-type p53 showed stabilization of p53, its translocation into the nucleus, and activation of several target genes in response to DNA-damaging agents, suggesting that p53 was functionally active. A high-molecular-weight protein localized in the cytoplasm and mimicking a p53 epitope was found in several cell lines. It was shown that the DO-1 epitope of this protein does not derive from p53, ruling out cytoplasmic retention of p53 in melanoma cell lines. A mechanism of camptothecin-induced stabilization of p53 by decreasing the level of the HDM2 mRNA was described for melanoma cells but not for normal melanocytes, which suggested a differential effect of camptothecin on tumor-derived and primary cells.     

p53 protein and p21 mRNA levels and caspase-3 activity are altered by zinc status in aortic endothelial cells

The influence ofzinc status on the levels of p53, as well as downstream targetsof p53 in cell repair and survival, was examined in human aorticendothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) wasused to deplete zinc over one passage. Other treatments includedzinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 µM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levelsin the ZA cells were not different, but levels in ZS cells weresignificantly higher (40%) than in ZN cells. No difference in p53 mRNAabundance was detected among all treatments; however, p53 nuclearprotein levels were >100% higher in the ZD and ZS cells and almost200% higher in the ZA cells than in ZN controls. In addition, p21 mRNAabundance, a downstream target of p53 protein, was increased in the ZScells compared with both the ZN control and ZD cells. In the ZS cells,bax and mcl-1 were also ~50% higher compared with ZN controls,whereas bcl-2 mRNA was increased compared with ZA cells. Moreover,caspase-3 activity of ZD cells was not different from that of ZNcontrols but was reduced to 83 and 69% of ZN controls in ZA and ZScells, respectively. Thus p53 protein and p53 downstream target genesappeared to be modulated by intracellular zinc status in HAECs.

     

p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

p53 at the crossroads between cancer and neurodegeneration

Aging, dementia, and cancer share a critical set of altered cellular functions in response to DNA damage, genotoxic stress, and other insults. Recent data suggest that the molecular machinery involved in maintaining neural function in neurodegenerative disease may be shared with oncogenic pathways. Cancer and neurodegenerative diseases may be influenced by common signaling pathways regulating the balance of cell survival versus death, a decision often governed by checkpoint proteins. This paper focuses on one such protein, p53, which represents one of the most extensively studied proteins because of its role in cancer prevention and which, furthermore, has been recently shown to be involved in aging and Alzheimer disease (AD). The contribution of a conformational change in p53 to aging and neurodegenerative processes has yet to be elucidated. In this review we discuss the multiple functions of p53 and how these correlate between cancer and neurodegeneration, focusing on various factors that may have a role in regulating p53 activity. The observation that aging and AD interfere with proteins controlling duplication and cell cycle may lead to the speculation that, in senescent neurons, aberrations in proteins generally dealing with cell cycle control and apoptosis could affect neuronal plasticity and functioning rather than cell duplication.