PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy

Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of the study is to investigate whether PUMA is involved in PHG by mediating ER stress apoptotic signaling. To identify whether PUMA is involved in PHG by mediating ER stress, gastric mucosal injury and apoptosis were studied in both PHG patients and PHG animal models using PUMA knockout (PUMA-KO) and PUMA wild-type (PUMA-WT) mice. The induction of PUMA expression and ER stress signaling were investigated, and the mechanisms of PUMA-mediated apoptosis were analyzed. GES-1 and SGC7901 cell lines were used to further identify whether PUMA-mediated apoptosis was induced by ER stress in vitro. Epithelial apoptosis and PUMA were markedly induced in the gastric mucosa of PHG patients and mouse PHG models. ER stress had a potent role in the induction of PUMA and apoptosis in PHG models, and the apoptosis was obviously attenuated in PUMA-KO mice. Although the targeted deletion of PUMA did not affect ER stress, mitochondrial apoptotic signaling was downregulated in mice. Meanwhile, PUMA knockdown significantly ameliorated ER stress-induced mitochondria-dependent apoptosis in vitro. These results indicate that PUMA mediates ER stress-induced mucosal epithelial apoptosis through the mitochondrial apoptotic pathway in PHG, and that PUMA is a potentially therapeutic target for PHG.     

PUMA Promotes Bax Translocation by Both Directly Interacting with Bax and by Competitive Binding to Bcl-XL during UV-induced Apoptosis

Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. p53 up-regulated modulator of apoptosis (PUMA) has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which PUMA regulates apoptosis, especially how PUMA activates Bcl-2-associated X protein (Bax) in response to UV irradiation is still controversial. In this study, by using real-time single-cell analysis and fluorescence resonance energy transfer, we investigated the tripartite nexus among PUMA, Bax, and Bcl-X_L in living human lung adenocarcinoma cells (ASTC-a-1) to illustrate how PUMA promotes Bax translocation to initiate apoptosis. Our results show that the interaction between PUMA and Bax increased gradually, with Bax translocating to mitochondria and colocalizing with PUMA after UV irradiation, indicating PUMA promotes Bax translocation directly. Simultaneously, the interaction increased markedly between PUMA and Bcl-X_L and decreased significantly between Bcl-X_L and Bax after UV treatment, suggesting PUMA competitively binds to Bcl-X_L to activate Bax indirectly. The above-mentioned results were further confirmed by coimmunoprecipitation experiments. In addition, pifithrin-α (a p53 inhibitor) and cycloheximide (a protein synthesis inhibitor) could inhibit PUMA-mediated Bax translocation and cell apoptosis. Together, these studies create an important conclusion that PUMA promotes Bax translocation by both by directly interacting with Bax and by competitive binding to Bcl-X_L in UV-induced apoptosis.     

PUMA-mediated tumor suppression: A tale of two stories

Several recent studies provided evidence that PUMA, a pro-apoptotic member of the BH3-only protein subgroup of the Bcl-2 family, is critical for restricting survival and recovery of different cell types, including those of the hematopoietic system, after g-irradiation (IR)-triggered DNA damage. According to its pro-apoptotic function downstream of p53, PUMA is considered to act as a tumor suppressor. While this expectation was met in a model of oncogene-driven lymphomagenesis or carcinogen-driven tumor formation in the gut, studies on IR-driven tumor formation revealed surprising new insights into the role of p53-triggered and PUMA-mediated apoptosis in tumorigenesis and stem cell homeostasis.     

PUMA regulates intestinal progenitor cell radiosensitivity and gastrointestinal syndrome

Radiation is one of the most effective cancer treatments. However, gastrointestinal (GI) syndrome is a major limiting factor in abdominal and pelvic radiotherapy. The loss of crypt stem cells or villus endothelial cells has been suggested to be responsible for radiation-induced intestinal damage. We report here a critical role of the BH3-only protein p53 upregulated modulator of apoptosis (PUMA) in the radiosensitivity of intestinal epithelium and pathogenesis of GI syndrome. PUMA was induced in a p53-dependent manner and mediated radiation-induced apoptosis via the mitochondrial pathway in the intestinal mucosa. PUMA-deficient mice exhibited blocked apoptosis in the intestinal progenitor and stem cells, enhanced crypt proliferation and regeneration, and prolonged survival following lethal doses of radiation. Unexpectedly, PUMA deficiency had little effect on radiation-induced intestinal endothelial apoptosis. Suppressing PUMA expression by antisense oligonucleotides provided significant intestinal radioprotection. Therefore, PUMA-mediated apoptosis in the progenitor and stem cell compartments is crucial for radiation-induced intestinal damage.     

p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation

p73, an important developmental gene, shares a high sequence homology with p53 and induces both G(1) cell cycle arrest and apoptosis. However, the molecular mechanisms through which p73 induces apoptosis are unclear. We found that p73-induced apoptosis is mediated by PUMA (p53 up-regulated modulator of apoptosis) induction, which, in turn, causes Bax mitochondrial translocation and cytochrome c release. Overexpression of p73 isoforms promotes cell death and bax promoter transactivation in a time-dependent manner. However, the kinetics of apoptosis do not correlate with the increase of Bax protein levels. Instead, p73-induced mitochondrial translocation of Bax is kinetically compatible with the induction of cell death. p73 is localized in the nucleus and remains nuclear during the induction of cell death, indicating that the effect of p73 on Bax translocation is indirect. The ability of p73 to directly transactivate PUMA and the direct effect of PUMA on Bax conformation and mitochondrial relocalization suggest a molecular link between p73 and the mitochondrial apoptotic pathway. Our data therefore indicate that PUMA-mediated Bax mitochondrial translocation, rather than its direct transactivation, correlates with cell death. Finally, human DeltaNp73, an isoform lacking the amino-terminal transactivation domain, inhibits TAp73-induced as well as p53-induced apoptosis. The DeltaNp73 isoforms seem therefore to act as dominant negatives, repressing the PUMA/Bax system and, thus, finely tuning p73-induced apoptosis. Our findings demonstrate that p73 elicits apoptosis via the mitochondrial pathway using PUMA and Bax as mediators.     

Puma, a critical mediator of cell death — one decade on from its discovery

PUMA (p53 upregulated modulator of apoptosis) is a pro-apoptotic member of the BH3-only subgroup of the Bcl-2 family. It is a key mediator of p53-dependent and p53-independent apoptosis and was identified 10 years ago. The PUMA gene is mapped to the long arm of chromosome 19, a region that is frequently deleted in a large number of human cancers. PUMA mediates apoptosis thanks to its ability to directly bind known anti-apoptotic members of the Bcl-2 family. It mainly localizes to the mitochondria. The binding of PUMA to the inhibitory members of the Bcl-2 family (Bcl-2-like proteins) via its BH3 domain seems to be a critical regulatory step in the induction of apoptosis. It results in the displacement of the proteins Bax and/or Bak. This is followed by their activation and the formation of pore-like structures on the mitochondrial membrane, which permeabilizes the outer mitochondrial membrane, leading to mitochondrial dysfunction and caspase activation. PUMA is involved in a large number of physiological and pathological processes, including the immune response, cancer, neurodegenerative diseases and bacterial and viral infections.     

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.     

PUMA cooperates with direct activator proteins to promote mitochondrial outer membrane permeabilization and apoptosis

The BCL-2 family of proteins regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP). Within the family there are numerous protein?protein interactions that influence MOMP; however, defining the ultimate signal that commits a cell to apoptosis remains controversial. We chose to examine the function of the BH3-only protein, p53 upregulated modulator of apoptosis (PUMA), to define its contribution to MOMP and cooperation with the direct activator proteins. PUMA is a potent regulator of MOMP and our data suggest that this function is attributed to two distinct mechanisms which both rely on PUMA binding to the anti-apoptotic BCL-2 proteins: de-repression and sensitization. Here we will define these interactions and discuss our experiments that suggest PUMA cooperates with direct activator proteins to efficiently induce MOMP and apoptosis.     

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.     

PUMA与心肌细胞凋亡的研究进展

p53上调的凋亡调节物(p53 upregulated modulator of apoptosis,PUMA)是新近发现的一种具有促凋亡作用的p53靶基因.与以往发现的其他p53靶基因比较,PUMA在促凋亡作用中有两个重要的特点:一是PUMA几乎介导p53依赖的所有凋亡信号;二是PUMA不仅介导p53依赖的凋亡信号,而且还可以介导p53非依赖的凋亡信号.也就是说,尽管PUMA是p53靶基因,但是其在p53非依赖细胞凋亡中也发挥重要作用.由此可见,PUMA是一个强大的促凋亡因子.在心肌细胞,PUMA参与缺血/再灌注、内质网应激、阿霉素等多种刺激诱导的细胞凋亡.因此,PUMA在心肌细胞凋亡中发挥重要作用.     

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53^-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53^-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.     

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase–1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi–1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.     

Fas/FasL mediates NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to drive liver fibrosis

Fas/Fas ligand (FasL)-mediated cell apoptosis involves a variety of physiological and pathological processes including chronic hepatic diseases, and hepatocytes apoptosis contributes to the development of liver fibrosis following various causes. However, the mechanism of the Fas/FasL signaling and hepatocytes apoptosis in liver fibrogenesis remains unclear. The Fas/FasL signaling and hepatocytes apoptosis in liver samples from both human sections and mouse models were investigated. NF-κBp65 wild-type mice (p65^f/f), hepatocytes specific NF-κBp65 deletion mice (p65Δhepa), p53-upregulated modulator of apoptosis (PUMA) wild-type (PUMA-WT) and PUMA knockout (PUMA-KO) littermate models, and primary hepatic stellate cells (HSCs) were also used. The mechanism underlying Fas/FasL-regulated hepatocytes apoptosis to drive HSCs activation in fibrosis was further analyzed. We found Fas/FasL promoted PUMA-mediated hepatocytes apoptosis via regulating autophagy signaling and NF-κBp65 phosphorylation, while inhibition of autophagy or PUMA deficiency attenuated Fas/FasL-modulated hepatocytes apoptosis and liver fibrosis. Furthermore, NF-κBp65 in hepatocytes repressed PUMA-mediated hepatocytes apoptosis via regulating the Bcl-2 family, while NF-κBp65 deficiency in hepatocytes promoted PUMA-mediated hepatocytes apoptosis and enhanced apoptosis-linked inflammatory response, which contributed to the activation of HSCs and liver fibrogenesis. These results suggest that Fas/FasL contributes to NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to enhance liver fibrogenesis, and this network could be a potential therapeutic target for liver fibrosis.Subject terms: Apoptosis, Extracellular signalling molecules