Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-α-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2−/− T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2−/− T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.

p53 expression is required for thymocyte apoptosis induced by adenosine deaminase deficiency.

Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous enzyme in the purine catabolic pathway. In contrast to the widespread tissue distribution of this enzyme, inherited ADA deficiency in human results in a tissue-specific severe combined immunodeficiency. To explain the molecular basis for this remarkable tissue specificity, we have used a genetic approach to study ADA deficiency. We demonstrate that ADA deficiency causes depletion of CD8low transitional and CD4+CD8+ double-positive thymocytes by an apoptotic mechanism. This effect is mediated by a p53-dependent pathway, since p53-deficient mice are resistant to the apoptosis induced by ADA deficiency. DNA damage, known to be caused by the abnormal accumulation of dATP in ADA deficiency, is therefore responsible for the ablation of T-cell development and for the immunodeficiency. The two thymocyte subsets most susceptible to apoptosis induced by ADA deficiency are also the two thymocyte subsets with the lowest levels of bcl-2 expression. We show that thymocytes from transgenic mice that overexpress bcl-2 in the thymus are rescued from apoptosis induced by ADA deficiency. Thus, the tissue specificity of the pathological effects of ADA deficiency is due to the low bcl-2 expression in CD8low transitional and CD4+CD8+ double-positive thymocytes.

Thymocyte-targeted overexpression of xiap transgene disrupts T lymphoid apoptosis and maturation

The X-linked inhibitor of apoptosis (XIAP) and other members of the inhibitor of apoptosis (IAP) family can suppress apoptosis induced by a diverse variety of triggers. Functional studies done to date have focused on tissue culture models and adenovirus overexpression of XIAP and other IAP proteins. Here we report the phenotype of an engineered transgenic mouse overexpressing a human IAP, as well as assessing the long-term consequence of IAP overexpression. We document the relative protein expression levels of the endogenous mouse homologue to XIAP, mouse inhibitor of apoptosis (MIAP 3), within thymocyte and T cell subpopulations. The consequence of lymphoid-targeted overexpression of XIAP in transgenic mice suggests a physiological role for the endogenous protein, MIAP3. Xiap-transgenic mice accumulated thymocytes and/or T cells in primary and secondary lymphoid tissue, T cell maturation was perturbed, and transgenic thymocytes resisted a variety of apoptotic triggers both in vitro and in vivo. These observations imply a possible key function for the intrinsic cellular inhibitor XIAP in maintaining the homeostasis of the immune system.

Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.

Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK−/− rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK−/− mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK−/− mice raised in constant darkness. One day of constant light caused the rods in the RK−/− mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.

Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the α1 and α2 helices (Δloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Δloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K→R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.

The p42 variant of ETS1 protein rescues defective Fas-induced apoptosis in colon carcinoma cells

ETS1 is a cellular homologue of the product of the viral ets oncogene of the E26 virus, and it functions as a tissue-specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. ETS1 controls the expression of critical genes involved in these processes by binding to ets binding sites present in the transcriptional regulatory regions. The ETS1 gene generates two proteins, p51 and a spliced variant, p42, lacking exon VII. In this paper we show that p42-ETS1 expression bypasses the damaged Fas-induced apoptotic pathway in DLD1 colon carcinoma cells by up-regulating interleukin 1β-converting enzyme (ICE)/caspase-1 and causes these cancer cells to become susceptible to the effects of the normal apoptosis activation system. ICE/caspase-1 is a redundant system in many cells and tissues, and here we demonstrate that it is important in activating apoptosis in cells where the normal apoptosis pathway is blocked. Blocking ICE/caspase-1 activity by using specific inhibitors of this protease prevents the p42-ETS1-induced apoptosis from occurring, indicating that the induced ICE/caspase-1 enzyme is responsible for killing the cancer cells. p42-ETS1 activates a critical alternative apoptosis pathway in cancer cells that are resistant to normal immune attack, and thus it may be useful as an anticancer therapeutic.

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N′-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine . By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.

Different mechanisms for suppression of apoptosis by cytokines and calcium mobilizing compounds

Overexpression of wild-type p53 in M1 myeloid leukemia cells induces apoptotic cell death that was suppressed by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin (TG). This suppression of apoptosis by A23187 or TG was associated with suppression of caspase activation but not with suppression of wild-type-p53-induced expression of WAF-1, mdm-2, or FAS. In contrast to suppression of apoptosis by the cytokines interleukin 6 (IL-6) and interferon γ, a protease inhibitor, or an antioxidant, suppression of apoptosis by A23187 or TG required extracellular Ca2+ and was specifically abolished by the calcineurin inhibitor cyclosporin A. IL-6 induced immediate early activation of junB and zif/268 (Egr-1) but A23187 and TG did not. A23187 and TG also suppressed induction of apoptosis by doxorubicin or vincristine in M1 cells that did not express p53 by a cyclosporin A-sensitive mechanism. Suppression of apoptosis by A23187 or TG was not associated with autocrine production of IL-6. Apoptosis induced in IL-6-primed M1 cells after IL-6 withdrawal was not suppressed by A23187 or TG but was suppressed by the cytokines IL-6, IL-3, or interferon γ. The results indicate that these Ca2+-mobilizing compounds can suppress some pathways of apoptosis suppressed by cytokines but do so by a different mechanism.

NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 → S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor β1 (TGF-β1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-β1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-β1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-β1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-β1 in vascular disease.

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.