Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Tax contributes apoptosis resistance to HTLV-1-infected T cells via suppression of Bid and Bim expression.

The human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). HTLV-1 Tax has been shown to have a prosurvival role in infected T cells by enhancing expression of the Bcl-2 family of antiapoptotic proteins. In this study, we show that the expression of proapoptotic BH3-only proteins Bim (Bcl-2-interacting mediator of cell death) and Bid (BH3-interacting domain death agonist) is diminished in HTLV-1-infected leukemic cells. Using a Tax-inducible system and a transient overexpression approach, we demonstrate that Tax downregulates Bid and Bim expression at the transcriptional level. We show that reinforced expression of Bim and Bid in HTLV-1-infected T-cell lines sensitizes CD95/TRAIL- and anticancer drug-induced apoptosis. Furthermore, we show that Tax suppresses Bid and Bim expression by enhancing hypoxia-inducible factor-1α (HIF-1α) protein expression. siRNA knockdown of HIF-1α or chemical inhibition of the transactivation activity of HIF-1α resulted in an increase in Bid and Bim expression and, consequently, in an increase in CD95/TRAIL- and anticancer drug-induced apoptosis in HTLV-1-infected leukemic T-cell lines. Our study provides evidence that besides upregulation of prosurvival Bcl-2 proteins, Tax may also confer apoptosis resistance to HTLV-1-infected T cells by suppressing the expression of the proapoptotic BH3-only proteins Bim and Bid.

The traditional Chinese medical compound Rocaglamide protects nonmalignant primary cells from DNA damage-induced toxicity by inhibition of p53 expression.

One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors.

AF-1 a novel regulator of the redox equilibrium during aging.

Multicellular organisms generally undergo qualitative changes within time that are associated with progressive degeneration of biological functions, increased susceptibility to diseases, and increased probability to die within a given time period (aging). One of the major factors contributing to aging is accumulation of oxidative damage. We show that Aging Factor-1 (AF-1) negatively influences the redox balance during aging. AF-1 expression is greatly enhanced in various human cell types of aged individuals which leads to decreased stress resistance towards oxidative damage. We used Drosophila melanogaster as an in vivo model system. Functional knockdown of AF-1 in Drosophila melanogaster reveals a gender-specific effect on lifespan. Whereas female flies show a prolonged healthy lifespan, males show no difference (or even a shortened lifespan). Altogether our data suggest AF-1 expression during aging to be a mechanism that affects healthy aging and age-related stress resistance depending on the gender of the fly.

TNF-α mediates mitochondrial uncoupling and enhances ROS-dependent cell migration via NF-κB activation in liver cells.

Development of hepatocellular carcinoma (HCC) is accompanied by a continuous increase in reactive oxygen species (ROS) levels. To investigate the primary source of ROS in liver cells, we used tumor necrosis factor-alpha (TNF-α) as stimulus. Applying inhibitors against the respiratory chain complexes, we identified mitochondria as primary source of ROS production. TNF-α altered mitochondrial integrity by mimicking a mild uncoupling effect in liver cells, as indicated by a 40% reduction in membrane potential and ATP depletion (35%). TNF-α-induced ROS production activated NF-κB 3.5-fold and subsequently enhanced migration up to 12.7-fold. This study identifies complex I and complex III of the mitochondrial respiratory chain as point of release of ROS upon TNF-α stimulation of liver cells, which enhances cell migration by activating NF-κB signalling.

Regulation of CD95/Fas signaling at the DISC.

CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation.

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP(L), and the short c-FLIP isoform, c-FLIP(R), inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1.

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression.

The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK-ERK-MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.

Modulation of CD4+ T-cell activation by CD95 co-stimulation.

CD95 is a dual-function receptor that exerts pro- or antiapoptotic effects depending on the cellular context, the state of activation, the signal threshold and the mode of ligation. In this study, we report that CD95 engagement modulates TCR/CD3-driven signaling pathways in resting T lymphocytes in a dose-dependent manner. While high doses of immobilized CD95 agonists silence T cells, lower concentrations augment activation and proliferation. We analyzed the co-stimulatory capacity of CD95 in detail in resting human CD4(+) T cells, and demonstrate that low-dose ligand-induced co-internalization of CD95 and TCR/CD3 complexes enables non-apoptotic caspase activation, the prolonged activation of MAP kinases, the upregulation of antiapoptotic proteins associated with apoptosis resistance, and the activation of transcription factors and cell-cycle regulators for the induction of proliferation and cytokine production. We propose that the levels of CD95L on antigen-presenting cells (APCs), neighboring T cells or epithelial cells regulate inhibitory or co-stimulatory CD95 signaling, which in turn is crucial for fine-tuning of primary T-cell activation.

Reduction of regulatory T cells in skin lesions but not in peripheral blood of patients with systemic scleroderma.

OBJECTIVE: To determine the frequency and suppressive capacity of regulatory T cells (T(reg)) and their association with clinical parameters in patients with systemic scleroderma (SSc). METHODS: Peripheral blood from 25 patients with SSc, 15 patients with localised scleroderma (LS) and 29 healthy controls (HC) was studied. Analysis of CD4(+) forkhead box P3 (Foxp3)(+) and CD4(+)CD25(++)Foxp3(+) T(reg) subpopulations was carried out by flow cytometry and cell proliferation was quantified by (3)H-thymidine incorporation. Quantitative analysis of T(reg) was further performed in skin biopsies from 17 patients with SSc and 21 patients with LS using anti-CD4 and anti-Foxp3 monoclonal antibodies for immunohistochemistry. RESULTS: The frequency of CD4(+)Foxp3(+) and CD4(+)CD25(++)Foxp3(+) T(reg) in peripheral blood from patients with SSc was not significantly different from that of patients with LS or HC. The suppressive capacity of CD4(+)CD25(++) T(reg) in SSc was also found to be similar to that of HC. Phenotypic and functional data revealed no significant difference between the limited or diffuse form of SSc. Moreover, therapy with bosentan showed no significant effect on the frequency of T(reg) during the course of the disease. However, the frequency of T(reg) in skin lesions from patients with SSc or LS, determined as the percentage of CD4(+) cells expressing Foxp3 in the inflammatory infiltrate, was significantly reduced compared with other inflammatory skin diseases. CONCLUSION: These results indicate that although the authors found no defect in the frequency or function of peripheral T(reg) subpopulations, the reduction of CD4(+)Foxp3(+) T(reg) in the skin of patients with SSc may be important in the pathogenesis of the disease.

Characterization of regulatory T cells in patients with dermatomyositis.

The purpose of this study was to characterize regulatory T cells (T(reg)) in skin lesions and peripheral blood from patients with dermatomyositis (DM) and to determine the serum levels of regulatory cytokines in the disease. In skin biopsy specimens from patients with DM, immunohistochemistry was performed for CD4(+), CD25(+), forkhead/winged helix transcription factor (FoxP3)(+), transforming growth factor (TGF)-ß(+) and interleukin (IL)-10(+) cells. Additionally, we defined the number of T(reg) subpopulations in peripheral blood by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RO, CD95, CCR4 and CLA. The levels of TGF-ß and IL-10 were also determined in serum samples from patients with DM by enzyme-linked immunosorbent assays. Controls included patients with cutaneous lupus erythematosus, psoriasis and atopic dermatitis (AD) as well as healthy donors. The frequency of FoxP3(+) cells was significantly reduced in skin lesions from patients with DM (p < 0.001) compared to psoriasis and AD. Moreover, the number of cells positive for TGF-ß was lower in DM than in psoriasis and AD, while IL-10(+) cells were significantly reduced only compared to psoriasis. The number of CD4(+)CD25(++)FoxP3(+) T(reg) in the peripheral blood of patients with DM was significantly reduced compared to healthy controls (p < 0.05), whereas other cell populations showed no significant differences. Finally, TGF-ß and IL-10 serum levels were significantly lower in patients with DM compared to healthy controls (p < 0.05). These data suggest that the depletion of T(reg) and their main effector cytokines in the skin and the serum of patients with DM may be an important factor in the pathogenesis of the disease.

Rocaglamide sensitizes leukemic T cells to activation-induced cell death by differential regulation of CD95L and c-FLIP expression.

Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid - the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.

Low number of regulatory T cells in skin lesions of patients with cutaneous lupus erythematosus.

OBJECTIVE: To define the phenotype and function of CD4+,CD25+ regulatory T cells (Treg) in patients with cutaneous lupus erythematosus (CLE), a heterogeneous autoimmune disease characterized primarily by inflammatory skin lesions. METHODS: The number of Treg in skin specimens obtained from patients with various subtypes of CLE was investigated by immunohistochemical analysis, using anti-Foxp3 and anti-CD4 monoclonal antibodies. Furthermore, characterization of peripheral blood CD4+,CD25+ Treg from normal healthy donors and patients with CLE was carried out by flow cytometry, analyzing the expression of Foxp3 and Treg subpopulations. We also purified CD4+,CD25(high) Treg obtained from patients with CLE and tested the sensitivity of these cells to CD95L-mediated apoptosis. RESULTS: Quantitative analysis of CD4+ T cells in skin lesions from patients with CLE revealed that the number was similar to that in lesions from patients with other chronic inflammatory diseases, but the number of Foxp3+ Treg in CLE was significantly reduced. There was no correlation between disease subtype and the frequency of Foxp3+ Treg in the skin of patients with CLE. In peripheral blood, no significant differences were observed in the number and phenotype of CD4+,CD25+ Treg or in the sensitivity to apoptosis of CD4+,CD25(high) Treg derived from patients with CLE and those derived from normal healthy donors. CONCLUSION: These data suggest that an organ-specific abnormality of Treg in the skin underscores the importance of analyzing Treg in the affected tissue. Such a local process might give insight into the pathogenic mechanisms of CLE and differs from a global peripheral dysfunction as reported for patients with a systemic manifestation of the disease.

Sustained JNK signaling by proteolytically processed HPK1 mediates IL-3 independent survival during monocytic differentiation.

We studied monocytic differentiation of primary mouse progenitor cells to understand molecular mechanisms of differentiation. We found a tightly controlled non-apoptotic activation of caspase-3 that correlated with differentiation. Although caspase activity was already detected during monocytic differentiation, a caspase-3 target has not been identified yet. We show that hematopoietic progenitor kinase 1 (HPK1) is processed towards its N- and C-terminal fragments during monocytic differentiation. While HPK1 is an immunoreceptor-proximal kinase in T and B cells, its role in myeloid cells is elusive. Here, we show that the N-terminal cleavage product, HPK1-N, comprising the kinase domain, confers progenitor cell survival independent of the growth factor IL-3. Furthermore, HPK1-N causes differentiation of progenitor cells towards the monocytic lineage. In contrast to full-length kinase, HPK1-N is constitutively active causing sustained JNK activation, Bad phosphorylation and survival. Blocking of caspase activity during differentiation of primary mouse progenitor cells leads to reduced HPK1-N levels, suppressed JNK activity and attenuated monocytic differentiation. Our work explains growth factor-independent survival during monocytic differentiation by caspase-mediated processing of HPK1 towards HPK1-N.

Pathophysiology of cutaneous lupus erythematosus--novel aspects.

The pathophysiology of cutaneous lupus erythematosus (CLE) has been investigated in numerous studies demonstrating that the combination of specific cellular and molecular events is leading to inflammation and tissue damage in this disease. However, a complete understanding of the diverse pathophysiological mechanisms and interactions does not exist. Various environmental factors influence the clinical expression of CLE and a striking relationship has emerged between sunlight exposure and the various subtypes of this disease. In the past years, photoprovocation tests with different ultraviolet (UV) wavelengths have been approved to be an optimal way to evaluate photosensitivity in patients with CLE. Furthermore, research on the pathogenetic mechanisms of UV-induced skin lesions has become an increasingly dynamic field and several new aspects of this disease could be identified. In this review, the impact of UV exposure that contributes to the manifestations of CLE is discussed and recently reported mechanisms in the pathophysiology of this disease are considered including the clearance of apoptotic cells, expression of inducible nitric oxide synthase, function of CD4(+)CD25(+) regulatory T cells, and the role of chemokines for lymphocyte recruitment. Elucidation of the relevant factors might lead to future development of effective strategies to prevent abnormal reactivity in patients with CLE.

Paucity of FOXP3+ cells in skin and peripheral blood distinguishes Sézary syndrome from other cutaneous T-cell lymphomas.

Cutaneous T-cell lymphomas (CTCL) are mainly comprised of two variants: mycosis fungoides (MF) with CD4(+) tumor cells confined to the skin and the leukemic Sézary syndrome with tumor cell spread to the blood. In this study, we investigated cutaneous expression of the regulatory T-cell (T(reg)) marker FOXP3 in 30 CTCL patients. Immunohistochemical analysis revealed significantly lower numbers of CD4(+)FOXP3(+) cells within the dermal lymphomononuclear infiltrate of Sézary patients (16% FOXP3(+) cells of CD4(+) cells) in contrast to MF (43% FOXP3(+) cells (P<0.05)) and rare types of CTCL (45% FOXP3(+) cells). Furthermore, CD4(+)FOXP3(+) T cells were also markedly reduced in the CD4(+) population within the peripheral blood of Sézary patients compared to controls as determined by fluorescence-activated cell sorter, quantitative PCR and functional analyses. The data support the conclusion that the neoplastic cells in CTCL do not express the T(reg) marker FOXP3. Our data also identify Sézary syndrome as, to our knowledge, the first reported neoplastic disease with a clear reduction in T(reg) numbers within the CD4(+) population. This lack of T(reg) might account for the more aggressive nature of Sézary syndrome compared with other CTCL.