AhR promotes phosphorylation of ARNT isoform 1 in human T cell malignancies as a switch for optimal AhR activity.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform­specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform­specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.

Burn-Induced Apoptosis in the Livers of Aged Mice Is Associated With Caspase Cleavage of Bcl-xL.

INTRODUCTION: Older adult burn victims have poorer outcomes than younger burn victims. The liver is critical for the recovery of patients with burns. Postburn hepatic apoptosis in young individuals compromises liver integrity; however, this pathway has not yet been studied in older individuals. Because aged animals with burns suffer significant liver damage, we hypothesized that apoptosis is altered in these animals and may affect liver function. Understanding postburn hepatic apoptosis and its effects on liver function in aged animals may help improve outcomes in older patients. METHODS: We compared the protein and gene expression levels in young and aged mice after a 15% total-body-surface-area burn. Liver and serum samples were collected at different time points after injury. RESULTS: Caspase-9 expression in liver tissue was downregulated by 47% in young animals and upregulated by 62% in aged animals 9 h postburn (P < 0.05). The livers of aged mice showed a Bcl-extra-large (Bcl-xL) transcription increase only after 6 h; however, the livers of young mice exhibited 4.3-fold, 14.4-fold, and 7.8-fold Bcl-xL transcription increases at 3, 6, and 9 h postburn, respectively (P < 0.05). The livers of young mice showed no changes in Caspase-9, Caspase-3, or Bcl-xL protein levels during the early postburn period. In contrast, the livers of aged mice contained cleaved caspase-9, reduced full-length caspase-3, and an accumulation of ΔN-Bcl-x at 6 and 9 h postburn (P < 0.05). p21 expression decreased in aged mice; however, it was significantly increased in the liver tissue of young mice postburn (P < 0.05). Serum amyloid A1 and serum amyloid A2 serum protein levels were 5.2- and 3.1-fold higher in young mice than in aged mice, respectively, at 6 and 9 h postburn (P < 0.05). CONCLUSIONS: Livers of aged mice exhibited different apoptotic processes compared to those of young mice early after burn injury. Collectively, burn-induced liver apoptosis in aged mice compromises hepatic serum protein production.

Transcriptomics, metabolomics, and in-silico drug predictions for liver damage in young and aged burn victims.

Burn induces a systemic response affecting multiple organs, including the liver. Since the liver plays a critical role in metabolic, inflammatory, and immune events, a patient with impaired liver often exhibits poor outcomes. The mortality rate after burns in the elderly population is higher than in any other age group, and studies show that the liver of aged animals is more susceptible to injury after burns. Understanding the aged-specific liver response to burns is fundamental to improving health care. Furthermore, no liver-specific therapy exists to treat burn-induced liver damage highlighting a critical gap in burn injury therapeutics. In this study, we analyzed transcriptomics and metabolomics data from the liver of young and aged mice to identify mechanistic pathways and in-silico predict therapeutic targets to prevent or reverse burn-induced liver damage. Our study highlights pathway interactions and master regulators that underlie the differential liver response to burn injury in young and aged animals.

Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms.

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.

Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies.

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways.

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.

BH3-only protein BIM mediates heat shock-induced apoptosis.

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax(-/-)Bak(-/-) cells and better than either Bid(-/-) or dominant-negative caspase-9-expressing cells. Only Bim(-/-) and Bax(-/-)Bak(-/-) cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid(-/-) cells, it readily did so in Bim(-/-) cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1(-/-) cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X(L) with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers.

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-kappaB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process.

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

Cleavage of the apoptosis inhibitor DIAP1 by the apical caspase DRONC in both normal and apoptotic Drosophila cells.

In Drosophila S2 cells, the apical caspase DRONC undergoes a low level of spontaneous autoprocessing. Unintended apoptosis is prevented by the inhibitor of apoptosis DIAP1, which targets the processed form of DRONC for degradation through its E3 ubiquitin protein ligase activity. Recent reports have demonstrated that shortly after the initiation of apoptosis in S2 cells, DIAP1 is cleaved following aspartate residue Asp-20 by the effector caspase DrICE. Here we report a novel caspase-mediated cleavage of DIAP1 in S2 cells. In both living and dying S2 cells, DIAP1 is cleaved by DRONC after glutamate residue Glu-205, located between the first and second BIR domains. The mutation of Glu-205 prevented the interaction of DIAP1 and processed DRONC but had no effect on the interaction with full-length DRONC. The mutation of Glu-205 also had a negative effect on the ability of overexpressed DIAP1 to prevent apoptosis stimulated by the proapoptotic protein Reaper or by UV light. These results expand our knowledge of the events that occur in the Drosophila apoptosome prior to and after receiving an apoptotic signal.

Silencing of the baculovirus Op-iap3 gene by RNA interference reveals that it is required for prevention of apoptosis during Orgyia pseudotsugata M nucleopolyhedrovirus infection of Ld652Y cells.

The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.

Mechanism of Dronc activation in Drosophila cells.

Proteolytic processing is required for the activation of most caspases. However, recent reports have suggested that the activation of the mammalian initiator caspase caspase-9 occurs during dimerization rather than after processing. Previously, we reported that, in normal living Drosophila S2 cells, the initiator caspase Dronc is continuously processed to a 40 kDa form we called Pr1 and that, during apoptosis, a second processed form of 37 kDa is also observed, which we called Pr2. In this study, we determined that Dronc Pr1 is the result of Dronc autoprocessing at amino acid E352, whereas Pr2 results from Drice cleaving full-length Dronc at amino acid D135. By using purified recombinant proteins and expressing Dronc cleavage mutants in S2 cells, we determined that autoprocessing at E352 is crucial for Dronc caspase activity, whereas Drice cleavage at D135 has little effect on Dronc activity. Suppression of the oligomerizing factor Dark by RNA interference revealed that Dark is required for Dronc autoprocessing at E352, whereas RNA interference of the effector caspase Drice revealed that Drice is also required for apoptosis in S2 cells. These results provide the first details of the mechanisms regulating initiator caspase activation in an invertebrate organism.

The Drosophila DIAP1 protein is required to prevent accumulation of a continuously generated, processed form of the apical caspase DRONC.

Although loss of the inhibitor of apoptosis (IAP) protein DIAP1 has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation is unknown. Depletion of the DIAP1 protein in Drosophila S2 cells or the Sf-IAP protein in Spodoptera frugiperda Sf21 cells by RNA interference (RNAi) or cycloheximide treatment resulted in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc or dark largely suppressed this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibited DRICE processing following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induced DRONC processing, which occurred in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulated when dark was silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 resulted in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing was observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase.