Differential apoptosis effects of primate lentiviral Vpr and Vpx in mammalian cells

The growth inhibitory effects of Vpr and Vpx are species- and cell type-dependent. HIV-1, HIV-2 and SIV Vpr are primarily cytostatic in mammalian cells and HIV-1 Vpr has been reported to induce apoptosis in human cells. Our previous studies have shown that HIV-1, HIV-2 and SIV Vpr and Vpx have differential cytostatic and cytotoxic effects in the yeast cells [Zhang et al.: Virology, 230:103-112; 1997]. Here, we further examined the apoptosis function of HIV-1 Vpr in different species of mammalian cells and investigated if other primate lentiviral Vpr and Vpx exert similar functions. Our results show that none of the primate lentiviral Vpr or Vpx we tested induces apoptosis in nonhuman species of mammalian cells. However, HIV-1 Vpr, but not HIV-2 or SIV Vpr and/or Vpx, induced apoptosis in different types of human cell lines. Further, the apoptotic effect of HIV-1 Vpr can be distinguished from that of the human interferon-gamma, a known proapoptotic protein, that HIV-1 Vpr shows little to no paracrine and/or bystander effect. When coexpressed with Bcl-2 or Bcl-X(L), the apoptotic effect of HIV-1 Vpr became markedly attenuated. These results indicate that the apoptotic effect of HIV-1 Vpr is species-dependent and is intracellularly modulated by the Bcl-2 family of proteins. Our study also suggests that the proapoptotic function of HIV-1 Vpr is developmentally associated with human but not nonhuman primate species.     

HIV-1 Vpr induces apoptosis through caspase 9 in T cells and peripheral blood mononuclear cells

Human immunodeficiency virus, type 1 (HIV-1), vpr gene encodes a 14-kDa virion-associated protein, which exhibits significant effects on human cells. One important property of Vpr is its ability to induce apoptosis during infection. Apoptotic induction is likely to play a role in the pathogenesis of AIDS. However, the pathway of apoptosis is not clearly defined. In this report we investigate the mechanism of apoptosis induced by HIV-1 Vpr using a Vpr pseudotype viral infection system or adeno delivery of Vpr in primary human lymphoid cells and T-cells. With either vector, HIV-1 Vpr induced cell cycle arrest at the G(2)/M phase and apoptosis in lymphoid target cells. Furthermore, we observed that with both vectors, caspase 9, but not caspase 8, was activated following infection of human peripheral blood mononuclear cell with either Vpr-positive HIV virions or adeno-delivered Vpr. Activation of the caspase 9 pathway resulted in caspase 3 activation and apoptosis in human primary cells. These effects were coincident with the disruption of the mitochondrial transmembrane potential and induction of cytochrome c release by Vpr. The Vpr-induced signaling pathway did not induce CD95 or CD95L expression. Bcl-2 overexpressing cells succumb to Vpr-induced apoptosis. These studies illustrate that Vpr induces a mitochondria-dependent apoptotic pathway that is distinct from apoptosis driven by the Fas-FasL pathway.     

PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector

Under iron limitation, the opportunistic human pathogen Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted into the extracellular environment, pyochelin complexes ferric ions and delivers them, via the outer membrane receptor FptA, to the bacterial cytoplasm. Extracellular pyochelin also acts as a signalling molecule, inducing the expression of pyochelin biosynthesis and uptake genes by a mechanism involving the AraC-type regulator PchR. We have identified a 32 bp conserved sequence element (PchR-box) in promoter regions of pyochelin-controlled genes and we show that the PchR-box in the pchR-pchDCBA intergenic region is essential for the induction of the pyochelin biosynthetic operon pchDCBA and the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin and iron. PchR-box mutations that interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. We conclude that pyochelin, probably in its iron-loaded state, is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation suggests that the siderophore can enter the cytoplasm.     

Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis

Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.     

Autophagy is a regulator of TRAIL-induced apoptosis in NSCLC A549 cells

Autophagy, a catabolic process by which cytoplasmic components are degraded in lysosomes, plays an important role in the maintenance of cellular homeostasis. Dysregulation of autophagy is associated with several diseases. However, few studies have addressed the role of autophagy in the lung, and its role in lung diseases remains unclear. In the present study, we examined the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on autophagy in A549 cells and explored the underlying mechanisms. We showed that TRAIL promoted autophagosome formation, as detected by the levels of LC3-II, and its effect on promoting autophagy was dependent on the expression of the autophagy related genes (ATGs) Atg5, Atg7, and beclin-1. TRAIL-induced ATG expression was attenuated by JNK silencing or treatment with the JNK inhibitor SP600125, indicating the involvement of the JNK pathway. Crosstalk between autophagy and apoptosis was demonstrated by silencing the autophagy related genes Atg5, Atg7, and beclin-1, and the dependence of TRAIL-induced apoptosis on autophagy-related gene expression. Taken together, our results indicate that TRAIL promotes autophagy in A549 cells via a mechanism involving the modulation of ATG expression through the JNK pathway. Inhibition of autophagy enhanced TRAIL-induced cell proliferative inhibition and apoptosis in A549 cells.     

A novel role for Vpr of human immunodeficiency virus type 1 as a regulator of the splicing of cellular pre-mRNA

Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.     

Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

Thioredoxin as a neurotrophic cofactor and an important regulator of neuroprotection

Oxidative stress has been implicated in the pathogenesis of a wide variety of neuronal diseases, including ischemic neuronal injury, Alzheimer’s disease, and Parkinson’s disease. Thioredoxin reduces exposed protein disulfides and couples with peroxiredoxin to scavenge reactive oxygen species. Nerve growth factor (NGF) has profound effects on neurons, including promotion of survival and differentiation via multiple signaling pathways. As for the NGF-induced neurite outgrowth, the CREB-cAMP responsive element (CRE) pathway is important to the activation of immediate-early genes such as c-fos. Thioredoxin is upregulated by NGF through ERK and the CREB-CRE pathway in PC12 cells. Thioredoxin is necessary for NGF signaling through CRE leading to c-fos expression and also plays a critical role in the NGF-mediated neurite outgrowth in PC12 cells. Therefore, thioredoxin appears to be a neurotrophic cofactor that augments the effect of NGF on neuronal differentiation and regeneration. NGF acts also as a neuronal survival factor. Previous reports showed that thioredoxin exerts a cytoprotective effect in the nervous system. The cytoprotective effect is mediated by enhancing the action of NGF, via the regulation of antiapoptotic signaling, or through its antioxidative stress activity.     

HIV ENV glycoprotein-mediated bystander apoptosis depends on expression of the CCR5 co-receptor at the cell surface and ENV fusogenic activity

HIV-1 infections lead to a progressive depletion of CD4 cells culminating in AIDS. The coreceptor usage by HIV varies from CCR5 (R5) tropic early in infection to CXCR4 (X4) tropic in later infections. Although the coreceptor switch from R5 to X4 tropic HIV is well associated with progression to AIDS, the role of CCR5 in disease progression especially in patients infected exclusively with R5 isolates throughout the disease remains enigmatic. To better understand the role of CCR5 and R5 tropic HIV envelope in AIDS pathogenesis, we asked whether the levels of CCR5 and/or HIV Env-mediated fusion determine apoptosis of bystander cells. We generated CD4(+) T cell lines expressing varying levels of CCR5 on the cell surface to show that CCR5 expression levels correlate with bystander apoptosis induction. The mechanism of apoptosis involved caspase-3 activation and mitochondrial depolarization and was dependent on gp41 fusion activity as confirmed by fusion-restricted gp41 point mutants and use of the fusion inhibitor T20. Interestingly, lower levels of CCR5 were able to support virus replication in the absence of bystander apoptosis. Our findings suggest that R5 HIV-1-mediated bystander apoptosis is dependent on both CCR5 expression levels as well as fusogenic activity of the Env glycoprotein.     

Scleroderma pathogenesis: a pivotal role for fibroblasts as effector cells

Scleroderma (systemic sclerosis; SSc) is characterised by fibrosis of the skin and internal organs in the context of autoimmunity and vascular perturbation. Overproduction of extracellular matrix components and loss of specialised epithelial structures are analogous to the process of scar formation after tissue injury. Fibroblasts are the resident cells of connective tissue that become activated at sites of damage and are likely to be important effector cells in SSc. Differentiation into myofibroblasts is a hallmark process, although the mechanisms and cellular origins of this important fibroblastic cell are still unclear. This article reviews fibroblast biology in the context of SSc and highlights the potentially important place of fibroblast effector cells in fibrosis. Moreover, the heterogeneity of fibroblast properties, multiplicity of regulatory pathways and diversity of origin for myofibroblasts may underpin clinical diversity in SSc, and provide novel avenues for targeted therapy.     

The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.