Transglutaminase 2 gene ablation protects against renal ischemic injury by blocking constant NF-κB activation

Transglutaminase 2 knockout (TGase2(-/-)) mice show significantly reduced inflammation with decreased myofibroblasts in a unilateral ureteral obstruction (UUO) model, but the mechanism remains to be clarified. Nuclear factor-κB (NF-κB) activation plays a major role in the progression of inflammation in an obstructive nephropathy model. However, the key factors extending the duration of NF-κB activation in UUO are not known. In several inflammatory diseases, we and others recently found that TGase 2 plays a key role in extending NF-κB activation, which contributes to the pathogenesis of disease. In the current study, we found that NF-κB activity in mouse embryogenic fibroblasts (MEFs) from TGase2(-/-) mice remained at the control level while the NF-κB activity of wild-type (WT) MEFs was highly increased under hypoxic stress. Using the obstructive nephropathy model, we found that NF-κB activity remained at the control level in TGase2(-/-) mouse kidney tissues, as measured by COX-2 expression, but was highly increased in WT tissues. We conclude that TGase 2 gene ablation reduces the duration of NF-κB activation in ischemic injury.     

Tissue transglutaminase protects against apoptosis by modifying the tumor suppressor protein p110 Rb

Tissue transglutaminase (TGase) is involved in the regulation of several biological events including cellular differentiation and apoptosis. The expression and activation of TGase are up-regulated in response to retinoic acid (RA), leading to the protection of several cell lines against N-(4-hydroxyphenyl)retinamide (HPR)-induced apoptosis. The anti-apoptotic mechanisms of TGase are poorly understood at this time. We examined the interaction of TGase with the retinoblastoma (Rb) protein, a substrate of TGase that is also implicated in cell survival functions. In cells undergoing HPR-induced apoptosis, Rb is degraded. This degradation is blocked when cells are pretreated with RA, an important regulator of TGase. In vitro studies revealed that TGase protects Rb from caspase-induced degradation in a transamidation-dependent manner. Experiments performed with fibroblasts from Rb(-/-) mice further demonstrated that the presence of Rb was required for TGase to exhibit anti-apoptotic activity in response to RA treatment. Microinjection of Rb(-/-) cells with a transamidation-defective TGase mutant and Rb afforded no protection from HPR-induced apoptosis. Taken together, these findings suggest that the ability of TGase to modify Rb via transamidation underlies the ability of TGase to provide protection against apoptotic insults and to ensure that cells remain viable during differentiation.     

Depletion of cathepsin D by transglutaminase 2 through protein cross-linking promotes cell survival

Transglutaminase 2 (TGase 2) promotes nuclear factor-κB (NF-κB) activity through depletion of the inhibitory subunit of NF-κB (I-κBα) via protein cross-linking, leading to resolution of inflammation. Increased expression of TGase 2 contributes to inflammatory disease pathogenesis via constitutive NF-κB activation. Conversely, TGase 2 inhibition often reverses inflammation in animal models. The role of TGase 2 in apoptosis remains less clear, as both pro- and anti-apoptotic functions of TGase 2 have been demonstrated under different experimental conditions. Apoptosis is intact in a TGase 2 knock out mouse (TGase2^?/?), which is phenotypically normal. However, upon exposure to tumor necrosis factor (TNF)-α-induced apoptotic stress, mouse embryonic fibroblasts (MEFs) from TGase2^?/? mice were more sensitive to cell death than MEFs from wild-type (TGase 2^+/+) mice. In the current study, to explore the role of TGase 2 in apoptosis, TGase 2-binding proteins were identified by LC/MS. TGase 2 was found to associate with cathepsin D (CTSD). Binding of TGase 2 to CTSD resulted in the depletion of CTSD via cross-linking in vitro as well as in MEFs, leading to decreased levels of apoptosis. Furthermore, cytoplasmic CTSD levels were higher in MEFs from TGase 2^?/? mice than in those from TGase 2^+/+ mice, as were caspase 3 activation and poly (ADP-ribose) polymerase (PARP) processes. These results suggest that TGase 2, while not previously implicated as a major regulatory factor in apoptosis, may regulate the balance between cell survival and cell death through the modulation of CTSD levels.     

Detection of in situ activation of transglutaminase during apoptosis: correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry

BACKGROUND: One of the hallmarks of apoptosis is activation of tissue transglutaminase (Tgase; also called transglutaminase type 2 [TGase 2]). Its activation causes cross-linking of cytoplasmic proteins, making them insoluble and presumably less immunogenic. Several biochemical and cytochemical methods to detect activity of TGase 2 exist, but none has been adapted for multiparameter flow or image cytometry. METHODS: Apoptosis of HL-60 or U-937 leukemic cells was induced by camptothecin, tumor necrosis factor alpha, hyperthermia, or the cytotoxic RNase onconase. Two different approaches to detect TGase 2 activation were developed: (a) the unfixed cells were treated with 4',6'-diamidino-2-phenylindole, and sulforhodamine 101 in solutions of nonionic detergents; (b) the TGase 2 substrate fluoresceinated polyamine cadaverine (F-CDV) was administered into the cultures for several hours before cell harvesting. The cells were then fixed and their DNA counterstained with propidium. Cellular fluorescence was measured by flow or laser scanning cytometry. RESULTS: (a) Exposure of nonapoptotic cells to detergents caused their full lysis, resulting in preparation of isolated nuclei devoid of cytoplasm. Conversely, the cross-linking of cytoplasmic protein by activated TGase 2 in apoptotic cells provided resistance to detergents: the nuclei or nuclear (chromatin) fragments of apoptotic cells remained attached to the cytoplasmic protein, embedded within the proteinaceous "shell." Such cells were identified by their high protein content: intensity of fluorescence after staining with the protein fluorochrome sulforhodamine 101 was markedly higher than that of isolated nuclei. (b) Activation of TGase 2 was also detected by virtue of intense cell labeling with fluoresceinated polyamine cadaverine. Interestingly, in many cells apoptosis progressed without evidence of activation of TGase 2, suggesting that this event may not be a prerequisite for completion of apoptosis. CONCLUSIONS: Activation of TGase 2 can be detected simply by cell resistance to detergents or in situ reactivity with F-CDV. Both methods allow one to correlate activation of TGase 2 with the cell cycle position. However, because activation of TGase 2 is not always detected during apoptosis, the lack of the activation cannot be considered a marker of nonapoptotic cells. Hence, an apoptotic index based solely on TGase 2 activation may underestimate incidence of apoptosis.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted inactivation of Gh/tissue transglutaminase II

The novel G-protein, G(h)/tissue transglutaminase (TGase II), has both guanosine triphosphatase and Ca(2+)-activated transglutaminase activity and has been implicated in a number of processes including signal transduction, apoptosis, bone ossification, wound healing, and cell adhesion and spreading. To determine the role of G(h) in vivo, the Cre/loxP site-specific recombinase system was used to develop a mouse line in which its expression was ubiquitously inactivated. Despite the absence of G(h) expression and a lack of intracellular TGase activity that was not compensated by other TGases, the Tgm2(-/-) mice were viable, phenotypically normal, and were born with the expected Mendelian frequency. Absence of G(h) coupling to alpha(1)-adrenergic receptor signaling in Tgm2(-/-) mice was demonstrated by the lack of agonist-stimulated [alpha-(32)P]GTP photolabeling of a 74-kDa protein in liver membranes. Annexin-V positivity observed with dexamethasone-induced apoptosis was not different in Tgm2(-/-) thymocytes compared with Tgm2(+/+) thymocytes. However, with this treatment there was a highly significant decrease in the viability (propidium iodide negativity) of Tgm2(-/-) thymocytes. Primary fibroblasts isolated from Tgm2(-/-) mice also showed decreased adherence with culture. These results indicate that G(h) may be importantly involved in stabilizing apoptotic cells before clearance, and in responses such as wound healing that require fibroblast adhesion mediated by extracellular matrix cross-linking.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Sporadic inclusion body myositis correlates with increased expression and cross-linking by transglutaminases 1 and 2

Sporadic inclusion body myositis (SIBM) is characterized by vacuolar degeneration of muscle fibers and intrafiber clusters of paired helical filaments with abnormal amyloid deposition. Because of their potential involvement in other degenerative disorders, we have examined the expression of transglutaminases (TGases) in normal and SIBM tissues. We report that at least two different enzymes, the ubiquitous TGase 2 as well as the TGase 1 enzyme, are present in muscle tissues. However, in comparison with normal tissue, the expression of TGases 1 and 2 was increased 2.5- and 4-fold in SIBM, accompanied by about a 20-fold higher total TGase activity. By immunohistochemical staining, in normal muscle, TGase 2 expression was restricted to some endomysial connective tissue elements, whereas TGase 1 and beta-amyloid proteins were not detectable. In SIBM muscle, both TGases 1 and 2 as well as amyloid proteins were brightly expressed and co-localized in the vacuolated muscle fibers, but none of these proteins colocalized with inflammatory cell markers. Next, we isolated high molecular weight insoluble proteins from SIBM muscle tissue and showed that they were cross-linked by about 6 residues/1000 residues of the isopeptide bond. Furthermore, by amino acid sequencing of solubilized tryptic peptides, they contain amyloid and skeletal muscle proteins. Together, these findings suggest that elevated expression of TGases 1 and 2 participate in the formation of insoluble amyloid deposits in SIBM tissue and in this way may contribute to progressive and debilitating muscle disease.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

IgE production in CD40/CD40L cross-talk of B and mast cells and mediator release via TGase 2 in mouse allergic asthma

TGase 2 is over-expressed in a variety of inflammatory diseases including allergic asthma. This study aimed to investigate the role of TGase 2 on IgE production and signaling pathways in mast cell activation related to OVA-induced allergic asthma. Bone marrow-derived mast cells (BMMCs) isolated from WT or TGase 2^?/? mice were activated with Ag/Ab (refer to act-WT-BMMCs and act-KO-BMMCs, respectively). B cells isolated from splenocytes were activated with anti-mouse IgM (act-B cells), and B cells were co-cultured with BMMCs. WT and TGase 2^?/? mice were sensitized and challenged with OVA adsorbed in alum hydroxide. Intracellular Ca^2 + ([Ca^2 +]_i) levels were determined by fluorescence intensity; IgE, mediators and TGase 2 activity by ELISA; the CD138 expression by FACS analyzer; cell surface markers and signal molecules by Western blot; NF-κB by EMSA; co-localization of mast cells and B cells by immunohistochemistry; Fcε RI-mediated mast cell activation by PCA test; expression of cytokines, MMPs, TIMPs, TLR2 and Fc?RI by RT-PCR. In vitro, act-KO-BMMCs reduced the [Ca^2 +]i levels, NF-κB activity, expression of CD40/CD40L, plasma cells, total IgE levels and TGase 2 activity in act-B cells co-cultured with act-BMMCs, expression of inflammatory cytokines and MMPs2/9, release of mediators (TNF-α, LTs and cytokines), and activities of signal molecules (PKCs, MAP kinases, I-κB and PLA2), which were all increased in act-WT-BMMCs. TGase 2 siRNA transfected/activated-BMMCs reduced all responses as same as those in act-KO-BMMCs. In allergic asthma model, TGase 2^?/? mice protected against PCA reaction, OVA-specific IgE production and AHR, and they reduced co-localization of mast cells and B cells or IgE in lung tissues, expression and co-localization of surface molecules in mast cells (c-kit and CD40L) and B cells (CD23 and CD40), inflammatory cells including mast cells, goblet cells, amounts of collagen and mediator release in BAL fluid and/or lung tissues, which were all increased in WT mice. TLR expression in TGase 2^?/? mice did not differ from those in WT mice. Our data suggest that TGase 2 expression and Ca^2 + influx required by bidirectional events in mast cell activation facilitate IgE production in B cells via up-regulating mast cell CD40L expression, and induce the expression of numerous signaling molecules associated with airway inflammation and remodeling in allergic asthma.     

"Tissue" transglutaminase in animal development

The "tissue" transglutaminase is a multifunctional enzyme that in its cross-linking configuration catalyzes Ca2+ -dependent reactions resulting in post-translational modification of proteins by establishing epsilon(gamma-glutamyl) lysine cross-links and/or covalent incorporation of biogenic amines (di- and poly-amines and histamine) into proteins. Several laboratories have shown that in Vertebrates, "tissue" transglutaminase (tTG) gene expression specifically characterizes cells undergoing apoptosis or programmed cell death (PCD). The Ca2+ -dependent activation of this enzyme leads to the formation of detergent-insoluble cross-linked protein polymers in cells undergoing PCD. This insoluble protein scaffold could stabilize the integrity of the dying cells before their clearance by phagocytosis, preventing the non-specific release of harmful intracellular components (e.g. lysosomal enzymes, nucleic acids, etc.) and consequently inflammatory responses and scar formation in bystander tissues. In this review we attempt to present an overview of the current knowledge on tTG expression and regulation in animal reproduction and development. The data available so far further strengthen the relationship existing between tTG expression and the induction of PCD.     

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.     

Effects of Tissue Transglutaminase on β -Amyloid1-42-Induced Apoptosis

Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in β-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that β-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced β-amyloid_1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for β-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of β-amyloid_1-42-induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with β-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis.     

Transglutaminase 2 mediates polymer formation of I-kappaBalpha through C-terminal glutamine cluster

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-kappaB (NF-kappaB) independently of I-kappaB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-kappaBalpha (I-kappaBalpha). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-kappaBalpha polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-kappaBalpha (amino acids 266-268) that appeared to play a key role in the formation of I-kappaBalpha polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-kappaBalpha. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266-268 reversed I-kappaBalpha polymerization in vitro. Furthermore, the depletion of free I-kappaBalpha in EcR/TG cells was completely rescued in vivo by transfection of mutant I-kappaBalphas in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-kappaB.     

Efficiency of homocysteine plus copper in inducing apoptosis is inversely proportional to gamma-glutamyl transpeptidase activity.

Hyperhomocysteinemia represents an independent risk factor for atherosclerosis, but the mechanisms leading to cellular dysfunctions remain unknown. Using ECV304 cells, we found that homocysteine (Hcy) plus copper (Cu2+) induced cytotoxic effects: loss of cell adhesion, increased permeability to PI, and the occurrence of morphologically apoptotic cells. This form of apoptosis, inhibited by Z-VAD-fmk, was associated with a loss of mitochondrial potential, a cytosolic release of cytochrome c, activation of caspase-3, degradation of poly(ADP-ribose)polymerase, and internucleosomal DNA fragmentation. However, the ability of Hcy plus Cu2+ to induce apoptosis decreased when the pretreatment culture time increased. As a positive correlation was found between the length of time of culture before treatment and the enhancement of gamma-glutamyl transpeptidase (gamma-GT) activity, we asked whether gamma-GT was involved in the control of Hcy plus Cu2+-induced apoptosis. Therefore, ECV304 cells were treated with either acivicin or dexamethasone, inhibiting and stimulating gamma-GT, respectively. In ECV304 cells and human umbilical venous endothelial cells, acivicin favored Hcy plus Cu2+-induced apoptosis whereas dexamethasone counteracted the apoptotic process. As acivicin and dexamethasone were also capable of modulating cell death in ECV304 cells treated with antitumoral drugs, our data emphasize that the involvement of gamma-GT in the control of apoptosis is not restricted to Hcy but also concerns other chemical compounds.     

Redox status of thioredoxin-1 （TRX1） determines the sensitivity of human liver carcinoma cells （HepG2） to arsenic trioxide-induced cell death

lntracellular redox homeostasis plays a critical role in determining tumor cells＇ sensitivity to drug-induced apoptosis. Here we investigated the role of thioredoxin-1 （TRX1）, a key component of redox regulation, in arsenic trioxide （AS2O3）-induced apoptosis. Over-expression of wild-type TRX1 in HepG2 cells led to the inhibition of As2O3-induced cytochrome c （cyto c） release, caspase activation and apoptosis, and down-regulation of TRX1 expression by RNAi sensitized HepG2 cells to As2O3-induced apoptosis. Interestingly, mutation of the active site of TRX1 from Cys^32/35 to Ser^32/35 converted this molecule from an apoptotic protector to an apoptotic promoter. In an effort to understand the mechanisms of this conversion, we used isolated mitochondria from mouse liver and found that recombinant wild-type TRX1 could protect mitochondria from the apoptotic changes. In contrast, the mutant form of TRX1 alone elicited mitochondria-related apoptotic changes, including the mitochondrial permeability transition pore （mPTP） opening, loss of mitochondrial membrane potential, and cyto c release from mitochondria. These apoptotic effects were inhibited by cyclosporine A （CsA）, indicating that mutant TRX1 targeted to mPTP. Alteration of TRX1 from its reduced form to oxidized form in vivo by 2,4-dinitrochlorobenzene （DNCB）, a specific inhibitor ofTRX reductase, also sensitized HepG2 cells to As203-induced apoptosis. These data suggest that TRX1 plays a central role in regulating apoptosis by blocking cyto c release, and inactivation of TRX1 by either mutation or oxidization of the active site cysteines may sensitize tumor cells to As2O3-induced apoptosis.     

Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.     

p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.